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Volume 15,Issue 2,2008 Table of Contents
2008, 15(2):101-104.
DOI: 10.3872/j.issn.1007-385X.2008.2.001
Abstract:
Abstract Great improvement has been made in the 5 year survival of patients with osteosarcoma due to application of novel chemo agent and progression in surgical techniques. However, the treatment outcome of osteosarcoma was bottlenecked by drug resistance and immuno escape mechanism. Recent studies showed that some osteosarcoma cells possess the characteristic of tumor stem cells: capable of self renewal and extensive proliferation. Meanwhile, SP cells, isolated from osteosarcoma cells, have high expression of ABCG2 and can form tumor in NOD/SCID mice, indicating that the drug resistance of osteosarcoma cells might be related to SP cells. Studies on the biomarkers of tumor stem cells and decrease of ABCG2 expression may contribute to the treatment outcome of osteosarcoma. Moreover, research on active and passive immunotherapy and Src protein tyrosine kinase targeted therapy may provide new pathways for treatment of osteosarcoma.
Abstract Great improvement has been made in the 5 year survival of patients with osteosarcoma due to application of novel chemo agent and progression in surgical techniques. However, the treatment outcome of osteosarcoma was bottlenecked by drug resistance and immuno escape mechanism. Recent studies showed that some osteosarcoma cells possess the characteristic of tumor stem cells: capable of self renewal and extensive proliferation. Meanwhile, SP cells, isolated from osteosarcoma cells, have high expression of ABCG2 and can form tumor in NOD/SCID mice, indicating that the drug resistance of osteosarcoma cells might be related to SP cells. Studies on the biomarkers of tumor stem cells and decrease of ABCG2 expression may contribute to the treatment outcome of osteosarcoma. Moreover, research on active and passive immunotherapy and Src protein tyrosine kinase targeted therapy may provide new pathways for treatment of osteosarcoma.
QIU Xiu-chun
,
SHAN Le-qun
,
JI Zhen-gang
,
YANG Tong-tao
,
LONG Hua
,
XU Yan-ming
,
ZHOU Yong
,
MA Bao-an
,
YANG An-gang
,
FAN Qing-yu
2008, 15(2):105-109.
DOI: 10.3872/j.issn.1007-385X.2008.2.002
Abstract:
Abstract Objective： To construct a fusion gene ScFv/tBid against HER2 and investigate its pro popotic effect on osteosarcoma cell line E10. Methods: HER2 expression on the surface of E10 cells was detected by immunofluorescent staining and flow cytometry (FCM)， then e23sFv fragment, a single chain HER2 antibody, was linked with a PE translocation domain (PE aa253-364) and tBid . The recombinant tBid gene was cloned into a pCMV plasmid to obtain pCMV ScFv/tBid, which was then transfected into E10 cells. Immunofluorescent staining was used to examine the expression of target protein and morphological changes of cells. Meanwhile, the pro apoptotic effect of ScFv/tBid gene was analyzed by Annexin V FITC staining and TUNEL staining. Results: Flow cytometry showed HER2 expression on cell surface, and the recombinant plasmid, pCMV ScFv/tBid, was successfully constructed and transfected into E10 cells. Overexpression of tBid protein was detected in E10 cells as revealed by immunofluorescent staining; and shrinkage and nuclear condensation were also noticed in E10 cells. Annexin V FITC staining and FCM revealed that the apoptosis rate of E10 cells was 161% after transfection with pCMV ScFv/tBid; the apoptosis rate in the control cells was 4.5%. TUNEL staining showed typical apoptosis characteristics of E10 cells after transfection. Conclusion: The recombinant anti HER2 fusion gene, ScFv/tBid, can be expressed in E10 cells transfected with pCMV ScFv/tBid, and subsequently induce apoptosis.
Abstract Objective： To construct a fusion gene ScFv/tBid against HER2 and investigate its pro popotic effect on osteosarcoma cell line E10. Methods: HER2 expression on the surface of E10 cells was detected by immunofluorescent staining and flow cytometry (FCM)， then e23sFv fragment, a single chain HER2 antibody, was linked with a PE translocation domain (PE aa253-364) and tBid . The recombinant tBid gene was cloned into a pCMV plasmid to obtain pCMV ScFv/tBid, which was then transfected into E10 cells. Immunofluorescent staining was used to examine the expression of target protein and morphological changes of cells. Meanwhile, the pro apoptotic effect of ScFv/tBid gene was analyzed by Annexin V FITC staining and TUNEL staining. Results: Flow cytometry showed HER2 expression on cell surface, and the recombinant plasmid, pCMV ScFv/tBid, was successfully constructed and transfected into E10 cells. Overexpression of tBid protein was detected in E10 cells as revealed by immunofluorescent staining; and shrinkage and nuclear condensation were also noticed in E10 cells. Annexin V FITC staining and FCM revealed that the apoptosis rate of E10 cells was 161% after transfection with pCMV ScFv/tBid; the apoptosis rate in the control cells was 4.5%. TUNEL staining showed typical apoptosis characteristics of E10 cells after transfection. Conclusion: The recombinant anti HER2 fusion gene, ScFv/tBid, can be expressed in E10 cells transfected with pCMV ScFv/tBid, and subsequently induce apoptosis.
2008, 15(2):110-114.
DOI: 10.3872/j.issn.1007-385X.2008.2.003
Abstract:
Abstract Objective: To explore the anti tumor effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) against giant cell tumor of bone (GCT), and to study the influence of IFN γ on the anti tumor effects of TRAIL and the possible mechanisms. MethodsFresh specimens were obtained from 9 patients who were pathologically diagnosed as having GCT in the orthopaedics department of No. 301 Hospital PLA from 2003 to 2006. The GCT cells from specimens were divided into 4 groups: control group, TRAIL group, IFN γ group and IFN γ plus TRAIL group. The cell inhibition was examined by CCK 8 assay. Cell apoptosis was detected by flow cytometric analysis and TUNEL assays. RT PCR was applied to semi quantitatively assay the mRNA expression of TRAIL, DR4 and DR5 in GCT cells before and after the treatment with IFN γ (500 U/ml). Results: The GCT cell survival rate was （98.64±0.31）% in the control group. TRAIL alone (100, 500,1 000 μg/L) had no obvious effect on tumor cell proliferation. After treatment with IFN γ at 500 and 1 000 U/ml , the tumor cell survival rates were (94.05±1.89) % and (90.47±2.66) %, respectively. After treatement with IFN γ (500 U /ml) and TRAIL (100,200 μg/L), the tumor cell survival rates were (84.65±246)% and (77.65±3.14)%, respectively; and FCM showed that their apoptosis rates were (2794±288)% and (38.65±2.46)%, respectively ; the apoptosis rate was (1.77±0.49)% in the control group. TUNEL revealed that the apoptosis rates were (1963±351)% and (29.28±4.80)% , respectively; and the rate was (1.1±0.17)% in the control group. RT PCR showed that the expression levels of TRAIL, DR4 and DR5 in tumor cells were up regulated after treatment with IFN γ (500 U /ml) for 24 h. Conclusion: GCT cells are not sensitive to TRAIL. IFN γ can up regulate the apoptosis rate of GCT cells induced by TRAIL, which might be associated with the up regulation of TRAIL, DR4 and DR5 mRNA expression by IFN γ.
Abstract Objective: To explore the anti tumor effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) against giant cell tumor of bone (GCT), and to study the influence of IFN γ on the anti tumor effects of TRAIL and the possible mechanisms. MethodsFresh specimens were obtained from 9 patients who were pathologically diagnosed as having GCT in the orthopaedics department of No. 301 Hospital PLA from 2003 to 2006. The GCT cells from specimens were divided into 4 groups: control group, TRAIL group, IFN γ group and IFN γ plus TRAIL group. The cell inhibition was examined by CCK 8 assay. Cell apoptosis was detected by flow cytometric analysis and TUNEL assays. RT PCR was applied to semi quantitatively assay the mRNA expression of TRAIL, DR4 and DR5 in GCT cells before and after the treatment with IFN γ (500 U/ml). Results: The GCT cell survival rate was （98.64±0.31）% in the control group. TRAIL alone (100, 500,1 000 μg/L) had no obvious effect on tumor cell proliferation. After treatment with IFN γ at 500 and 1 000 U/ml , the tumor cell survival rates were (94.05±1.89) % and (90.47±2.66) %, respectively. After treatement with IFN γ (500 U /ml) and TRAIL (100,200 μg/L), the tumor cell survival rates were (84.65±246)% and (77.65±3.14)%, respectively; and FCM showed that their apoptosis rates were (2794±288)% and (38.65±2.46)%, respectively ; the apoptosis rate was (1.77±0.49)% in the control group. TUNEL revealed that the apoptosis rates were (1963±351)% and (29.28±4.80)% , respectively; and the rate was (1.1±0.17)% in the control group. RT PCR showed that the expression levels of TRAIL, DR4 and DR5 in tumor cells were up regulated after treatment with IFN γ (500 U /ml) for 24 h. Conclusion: GCT cells are not sensitive to TRAIL. IFN γ can up regulate the apoptosis rate of GCT cells induced by TRAIL, which might be associated with the up regulation of TRAIL, DR4 and DR5 mRNA expression by IFN γ.
2008, 15(2):115-119.
DOI: 10.3872/j.issn.1007-385X.2008.2.004
Abstract:
To construct an adenovirus mediated mouse endostatin vector (Ad mEndo) and to observe its inhibitory effect on the pulmonary metastasis of osteosarcoma in nude mice, so as to discuss the relationship between ES expression and the pulmonary metastasis of osteosarcoma. Methods: Recombinant adenovirus plasmid pDC315 mEndo was constructed and used to prepare recombinant Ad mEndo. Osteosarcoma MG 63 cells were subcutaneously injected into the right fore limbs to establish nude mouse model of osteosarcoma; and the models were randomly divided into 3 groups: Ad mEndo group, Ad EGFP group and PBS group; animals receiving no transplantation served as blank control. The corresponding agents were injected (20 μl per time) for a consecutive of 5 times on a weekly basis. The tumor volumes, histopathological characteristics were observed; ELISA was employed to examine the serum ES level. Animals were sacrificed 7 weeks later and the pulmonary metastasis was observed. Results: Sixteen days later，the tumor volume was (1.53±005)cm 3 in Ad EGFP group, (1.56±0.07)cm 3 in the PBS group, and (0.91±0.03)cm 3 in the Ad mEndo group, with the tumor inhibitory rate being 40.7％ in the last group. The serum ES level in the Ad mEndo group was significantly higher than that of the other groups ( P <0.05). No pulmonary metastasis was noticed in the Ad mEndo group, but extensive metastases were seen in the lungs of the other 2 groups, with the metastasis rates being 80% and 90%. Mice without metastasis had significantly higher levels of ES compared with those with metastasis ( P <0.05). Conclusion: Adenovirus mediated mouse endostatin can significantly inhibit the pulmonary metastasis of osteosarcoma in nude mice, indicating a directly link between expression of ES and pulmonary metastasis.
To construct an adenovirus mediated mouse endostatin vector (Ad mEndo) and to observe its inhibitory effect on the pulmonary metastasis of osteosarcoma in nude mice, so as to discuss the relationship between ES expression and the pulmonary metastasis of osteosarcoma. Methods: Recombinant adenovirus plasmid pDC315 mEndo was constructed and used to prepare recombinant Ad mEndo. Osteosarcoma MG 63 cells were subcutaneously injected into the right fore limbs to establish nude mouse model of osteosarcoma; and the models were randomly divided into 3 groups: Ad mEndo group, Ad EGFP group and PBS group; animals receiving no transplantation served as blank control. The corresponding agents were injected (20 μl per time) for a consecutive of 5 times on a weekly basis. The tumor volumes, histopathological characteristics were observed; ELISA was employed to examine the serum ES level. Animals were sacrificed 7 weeks later and the pulmonary metastasis was observed. Results: Sixteen days later，the tumor volume was (1.53±005)cm 3 in Ad EGFP group, (1.56±0.07)cm 3 in the PBS group, and (0.91±0.03)cm 3 in the Ad mEndo group, with the tumor inhibitory rate being 40.7％ in the last group. The serum ES level in the Ad mEndo group was significantly higher than that of the other groups ( P <0.05). No pulmonary metastasis was noticed in the Ad mEndo group, but extensive metastases were seen in the lungs of the other 2 groups, with the metastasis rates being 80% and 90%. Mice without metastasis had significantly higher levels of ES compared with those with metastasis ( P <0.05). Conclusion: Adenovirus mediated mouse endostatin can significantly inhibit the pulmonary metastasis of osteosarcoma in nude mice, indicating a directly link between expression of ES and pulmonary metastasis.
2008, 15(2):120-124.
DOI: 10.3872/j.issn.1007-385X.2008.2.005
Abstract:
To investigate the inhibitory effects of endostatin ( ES ) on the growth and metastasis of transplanted osteosarcoma UMR106 cells in nude mice. Methods： pBudCE4.1/ES was amplified and transfected into osteosarcoma cell line UMR106 through lipidosome; the non transfected UMR106 cells were eliminated by Zeocin. The proliferation of ES UMR106 cells was examined by MTT assay and the production of ES by ES UMR106 cells was investigated by ELISA. The effects of ES on the in vitro proliferation of vascular endothelial cells were observed by MTT assay. Sixteen nude mice were randomly divided into 2 groups: one group was transplanted with UMR106 cells and the other with ES UMR106 cells. The tumor size, pathological observation, tumor angiogenesis, and the pulmonary metastasis were observed. Results： The proliferation of UMR106 cells was not affected by ES transfection. ES UMR106 cells expressed bioactive ES, and the ES level in the medium was higher than 350 ng/ml 48 h after transfection. ES produced by ES UMR106 cells significantly inhibited the proliferation of vascular endothelial cells. Compared with UMR106 transplanted tumor, ES UMR106 transplanted tumor grew slowly in nude mice, and the formed tumors was well margined and had less angiogenesis and mass necrosis. Two of the 8 mice transplanted with UMP106 cells had pulmonary metastasis and no metastasis was found in ES UMR106 transplanted group. Conclusion: Transfection with recombinant endostatin plasmid can inhibit the growth and metastasis of transplanted osteosarcoma in nude mice by inhibiting vascular endothelial cell proliferation.
To investigate the inhibitory effects of endostatin ( ES ) on the growth and metastasis of transplanted osteosarcoma UMR106 cells in nude mice. Methods： pBudCE4.1/ES was amplified and transfected into osteosarcoma cell line UMR106 through lipidosome; the non transfected UMR106 cells were eliminated by Zeocin. The proliferation of ES UMR106 cells was examined by MTT assay and the production of ES by ES UMR106 cells was investigated by ELISA. The effects of ES on the in vitro proliferation of vascular endothelial cells were observed by MTT assay. Sixteen nude mice were randomly divided into 2 groups: one group was transplanted with UMR106 cells and the other with ES UMR106 cells. The tumor size, pathological observation, tumor angiogenesis, and the pulmonary metastasis were observed. Results： The proliferation of UMR106 cells was not affected by ES transfection. ES UMR106 cells expressed bioactive ES, and the ES level in the medium was higher than 350 ng/ml 48 h after transfection. ES produced by ES UMR106 cells significantly inhibited the proliferation of vascular endothelial cells. Compared with UMR106 transplanted tumor, ES UMR106 transplanted tumor grew slowly in nude mice, and the formed tumors was well margined and had less angiogenesis and mass necrosis. Two of the 8 mice transplanted with UMP106 cells had pulmonary metastasis and no metastasis was found in ES UMR106 transplanted group. Conclusion: Transfection with recombinant endostatin plasmid can inhibit the growth and metastasis of transplanted osteosarcoma in nude mice by inhibiting vascular endothelial cell proliferation.
YANG Tong-tao
,
LI Cun-xiao
,
GAO Jie
,
HUANG Li-jun
,
ZHANG Yong
,
ZHOU Yong
,
FAN Qing-yu
,
MA Bao-an
2008, 15(2):125-128.
DOI: 10.3872/j.issn.1007-385X.2008.2.006
Abstract:
To explore the inhibitory effect of survivin targeted siRNA on human osteosarcoma cell line MG63 in vivo and in vitro. Methods: According to the survivin cDNA sequence, the specific RNA interference (RNAi) fragments were designed and synthesized, which were then cloned into pSilencer 3.0 H1 neo plasmid vector to construct pSiS vector. MG63 cells were transfected with RNAi vectors and negative control vector separately; the stably transfected cell strains were selected by G418; and cell growth was assessed by cell counting. Expression of survivin protein was investigated by Western blotting. Apoptosis analysis was examined by AnnexinⅤ method. Tumorigenesis assay was conducted in nude mice. Results: The specific survivin targeted siRNA eukaryotic vector pSiS was successfully constructed, and transfectants were obtained. Compared with blank vector transfected cells and un transfected cells, MG63/pSiS cells had a slow growth ( P <0.01). Expression of survivin protein was significantly inhibited in MG63/pSiS cells, which had an increased apoptotic rate ( P <0.01). The tumorigenesis of transplanted MG63/pSiS was greatly inhibited. Conclusion：Survivin targeted siRNA can promote the apoptosis of MG63 cells, and inhibit the in vitro growth and in vivo tumor formation in nude mice.
To explore the inhibitory effect of survivin targeted siRNA on human osteosarcoma cell line MG63 in vivo and in vitro. Methods: According to the survivin cDNA sequence, the specific RNA interference (RNAi) fragments were designed and synthesized, which were then cloned into pSilencer 3.0 H1 neo plasmid vector to construct pSiS vector. MG63 cells were transfected with RNAi vectors and negative control vector separately; the stably transfected cell strains were selected by G418; and cell growth was assessed by cell counting. Expression of survivin protein was investigated by Western blotting. Apoptosis analysis was examined by AnnexinⅤ method. Tumorigenesis assay was conducted in nude mice. Results: The specific survivin targeted siRNA eukaryotic vector pSiS was successfully constructed, and transfectants were obtained. Compared with blank vector transfected cells and un transfected cells, MG63/pSiS cells had a slow growth ( P <0.01). Expression of survivin protein was significantly inhibited in MG63/pSiS cells, which had an increased apoptotic rate ( P <0.01). The tumorigenesis of transplanted MG63/pSiS was greatly inhibited. Conclusion：Survivin targeted siRNA can promote the apoptosis of MG63 cells, and inhibit the in vitro growth and in vivo tumor formation in nude mice.
2008, 15(2):129-133.
DOI: 10.3872/j.issn.1007-385X.2008.2.007
Abstract:
To prepare Bifidobacterium exopolysaccharide loaded nanoparticles（ B. EPS NPs）and to investigate the effect of B. EPS NPs on the proliferation and apoptosis of human gastric cancer cells transplanted in nude mice. Methods: B. EPS and nanoparticles of Fe3O4 were used to prepare B. EPS NPs by the method of emulsion polymerization. Transplant models of human gastric cancer cells BGC 823 were established in nude mice and were divided into 5 groups after 6 days, namely, the normal saline group, nanoparticle group(NPs), cytoxan (CTX) group, B. EPS group, and B. EPS NPs group. The corresponding agents were used for gastric lavage. The growth of tumor was observed and cell apoptosis was detected by TUNEL. The ultra microstructure of tumor cells was observed under TEM, expression of proli ferating cell nuclear antigen (PCNA), bcl 2 and bax was examined by immunohistochemistry method. Results: The diameters of the empty loaded NPs and B. EPS NPs were (560±21）nm and （960±32）nm, respectively; the drug loading capacity of B. EPS was 30%. The growth of the transplanted tumor was inhibited in B. EPS NPs group, with the inhibitory rate being （46.4±2.94）%, which was higher than that of the B. EPS group(\[320±1.62\]％) and NPs group \[(4.9±0.98\]％). The apoptosis index in B. EPS NPs group (\[66.8±5.58\]％) was significantly higher than that of the B. EPS group（ \[41.3±4.26\]％, P <001）. TEM showed typical apoptotic structures in B. EPS NPs group. Compared with B. EPS group， B. EPS NPs group had markedly lower expression of PCNA and bcl 2 ( P <001) and higher expression of bax ( P <0.01). Conclusion: Nanoparticles can enhance the inhibitory effect and pro apoptosis effect of B. EPS for human gastric cancer (BGC 823) transplanted tumor.
To prepare Bifidobacterium exopolysaccharide loaded nanoparticles（ B. EPS NPs）and to investigate the effect of B. EPS NPs on the proliferation and apoptosis of human gastric cancer cells transplanted in nude mice. Methods: B. EPS and nanoparticles of Fe3O4 were used to prepare B. EPS NPs by the method of emulsion polymerization. Transplant models of human gastric cancer cells BGC 823 were established in nude mice and were divided into 5 groups after 6 days, namely, the normal saline group, nanoparticle group(NPs), cytoxan (CTX) group, B. EPS group, and B. EPS NPs group. The corresponding agents were used for gastric lavage. The growth of tumor was observed and cell apoptosis was detected by TUNEL. The ultra microstructure of tumor cells was observed under TEM, expression of proli ferating cell nuclear antigen (PCNA), bcl 2 and bax was examined by immunohistochemistry method. Results: The diameters of the empty loaded NPs and B. EPS NPs were (560±21）nm and （960±32）nm, respectively; the drug loading capacity of B. EPS was 30%. The growth of the transplanted tumor was inhibited in B. EPS NPs group, with the inhibitory rate being （46.4±2.94）%, which was higher than that of the B. EPS group(\[320±1.62\]％) and NPs group \[(4.9±0.98\]％). The apoptosis index in B. EPS NPs group (\[66.8±5.58\]％) was significantly higher than that of the B. EPS group（ \[41.3±4.26\]％, P <001）. TEM showed typical apoptotic structures in B. EPS NPs group. Compared with B. EPS group， B. EPS NPs group had markedly lower expression of PCNA and bcl 2 ( P <001) and higher expression of bax ( P <0.01). Conclusion: Nanoparticles can enhance the inhibitory effect and pro apoptosis effect of B. EPS for human gastric cancer (BGC 823) transplanted tumor.
2008, 15(2):134-138.
DOI: 10.3872/j.issn.1007-385X.2008.2.008
Abstract:
To construct a recombinant lentivirus plasmid of RNA interference targeting ( MSLN ) gene and to observe its effect on MSLN expression in human ovarian cancer cell line OVCAR 3 and its effect on cell proliferation. Methods: According to the Genbank information of MSLN, four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pRNAT U6.2/Lenti and 5 kinds of plasmids were packaged: LV MSLN negative,LV MSLN shRNA1, LV MSLN shRNA2, LV MSLN shRNA3, and LV MSLN shRNA4; and they were used to transfect OVCAR 3 cells. Western blotting and indirect immunofluorescence were then used to investigate the interfering efficiency. The plasmid with high interfering efficiency was packaged. The cell proliferation test and clone forming test was used to assess the changes in cell proliferation. Results: DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct. Lentivirus packaging was successfully done. Western blotting analysis confirmed that LV MSLN shRNA4 had the highest interfering efficiency (90%). MSLN specifically bound to cytomembrane of OVCAR 3 cells. Expression of MSLN in the interfered cells (OVC shRNA) was weaker than that in the control cells (OVC neg,OVC). OVC shRNA cells(\[11.2±1.3\]×10 5) grew slowly compared to OVC neg cells(\[20.5±2.5\]×10 5) and OVC cells(\[219±23\]×10 5) ( P ＜005）. There was a significant reduction in clone forming rate of OVC shRNA cells (152±2.1)% in comparison with OVC neg cells（\[27.9±2.5\]%) and OVC cells（\[288±31\]%）（ P ＜0.05）.Conclusions: We have successfully constructed MSLN RNAi recombination plasmid LV MSLN shRNA4, which can effectively inhibit MSLN expression and cell proliferation, which paves a way for studying MSLN function and gene therapy.
To construct a recombinant lentivirus plasmid of RNA interference targeting ( MSLN ) gene and to observe its effect on MSLN expression in human ovarian cancer cell line OVCAR 3 and its effect on cell proliferation. Methods: According to the Genbank information of MSLN, four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pRNAT U6.2/Lenti and 5 kinds of plasmids were packaged: LV MSLN negative,LV MSLN shRNA1, LV MSLN shRNA2, LV MSLN shRNA3, and LV MSLN shRNA4; and they were used to transfect OVCAR 3 cells. Western blotting and indirect immunofluorescence were then used to investigate the interfering efficiency. The plasmid with high interfering efficiency was packaged. The cell proliferation test and clone forming test was used to assess the changes in cell proliferation. Results: DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct. Lentivirus packaging was successfully done. Western blotting analysis confirmed that LV MSLN shRNA4 had the highest interfering efficiency (90%). MSLN specifically bound to cytomembrane of OVCAR 3 cells. Expression of MSLN in the interfered cells (OVC shRNA) was weaker than that in the control cells (OVC neg,OVC). OVC shRNA cells(\[11.2±1.3\]×10 5) grew slowly compared to OVC neg cells(\[20.5±2.5\]×10 5) and OVC cells(\[219±23\]×10 5) ( P ＜005）. There was a significant reduction in clone forming rate of OVC shRNA cells (152±2.1)% in comparison with OVC neg cells（\[27.9±2.5\]%) and OVC cells（\[288±31\]%）（ P ＜0.05）.Conclusions: We have successfully constructed MSLN RNAi recombination plasmid LV MSLN shRNA4, which can effectively inhibit MSLN expression and cell proliferation, which paves a way for studying MSLN function and gene therapy.
2008, 15(2):139-143.
DOI: 10.3872/j.issn.1007-385X.2008.2.009
Abstract:
To investigate whether PUMA gene transfection can increase sensitivity of pancreatic cancer cells (PC) to 5 FU induced apoptosis. Methods: PUMA pCEP4 containing full length PUMA cDNA or pCEP4 was transfected into human pancreatic cancer cell line AsPC 1 by lipofectamine transfection, G418 selection was used to select positive cells. AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were separately treated with serial concentrations of 5 FU（0.01 100 μmol/L）. MTT assay was used to determine the cell survival rate in each group and IC 50 of 5 FU was calculated. TUNEL，FCM and DNA ladder observation were employed to study cell apoptosis. Western blotting was performed to detect the expression of PUMA protein. Results: The 5 FU IC 50 values of AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were (12±1.9)μmol/L,（1.6±0.4）μmol/L and (10.4±1.6) μmol/L, respectively, with the sensitivity of AsPC 1/PUMA cells increased by 7.5 folds. 5 FU induced cell apoptosis of AsPC 1 cells in a dose dependent manner, with the apoptosis of AsPC 1/PUMA cells more prominent than those of AsPC 1 and AsPC 1/pCEP4 cells. Low concentration of 5 FU (0.1 μmol/L) induced few apoptosis of AsPC 1/pCEP4 cells（\[1.14±0.28\]%) and AsPC 1 cells (\[0.9±023\]%), and induced apoptosis in AsPC 1/PUMA cells（\[6.47±1.42\]%). High concentration of 5 FU (1.0 μmol/L ) induced apoptosis in all groups, with that in AsPC 1/PUMA cells（\[34.54±9.36\]%) significantly higher than those in AsPC 1/pCEP4 cells（\[15.8±5.15\]%) and AsPC 1 cells (\[12.8±3.74\]%, both P <0.01) . FCM and electrophoresis showed the same results. Expression of PUMA protein in AsPC 1/PUMA cells was significantly higher than those in AsPC 1 and AsPC 1pCEP4 cells. Conclusion: PUMA gene transfection greatly enhances the sensitivity of AsPC 1 cells to 5 FU induced apoptosis.
To investigate whether PUMA gene transfection can increase sensitivity of pancreatic cancer cells (PC) to 5 FU induced apoptosis. Methods: PUMA pCEP4 containing full length PUMA cDNA or pCEP4 was transfected into human pancreatic cancer cell line AsPC 1 by lipofectamine transfection, G418 selection was used to select positive cells. AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were separately treated with serial concentrations of 5 FU（0.01 100 μmol/L）. MTT assay was used to determine the cell survival rate in each group and IC 50 of 5 FU was calculated. TUNEL，FCM and DNA ladder observation were employed to study cell apoptosis. Western blotting was performed to detect the expression of PUMA protein. Results: The 5 FU IC 50 values of AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were (12±1.9)μmol/L,（1.6±0.4）μmol/L and (10.4±1.6) μmol/L, respectively, with the sensitivity of AsPC 1/PUMA cells increased by 7.5 folds. 5 FU induced cell apoptosis of AsPC 1 cells in a dose dependent manner, with the apoptosis of AsPC 1/PUMA cells more prominent than those of AsPC 1 and AsPC 1/pCEP4 cells. Low concentration of 5 FU (0.1 μmol/L) induced few apoptosis of AsPC 1/pCEP4 cells（\[1.14±0.28\]%) and AsPC 1 cells (\[0.9±023\]%), and induced apoptosis in AsPC 1/PUMA cells（\[6.47±1.42\]%). High concentration of 5 FU (1.0 μmol/L ) induced apoptosis in all groups, with that in AsPC 1/PUMA cells（\[34.54±9.36\]%) significantly higher than those in AsPC 1/pCEP4 cells（\[15.8±5.15\]%) and AsPC 1 cells (\[12.8±3.74\]%, both P <0.01) . FCM and electrophoresis showed the same results. Expression of PUMA protein in AsPC 1/PUMA cells was significantly higher than those in AsPC 1 and AsPC 1pCEP4 cells. Conclusion: PUMA gene transfection greatly enhances the sensitivity of AsPC 1 cells to 5 FU induced apoptosis.
2008, 15(2):144-149.
DOI: 10.3872/j.issn.1007-385X.2008.2.010
Abstract:
To explore the immunotherapeutic effect of Ag85A DNA vaccine and Ag85B DNA vaccine in treatment of bladder tumor in rats. Methods: The bladders of female Wistar rats were irrigated with carcinogen methyl nitrosourea (MNU) to create the model of bladder tumor. Totally 48 model rats were evenly randomized into 6 groups: normal saline (NS), pcDNA3.1, bacille Calmette Guérin(BCG), Ag85A DNA vaccine, Ag85B DNA vaccine and Ag85A+ Ag85B DNA vaccine groups. The corresponding drugs were injected into the right hind limbs of rats intramuscularly on day 7, 14, and 21 after model establishment. Animals were sacrificed on day 28, and the spleens were removed aseptically. The percentages of CD4 +T cells and CD8 +T cells in splenocytes were measured by flow cytometry and the value of CD4 +/CD8 + was calculated. Level of serum IFN γ was assayed by ELISA and pathological examination was done. Results: The rat model of bladder tumor was successfully constructed, with a tumorigenesis rate of 100%. The bladder tumor sizes in Ag85A DNA vaccine group, Ag85B DNA vaccine group and Ag85A+ Ag85B DNA vaccine group were decreased after treatment, and the tumor pathological grades were also improved, but the outcomes were not better than those of the BCG group. The percentages of CD4 +T cells in the 4 groups were (17.27±2.95)%， (23.15±156)%， (3080±183)%， (38.05±1.48)%，respectively; and the percentages of CD8 +T cells were (9.03±106)%, (1028±039)%, (11.29±0.74)%, (1314±1.24)%, respectively; the relative values of CD4 +/CD8 + were 1.90±0.10, 2.25±0.08, 2.73±0.19, 297±0.23, respectively; the production of IFN γ in Ag85A DNA vaccine group, Ag85B DNA vaccine group, Ag85A+ Ag85B DNA vaccine group, BCG group were (96.94±1238), (131.03±26.68) , (179.20±28.88)，(240.53±32.17) pg/ml, respectively. The above 4 parameters in the Ag85A DNA vaccine group, Ag85B DNA vaccine group and Ag85A+ Ag85B DNA vaccine group were obviously improved compared with the pcDNA3.1 and NS groups, but were still poorer than those of the BCG group ( P<0.01). The effects of Ag85A+ Ag85B DNA vaccine group were better than those of the Ag85A and Ag85B DNA vaccine group( P< 0.05). Conclusion: Ag85A DNA vaccine and Ag85B DNA vaccine can improve the immune response of rats with bladder tumor; a combination of both has better outcome than they are used alone, but even the combination can not reach the effect of BCG.
To explore the immunotherapeutic effect of Ag85A DNA vaccine and Ag85B DNA vaccine in treatment of bladder tumor in rats. Methods: The bladders of female Wistar rats were irrigated with carcinogen methyl nitrosourea (MNU) to create the model of bladder tumor. Totally 48 model rats were evenly randomized into 6 groups: normal saline (NS), pcDNA3.1, bacille Calmette Guérin(BCG), Ag85A DNA vaccine, Ag85B DNA vaccine and Ag85A+ Ag85B DNA vaccine groups. The corresponding drugs were injected into the right hind limbs of rats intramuscularly on day 7, 14, and 21 after model establishment. Animals were sacrificed on day 28, and the spleens were removed aseptically. The percentages of CD4 +T cells and CD8 +T cells in splenocytes were measured by flow cytometry and the value of CD4 +/CD8 + was calculated. Level of serum IFN γ was assayed by ELISA and pathological examination was done. Results: The rat model of bladder tumor was successfully constructed, with a tumorigenesis rate of 100%. The bladder tumor sizes in Ag85A DNA vaccine group, Ag85B DNA vaccine group and Ag85A+ Ag85B DNA vaccine group were decreased after treatment, and the tumor pathological grades were also improved, but the outcomes were not better than those of the BCG group. The percentages of CD4 +T cells in the 4 groups were (17.27±2.95)%， (23.15±156)%， (3080±183)%， (38.05±1.48)%，respectively; and the percentages of CD8 +T cells were (9.03±106)%, (1028±039)%, (11.29±0.74)%, (1314±1.24)%, respectively; the relative values of CD4 +/CD8 + were 1.90±0.10, 2.25±0.08, 2.73±0.19, 297±0.23, respectively; the production of IFN γ in Ag85A DNA vaccine group, Ag85B DNA vaccine group, Ag85A+ Ag85B DNA vaccine group, BCG group were (96.94±1238), (131.03±26.68) , (179.20±28.88)，(240.53±32.17) pg/ml, respectively. The above 4 parameters in the Ag85A DNA vaccine group, Ag85B DNA vaccine group and Ag85A+ Ag85B DNA vaccine group were obviously improved compared with the pcDNA3.1 and NS groups, but were still poorer than those of the BCG group ( P<0.01). The effects of Ag85A+ Ag85B DNA vaccine group were better than those of the Ag85A and Ag85B DNA vaccine group( P< 0.05). Conclusion: Ag85A DNA vaccine and Ag85B DNA vaccine can improve the immune response of rats with bladder tumor; a combination of both has better outcome than they are used alone, but even the combination can not reach the effect of BCG.
2008, 15(2):150-154.
DOI: 10.3872/j.issn.1007-385X.2008.2.011
Abstract:
To investigate the reversing effect of recombinant mutant human tumor necrosis factor (rmh TNF) on cisplatin(DDP) resistant human ovarian cancer cell line SKOV3/DDP in vitro and the related mechanism. Methods: DDP resistant human ovarian cancer cell line SKOV3/DDP was cultured in vitro. The cytotoxic effect of rmh TNF to SKOV3/DDP cells was examined by MTT assay and the nontoxic dose of rmh TNF was identified. The changes of DDP resistance was observed after cells were treated with nontoxic dose of rmh TNF by MTT assay. The expre ssion of GST π protein was examined by flow cytometry at different periods after rmh TNF intervention; RT PCR was used to analyze the expression of MDR1 gene in SKOV3/DDP cells before and after rmh TNF treatment. Results:（1）rmh TNF at 50 122.34 U/ml showed no evident inhibitory effect on the growth of SKOV3/DDP cells (the cell survival rate higher than 90%); and 100 U/ml was chosen for the reversing experiment ( nontoxic dose).（2）IC 50 values of SKOV3/DDP cells were (2329±0.43), (8.97±0.69) and (6.43±0.79) μg/ml after treatment with DDP for 24, 48 and 72 h, respectively; and the values decreased to （19.50±0.50）,（4.34±0.43） and （2.44±0.02）μg/ml after combined treatment with 100 U/ml rmh TNF, respectively.（3）Expression of GST π protein and MDR1 gene decreased with the prolongation of rmh TNF treatment. Conclusion: rmh TNF has reversal effect on the DDP resistant cell line SKOV3/DDP, and the mechanism may be associated with the down regulation of GST π protein and MDR1 gene expression.
To investigate the reversing effect of recombinant mutant human tumor necrosis factor (rmh TNF) on cisplatin(DDP) resistant human ovarian cancer cell line SKOV3/DDP in vitro and the related mechanism. Methods: DDP resistant human ovarian cancer cell line SKOV3/DDP was cultured in vitro. The cytotoxic effect of rmh TNF to SKOV3/DDP cells was examined by MTT assay and the nontoxic dose of rmh TNF was identified. The changes of DDP resistance was observed after cells were treated with nontoxic dose of rmh TNF by MTT assay. The expre ssion of GST π protein was examined by flow cytometry at different periods after rmh TNF intervention; RT PCR was used to analyze the expression of MDR1 gene in SKOV3/DDP cells before and after rmh TNF treatment. Results:（1）rmh TNF at 50 122.34 U/ml showed no evident inhibitory effect on the growth of SKOV3/DDP cells (the cell survival rate higher than 90%); and 100 U/ml was chosen for the reversing experiment ( nontoxic dose).（2）IC 50 values of SKOV3/DDP cells were (2329±0.43), (8.97±0.69) and (6.43±0.79) μg/ml after treatment with DDP for 24, 48 and 72 h, respectively; and the values decreased to （19.50±0.50）,（4.34±0.43） and （2.44±0.02）μg/ml after combined treatment with 100 U/ml rmh TNF, respectively.（3）Expression of GST π protein and MDR1 gene decreased with the prolongation of rmh TNF treatment. Conclusion: rmh TNF has reversal effect on the DDP resistant cell line SKOV3/DDP, and the mechanism may be associated with the down regulation of GST π protein and MDR1 gene expression.
2008, 15(2):155-158.
DOI: 10.3872/j.issn.1007-385X.2008.2.012
Abstract:
Objective:To investigate the therapeutic effects of anti CD3 monoclonal antibody activated autologous killer cells (CD3AK) in treatment of advanced malignant tumors and their influence on the immune function of patients.Methods: Fifty five patients with solid tumors and 12 with lymphoma, who were admitted to the Qianfoshan Hospital, Shandong University, were treated with CD3AK cells. IL 2 and CD3 Ab were used to induce autologous CD3AK cells from the peripheral blood mono nuclear cells (PBMC) from 67 patients. T cells subsets and the level of NK cells（CD16 +CD56 +）were determined before and after the treatment. Results: The effective rate of autologous CD3AK cells was 2545% for the solid tumors and the clinical beneficial rate was 74.54%; the effective rate was 83.33% for lymphoma and the clinical beneficial rate was 91.67%. The CD8 +cell subsets was decreased after treatment（ P <0.05); the CD3 +，CD4 +cell subsets and CD4 +/CD8 + ratio were significantly increased（ P <0.05）after one course of treatment with CD3AK cells. Conclusion: Autologous CD3AK cells can improve the unbalanced T cell subsets in patients with advanced malignant tumors, upgrade the quality of life of patients, and effectively control tumor growth, with better safety and without adverse effect.
Objective:To investigate the therapeutic effects of anti CD3 monoclonal antibody activated autologous killer cells (CD3AK) in treatment of advanced malignant tumors and their influence on the immune function of patients.Methods: Fifty five patients with solid tumors and 12 with lymphoma, who were admitted to the Qianfoshan Hospital, Shandong University, were treated with CD3AK cells. IL 2 and CD3 Ab were used to induce autologous CD3AK cells from the peripheral blood mono nuclear cells (PBMC) from 67 patients. T cells subsets and the level of NK cells（CD16 +CD56 +）were determined before and after the treatment. Results: The effective rate of autologous CD3AK cells was 2545% for the solid tumors and the clinical beneficial rate was 74.54%; the effective rate was 83.33% for lymphoma and the clinical beneficial rate was 91.67%. The CD8 +cell subsets was decreased after treatment（ P <0.05); the CD3 +，CD4 +cell subsets and CD4 +/CD8 + ratio were significantly increased（ P <0.05）after one course of treatment with CD3AK cells. Conclusion: Autologous CD3AK cells can improve the unbalanced T cell subsets in patients with advanced malignant tumors, upgrade the quality of life of patients, and effectively control tumor growth, with better safety and without adverse effect.
2008, 15(2):159-162.
DOI: 10.3872/j.issn.1007-385X.2008.2.013
Abstract:
To establish a method for isolating and culturing endothelial progenitor cells（EPCs), which have high potential of proliferation, migration and angiogenesis, from cord blood. Methods: CD133 +cells were selected from fresh cord blood mononuclear cells by magnetic activated cell sorting system (MACS), and were cultured in EGM 2MV medium. EPCs were identified by examining the morphology, cell markers and functions. And the EPCs were compared with human umbilical vein endothelial cells (HUVECs). Results: On the 7 th day, the adherent cells exhibited the small colonies; and on the 21 th day,the colonies were expanded，confluenced and displayed a typical “cobblestone” morphology. On the 14 th day, 90% attached cells took up Dil ac LDL, and bound FITC UEA 1 (double positive fluorescence). The cell markers of CD133 and CD34 decreased from 86.04% to 2.96% and 90.88% to 2.99%, respectively, while CD31 increased from 1.12% to 99.88%. Compared with HUVECS, EPCs had more potent potential of proliferation, migration and angiogenesis( P <0.05). Conclusion: CD133 + MACS can be used to isolate EPCs, with high capacity of proliferation, angiogenesis and migration, from cord blood mononuclear cells.
To establish a method for isolating and culturing endothelial progenitor cells（EPCs), which have high potential of proliferation, migration and angiogenesis, from cord blood. Methods: CD133 +cells were selected from fresh cord blood mononuclear cells by magnetic activated cell sorting system (MACS), and were cultured in EGM 2MV medium. EPCs were identified by examining the morphology, cell markers and functions. And the EPCs were compared with human umbilical vein endothelial cells (HUVECs). Results: On the 7 th day, the adherent cells exhibited the small colonies; and on the 21 th day,the colonies were expanded，confluenced and displayed a typical “cobblestone” morphology. On the 14 th day, 90% attached cells took up Dil ac LDL, and bound FITC UEA 1 (double positive fluorescence). The cell markers of CD133 and CD34 decreased from 86.04% to 2.96% and 90.88% to 2.99%, respectively, while CD31 increased from 1.12% to 99.88%. Compared with HUVECS, EPCs had more potent potential of proliferation, migration and angiogenesis( P <0.05). Conclusion: CD133 + MACS can be used to isolate EPCs, with high capacity of proliferation, angiogenesis and migration, from cord blood mononuclear cells.
2008, 15(2):163-168.
DOI: 10.3872/j.issn.1007-385X.2008.2.014
Abstract:
To investigate P53 gene mutations in 14 tumor cell lines. Methods: Primers were designed according to the intron sequences locating between exons 5 8 of P53 gene for amplification of P53 gene exon 5 8 of the 14 tumor cell lines from 9 kinds of human carcinoma, namely, the lung cancer, gloma, hepatocarcinoma, gastric carcinoma, prostate carcinoma, mammary carcinoma, colonic carcinoma, choroidal melanoma, and retinoblastoma. The PCR products were subjected to electrophoresis; DNA sequencing was performed for the amplified product and the results were compared with wild type P53 gene. Meanwhile, the P53 protein of 14 tumor cell lines was extracted for Western blotting analysis. Results: The products of exons 5 8 amplified by PCR were identical to that expected. The results of DNA sequencing showed that 8 of 14 tumor cell lines had P53 gene mutation in exons 5 8. Novel mutations were found in 4 tumor cell lines, including human lung cancer cell H1299, hepatocacinoma cell Hep3B and 7721, and choroidal melanoma cell OCM 1. The mutations mainly existed in the coding areas of the exons; most of them were missense mutations due to single base replacement; some others were silent mutations; and no mutations were found in the 2 normal cell lines. The results of Western blotting showed that only 6 of 8 mutant lines had P53 protein expression, which was not found in the non mutant lines and the normal cells. Conclusion: We have identified 8 lines with mutant P53 genes among the 14 tumor cell lines, novel mutations are found in 4 tumor cell lines and none in the two normal cell lines.
To investigate P53 gene mutations in 14 tumor cell lines. Methods: Primers were designed according to the intron sequences locating between exons 5 8 of P53 gene for amplification of P53 gene exon 5 8 of the 14 tumor cell lines from 9 kinds of human carcinoma, namely, the lung cancer, gloma, hepatocarcinoma, gastric carcinoma, prostate carcinoma, mammary carcinoma, colonic carcinoma, choroidal melanoma, and retinoblastoma. The PCR products were subjected to electrophoresis; DNA sequencing was performed for the amplified product and the results were compared with wild type P53 gene. Meanwhile, the P53 protein of 14 tumor cell lines was extracted for Western blotting analysis. Results: The products of exons 5 8 amplified by PCR were identical to that expected. The results of DNA sequencing showed that 8 of 14 tumor cell lines had P53 gene mutation in exons 5 8. Novel mutations were found in 4 tumor cell lines, including human lung cancer cell H1299, hepatocacinoma cell Hep3B and 7721, and choroidal melanoma cell OCM 1. The mutations mainly existed in the coding areas of the exons; most of them were missense mutations due to single base replacement; some others were silent mutations; and no mutations were found in the 2 normal cell lines. The results of Western blotting showed that only 6 of 8 mutant lines had P53 protein expression, which was not found in the non mutant lines and the normal cells. Conclusion: We have identified 8 lines with mutant P53 genes among the 14 tumor cell lines, novel mutations are found in 4 tumor cell lines and none in the two normal cell lines.
2008, 15(2):169-172.
DOI: 10.3872/j.issn.1007-385X.2008.2.015
Abstract:
To study the expression of Shh, patched(PTCH), smoothened (Smo) and Gli 1 genes，four components of the Hedgehog signaling pathway, in colonic cancer cell lines, pancreatic cancer cell line and colonic adenoma tissues, and to discuss the influence of cyclopamine, a Smo receptor specific inhibitor, on the growth of these tumor cells. Methods: The expression of Shh, PTCH, Smo and Gli 1 were investigated using RT PCR in 4 colonic cancer cell lines (LS174T, HCT116, SW116 and CT26), pancreatic cancer cell line BxPC3 and 2 colonic adenoma tissues. MTT method was used to study the inhibitory effect of cyclopamine on the growth of these cancer cell lines in vitro . Results: Shh, PTCH, Smo and Gli 1 genes were expressed in 2 of colonic adenoma tissues and SW116, CT26 and BxPC3 cells. The mRNA of Smo and PTCH genes were not found in LS174T and HCT116 cells; the expression of Shh and Gli 1 mRNA were significantly up regulated. Cyclopamine inhibited the growth of SW116, CT 26 and BxPC3, expecially the positive cells of Smo gene. Conclusion: Shh, PTCH, Smo and Gli 1 genes are expressed in colonic cancer cell lines, pancreatic cancer cell line and colonic adenoma tissues, Cyclopamine has obvious inhibitory effect on cells with overexpression of Smo , which implies that Hedgehog signaling pathway might be activated in some tumor cells of digestive system.
To study the expression of Shh, patched(PTCH), smoothened (Smo) and Gli 1 genes，four components of the Hedgehog signaling pathway, in colonic cancer cell lines, pancreatic cancer cell line and colonic adenoma tissues, and to discuss the influence of cyclopamine, a Smo receptor specific inhibitor, on the growth of these tumor cells. Methods: The expression of Shh, PTCH, Smo and Gli 1 were investigated using RT PCR in 4 colonic cancer cell lines (LS174T, HCT116, SW116 and CT26), pancreatic cancer cell line BxPC3 and 2 colonic adenoma tissues. MTT method was used to study the inhibitory effect of cyclopamine on the growth of these cancer cell lines in vitro . Results: Shh, PTCH, Smo and Gli 1 genes were expressed in 2 of colonic adenoma tissues and SW116, CT26 and BxPC3 cells. The mRNA of Smo and PTCH genes were not found in LS174T and HCT116 cells; the expression of Shh and Gli 1 mRNA were significantly up regulated. Cyclopamine inhibited the growth of SW116, CT 26 and BxPC3, expecially the positive cells of Smo gene. Conclusion: Shh, PTCH, Smo and Gli 1 genes are expressed in colonic cancer cell lines, pancreatic cancer cell line and colonic adenoma tissues, Cyclopamine has obvious inhibitory effect on cells with overexpression of Smo , which implies that Hedgehog signaling pathway might be activated in some tumor cells of digestive system.
2008, 15(2):173-175.
DOI: 10.3872/j.issn.1007-385X.2008.2.016
Abstract:
观察西妥昔单抗联合mIFL治疗15例奥沙利铂耐药性转移性结直肠癌的疗效及安全性。方法:15例经病理或细胞学诊断的奥沙利铂治疗无效或失败的晚期结直肠癌患者,西妥昔单抗首剂400mg/m2,第1周,随后每周1次250mg/m2,3周为一疗程,西妥昔单抗先于化疗药物使用,均联合mIFL方案治疗。结果:CR2例,PR3例,SD6例,PD4例,RR为33.33%,DCR为73.33%。TTP为3~48周,中位TTP17周。最常见的毒性反应为痤疮样皮疹、腹泻、恶心、呕吐。结论:西妥昔单抗联合mIFL治疗奥沙利铂耐药性转移性结直肠癌具有较高的总体疾病控制率,且耐受性好,不良反应多为轻中度,可作为一线奥沙利铂治疗失败转移性结直肠癌的推荐方案。
观察西妥昔单抗联合mIFL治疗15例奥沙利铂耐药性转移性结直肠癌的疗效及安全性。方法:15例经病理或细胞学诊断的奥沙利铂治疗无效或失败的晚期结直肠癌患者,西妥昔单抗首剂400mg/m2,第1周,随后每周1次250mg/m2,3周为一疗程,西妥昔单抗先于化疗药物使用,均联合mIFL方案治疗。结果:CR2例,PR3例,SD6例,PD4例,RR为33.33%,DCR为73.33%。TTP为3~48周,中位TTP17周。最常见的毒性反应为痤疮样皮疹、腹泻、恶心、呕吐。结论:西妥昔单抗联合mIFL治疗奥沙利铂耐药性转移性结直肠癌具有较高的总体疾病控制率,且耐受性好,不良反应多为轻中度,可作为一线奥沙利铂治疗失败转移性结直肠癌的推荐方案。
2008, 15(2):176-178.
DOI: 10.3872/j.issn.1007-385X.2008.2.017
Abstract:
构建一种携带抗肿瘤血管生成基因Endostatin的新型肿瘤基因-病毒治疗系统CNHK200-hEndo，观察其对人肺癌细胞裸鼠移植瘤的治疗效果。方法：克隆人抗血管生成基因Endostatin, 利用病毒重组技术将该基因插入肿瘤特异性增殖腺病毒CNHK200的基因组中，扩增并纯化CNHK200-hEn
构建一种携带抗肿瘤血管生成基因Endostatin的新型肿瘤基因-病毒治疗系统CNHK200-hEndo，观察其对人肺癌细胞裸鼠移植瘤的治疗效果。方法：克隆人抗血管生成基因Endostatin, 利用病毒重组技术将该基因插入肿瘤特异性增殖腺病毒CNHK200的基因组中，扩增并纯化CNHK200-hEn
2008, 15(2):179-181.
DOI: 10.3872/j.issn.1007-385X.2008.2.018
Abstract:
构建含人P21WAF1/CIP1基因启动子的表达载体pGL3-P21p。方法:以全血总RNA为模板,经RT-PCR扩增P21WAF1/CIP1基因启动子,并将其克隆到T-easy载体,测序分析,然后经酶切再克隆到pGL3-basic,构成重组真核表达载体pGL3-P21p。将pGL3-P21p转染到乳腺癌细胞株MCF7中,并检测细胞中荧光素酶的活性。结果:经测序、酶切等检测证实重组真核表达载体pGL3-P21p构建成功,荧光素酶活性检测显示P21WAF1/CIP1基因启动子对荧光酶基因表达起到了正调控作用。结论:P21WAF1/CIP1启动子荧光素酶真核表达载体的成功构建为进一步研究P21WAF1/CIP1基因启动子的表达调控奠定了基础。
构建含人P21WAF1/CIP1基因启动子的表达载体pGL3-P21p。方法:以全血总RNA为模板,经RT-PCR扩增P21WAF1/CIP1基因启动子,并将其克隆到T-easy载体,测序分析,然后经酶切再克隆到pGL3-basic,构成重组真核表达载体pGL3-P21p。将pGL3-P21p转染到乳腺癌细胞株MCF7中,并检测细胞中荧光素酶的活性。结果:经测序、酶切等检测证实重组真核表达载体pGL3-P21p构建成功,荧光素酶活性检测显示P21WAF1/CIP1基因启动子对荧光酶基因表达起到了正调控作用。结论:P21WAF1/CIP1启动子荧光素酶真核表达载体的成功构建为进一步研究P21WAF1/CIP1基因启动子的表达调控奠定了基础。
2008, 15(2):182-184.
DOI: 10.3872/j.issn.1007-385X.2008.2.019
Abstract:
观察硼替佐米(bortezomib)治疗恶性浆细胞病的临床疗效和不良反应。方法:2007年2-10月复旦大学附属华山医院血液科收治的10例恶性浆细胞病患者,接受VD方案(硼替佐米1.1~1.3mg/m2,第1、4、8、11天;地塞米松3mg/d,第1~4天,第8~11天)、VDT方案(VD方案加沙利度胺100~150mg/d,第1~28天)或PVADT方案(硼替佐米1.1~1.3mg/m2,第1、4、8、11天;地塞米松30mg/d,第1~4天,第8~11天;长春地辛1mg/d,第1~4天,脂质体多柔比星20mg第1、3天;沙利度胺100mg/d,第1~28天)化疗,4周为1疗程。结果:10例患者均在第1疗程后显示治疗反应,1例获得非常好的部分缓解(very good partial remission,VGPR)、9例获得部分缓解(partial remission,PR),总有效率为100%;坚持治疗的9例患者,现2例处于完全缓解(complete remission,CR)、1例VGPR、6例PR,总有效率为100%。不良反应主要包括末梢神经炎、血小板减少、胃肠道反应、乏力以及潜在的免疫抑制作用。结论:硼替佐米对于恶性浆细胞病有显著疗效;存在一些不良反应,但患者耐受良好。
观察硼替佐米(bortezomib)治疗恶性浆细胞病的临床疗效和不良反应。方法:2007年2-10月复旦大学附属华山医院血液科收治的10例恶性浆细胞病患者,接受VD方案(硼替佐米1.1~1.3mg/m2,第1、4、8、11天;地塞米松3mg/d,第1~4天,第8~11天)、VDT方案(VD方案加沙利度胺100~150mg/d,第1~28天)或PVADT方案(硼替佐米1.1~1.3mg/m2,第1、4、8、11天;地塞米松30mg/d,第1~4天,第8~11天;长春地辛1mg/d,第1~4天,脂质体多柔比星20mg第1、3天;沙利度胺100mg/d,第1~28天)化疗,4周为1疗程。结果:10例患者均在第1疗程后显示治疗反应,1例获得非常好的部分缓解(very good partial remission,VGPR)、9例获得部分缓解(partial remission,PR),总有效率为100%;坚持治疗的9例患者,现2例处于完全缓解(complete remission,CR)、1例VGPR、6例PR,总有效率为100%。不良反应主要包括末梢神经炎、血小板减少、胃肠道反应、乏力以及潜在的免疫抑制作用。结论:硼替佐米对于恶性浆细胞病有显著疗效;存在一些不良反应,但患者耐受良好。
2008, 15(2):185-188.
DOI: 10.3872/j.issn.1007-385X.2008.2.020
Abstract:
肿瘤干细胞理论的提出使靶向性杀伤肿瘤干细胞成为可能,而肿瘤干细胞的研究关键是寻找特异性表面分子标志物。表面黏附分子CD133是一种跨膜糖蛋白,其表达的一个显著特点就是,随着细胞的分化迅速下调,这使得它成为一个分离和鉴定干细胞和祖细胞的独特分子标志。作为理想的分子标志,CD133在脑肿瘤、前列腺癌、结肠癌、喉癌和肝癌等多种实体肿瘤干细胞分离研究中发挥着重要作用。多种实验证实,CD133+细胞与肿瘤信号转导、肿瘤免疫逃逸、药物耐受和放疗抵抗均有相关性,CD133+细胞可望成为肿瘤治疗的新靶点。
肿瘤干细胞理论的提出使靶向性杀伤肿瘤干细胞成为可能,而肿瘤干细胞的研究关键是寻找特异性表面分子标志物。表面黏附分子CD133是一种跨膜糖蛋白,其表达的一个显著特点就是,随着细胞的分化迅速下调,这使得它成为一个分离和鉴定干细胞和祖细胞的独特分子标志。作为理想的分子标志,CD133在脑肿瘤、前列腺癌、结肠癌、喉癌和肝癌等多种实体肿瘤干细胞分离研究中发挥着重要作用。多种实验证实,CD133+细胞与肿瘤信号转导、肿瘤免疫逃逸、药物耐受和放疗抵抗均有相关性,CD133+细胞可望成为肿瘤治疗的新靶点。
2008, 15(2):189-192.
DOI: 10.3872/j.issn.1007-385X.2008.2.021
Abstract:
异基因干细胞移植用于实体瘤的治疗有很大的潜力。传统的清髓性造血干细胞移植采用大剂量化疗药物对受者进行预处理,虽然起到一定的杀伤肿瘤细胞作用,但增加了移植相关死亡率(transplant-related mortality,TRM)。随着对非清髓性预处理的深入研究,人们将非清髓性造血干细胞移植用于实体瘤的治疗。与清髓性干细胞移植比较,非清髓性移植减轻了TRM,且拓宽了移植适应证。移植物抗宿主病(graft versus host disease,GVHD)是异基因移植后出现的毒性反应,甚至成为致命性的并发症。移植物抗肿瘤(graft versus tumor,GVT)效应是异基因移植治疗实体瘤的关键,而GVT效应常伴随GVHD的出现。因此,如何在保留GVT效应的同时降低GVHD是我们所面临挑战。目前,通过改变预处理方案、加强对移植物的处理、改变免疫抑制疗法等3种策略使用于GVHD的防治,取得了一定效果,为异基因造血干细胞移植治疗实体瘤带来了广阔前景。
异基因干细胞移植用于实体瘤的治疗有很大的潜力。传统的清髓性造血干细胞移植采用大剂量化疗药物对受者进行预处理,虽然起到一定的杀伤肿瘤细胞作用,但增加了移植相关死亡率(transplant-related mortality,TRM)。随着对非清髓性预处理的深入研究,人们将非清髓性造血干细胞移植用于实体瘤的治疗。与清髓性干细胞移植比较,非清髓性移植减轻了TRM,且拓宽了移植适应证。移植物抗宿主病(graft versus host disease,GVHD)是异基因移植后出现的毒性反应,甚至成为致命性的并发症。移植物抗肿瘤(graft versus tumor,GVT)效应是异基因移植治疗实体瘤的关键,而GVT效应常伴随GVHD的出现。因此,如何在保留GVT效应的同时降低GVHD是我们所面临挑战。目前,通过改变预处理方案、加强对移植物的处理、改变免疫抑制疗法等3种策略使用于GVHD的防治,取得了一定效果,为异基因造血干细胞移植治疗实体瘤带来了广阔前景。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.