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Volume 13,Issue 6,2006 Table of Contents
2006, 13(6):401-403.
DOI: 10.3872/j.issn.1007-385X.2006.6.001
Abstract:
2006, 13(6):404-411.
DOI: 10.3872/j.issn.1007-385X.2006.6.002
Abstract:
WANG Jun
,
ZHANG Ling
,
GU Hong tao
,
GUO Ning
,
SHI Ming
,
SHEN Bei fen
,
MAO Hai ting
,
LI Cui ling
,
WEN Pei e
2006, 13(6):412-416.
DOI: 10.3872/j.issn.1007-385X.2006.6.003
Abstract:
To elucidate the mechanisms by which anti-P185 erbB2 scFv-Fc-IL-2 (HFI) modulates tumor surface molecules and activates immune effector cells in vitro. Methods: Ovarian Cancer Cell Line SKOV3 and human peripheral blood monocyte peripheral blood mononuclear cells (PBMC) were treated with HFI. MTT assay was used to detect the proliferation and cytotoxicity; flow cytometry assay was used to examine expression of surface molecules; bioassay was used to examine cytotoxicity of membrane-associated TNF; and perforin mRNA level was analyzed by RT-PCR assay. Results: HFI had no direct inhibitory effect on proliferation of SKOV3 cells. After HFI treatment, the expression levels of ICAM-1 and Fas on SKOV3 cells rose from 24.85% and 0.53% to 85.36% and 59.19%, respectively （ P <0.01). Besides, HFI significantly enhanced the proliferation of human PBMC. CD3+CD8+ T cells and CD3-CD16+CD56+ T cells rose from 2437% and 6.90% to 38.80% and 13.45%, respectively （ P <0.01). Expression levels of CD25, LFA-1, and FasL significantly increased from 3.99%, 86.52%, and 5.02% to 12.96%, 99.06%, and 16.19%, respectivly（P<0.01). Expression levels of Perforin and membrane-associated TNF were also up-regulated. Cytotoxicities of LAK and NK were both improved（P<0.01). Conclusion: HFI can enhance the expression of ICAM-1 and Fas in SKOV3 cells and has obvious activating effect on PBMC. HFI can up-regulate the expression level of LFA-1/ ICAM-1，Fas/ FasL, promote the release of TNF and perforin, and improve LAK and NK cytotoxicity.
To elucidate the mechanisms by which anti-P185 erbB2 scFv-Fc-IL-2 (HFI) modulates tumor surface molecules and activates immune effector cells in vitro. Methods: Ovarian Cancer Cell Line SKOV3 and human peripheral blood monocyte peripheral blood mononuclear cells (PBMC) were treated with HFI. MTT assay was used to detect the proliferation and cytotoxicity; flow cytometry assay was used to examine expression of surface molecules; bioassay was used to examine cytotoxicity of membrane-associated TNF; and perforin mRNA level was analyzed by RT-PCR assay. Results: HFI had no direct inhibitory effect on proliferation of SKOV3 cells. After HFI treatment, the expression levels of ICAM-1 and Fas on SKOV3 cells rose from 24.85% and 0.53% to 85.36% and 59.19%, respectively （ P <0.01). Besides, HFI significantly enhanced the proliferation of human PBMC. CD3+CD8+ T cells and CD3-CD16+CD56+ T cells rose from 2437% and 6.90% to 38.80% and 13.45%, respectively （ P <0.01). Expression levels of CD25, LFA-1, and FasL significantly increased from 3.99%, 86.52%, and 5.02% to 12.96%, 99.06%, and 16.19%, respectivly（P<0.01). Expression levels of Perforin and membrane-associated TNF were also up-regulated. Cytotoxicities of LAK and NK were both improved（P<0.01). Conclusion: HFI can enhance the expression of ICAM-1 and Fas in SKOV3 cells and has obvious activating effect on PBMC. HFI can up-regulate the expression level of LFA-1/ ICAM-1，Fas/ FasL, promote the release of TNF and perforin, and improve LAK and NK cytotoxicity.
2006, 13(6):417-422.
DOI: 10.3872/j.issn.1007-385X.2006.6.004
Abstract:
To prepare human high affinity antibody fragment scFv that specifically binds to cell receptor Met by phage display technique. Methods: The variable regions were amplified from VH and VL of anti-Met Fab genes screened from the natural immune Fab antibody phage display library. The scFv genes were amplified by overlap PCR with different VH and VL library genes assembled by pairs of different CDR mutation primers. scFv DNA was purified and digested with SfiI and was then inserted into pComb3XSS. Positive phage-displayed antibodies with high affinity were selected on live cell lines. Results: After 5 cycles of cell screening and 2 cycles of antigen screening, 60 positive clones were subjected to ELISA. The selected high affinity scFv gene was cloned into pBAD/gIII for expression and was then studied by SDS-PAGE and Western blotting; a band was showed at about 30 000 as expected. To analyze the immunological characters of scFv for Met binding, flow cytometry and immunoprecipitation assays were carried out with S114, MKN45 and NIH3T3 cell lines. The results of flow cytometry and immunoprecipitation demonstrated scFv could bind to natural Met specifically on the surface of S114 and MKN45 cells (Kd=4.763×10 -8 mol/L), and the affinity was about 100 times higher than before mutation (Kd= 5.24×10 -6 mol/L). The results of ELISA showed that there was no change in the binding site of antigen after maturation of the affinity. Conclusion: It is indicated that anti-Met scFv antibody fragment can recognize Met extracellular domain in natural conformation with relatively high affinity, and it may be a potential candidate for clinical diagnosis and therapy.
To prepare human high affinity antibody fragment scFv that specifically binds to cell receptor Met by phage display technique. Methods: The variable regions were amplified from VH and VL of anti-Met Fab genes screened from the natural immune Fab antibody phage display library. The scFv genes were amplified by overlap PCR with different VH and VL library genes assembled by pairs of different CDR mutation primers. scFv DNA was purified and digested with SfiI and was then inserted into pComb3XSS. Positive phage-displayed antibodies with high affinity were selected on live cell lines. Results: After 5 cycles of cell screening and 2 cycles of antigen screening, 60 positive clones were subjected to ELISA. The selected high affinity scFv gene was cloned into pBAD/gIII for expression and was then studied by SDS-PAGE and Western blotting; a band was showed at about 30 000 as expected. To analyze the immunological characters of scFv for Met binding, flow cytometry and immunoprecipitation assays were carried out with S114, MKN45 and NIH3T3 cell lines. The results of flow cytometry and immunoprecipitation demonstrated scFv could bind to natural Met specifically on the surface of S114 and MKN45 cells (Kd=4.763×10 -8 mol/L), and the affinity was about 100 times higher than before mutation (Kd= 5.24×10 -6 mol/L). The results of ELISA showed that there was no change in the binding site of antigen after maturation of the affinity. Conclusion: It is indicated that anti-Met scFv antibody fragment can recognize Met extracellular domain in natural conformation with relatively high affinity, and it may be a potential candidate for clinical diagnosis and therapy.
2006, 13(6):423-428.
DOI: 10.3872/j.issn.1007-385X.2006.6.005
Abstract:
To express vascular basement membrane-derived multiple peptide (VBMDMP) in Pichia pastoris and to identify its bioactivity. Methods: PCR technique was used to obtain the target fragment GST-VBMDMP, which was then cloned into pPIC9K vector. The resultant vector pPIC9K-GST-VBMDMP was transfected into Pichia pastoris cells with spheroplast and the positive recons was identified. The His + Muts transformant was shake-cultured in MGY at 30 ℃. The culture supernatant (1 ml) was collected for preservation at -70 ℃ every 24 hours while maintaining the concentration of methanol at 0.5% in the culture medium. SDS-PAGE analysis was used to detect the protein expression after 8 days and then animal and cell experiments were performed with GST-VBMDMP. Results: SDS-PAGE analysis found that protein express increased during 1-7 days and decreased after 8 days in MGY medium; GST-VBMDMP fused protein was obtained by Glutathione Sepharose 4B and it inhibited the artery endothelial cell tube structure formation in C57BL／6 mouse; GST-VBMDMP（ 2, 6, 10 mg/kg）also significantly inhibited the primary cancer Lewis mouse （tumor inhibitor rates being 96.6%, 82.1%, and 61.2%, respectively）and metastatic lung tumors（tumor inhibitor rates being 96.8 %, 87.9 %, and 75.3%, respectively）（P<0.01）. Conclusion: VBMDMP can inhibit mouse artery endothelial cell tube structure formation and Lewis mouse primary, metastatic lung tumors.
To express vascular basement membrane-derived multiple peptide (VBMDMP) in Pichia pastoris and to identify its bioactivity. Methods: PCR technique was used to obtain the target fragment GST-VBMDMP, which was then cloned into pPIC9K vector. The resultant vector pPIC9K-GST-VBMDMP was transfected into Pichia pastoris cells with spheroplast and the positive recons was identified. The His + Muts transformant was shake-cultured in MGY at 30 ℃. The culture supernatant (1 ml) was collected for preservation at -70 ℃ every 24 hours while maintaining the concentration of methanol at 0.5% in the culture medium. SDS-PAGE analysis was used to detect the protein expression after 8 days and then animal and cell experiments were performed with GST-VBMDMP. Results: SDS-PAGE analysis found that protein express increased during 1-7 days and decreased after 8 days in MGY medium; GST-VBMDMP fused protein was obtained by Glutathione Sepharose 4B and it inhibited the artery endothelial cell tube structure formation in C57BL／6 mouse; GST-VBMDMP（ 2, 6, 10 mg/kg）also significantly inhibited the primary cancer Lewis mouse （tumor inhibitor rates being 96.6%, 82.1%, and 61.2%, respectively）and metastatic lung tumors（tumor inhibitor rates being 96.8 %, 87.9 %, and 75.3%, respectively）（P<0.01）. Conclusion: VBMDMP can inhibit mouse artery endothelial cell tube structure formation and Lewis mouse primary, metastatic lung tumors.
WANG Hai juan
,
LI Yun feng
,
QIAN Hai li
,
ZHANG Xue yan
,
FU Ming
,
LIANG Xiao
,
ZHAN Qi min
,
LIN Chen
2006, 13(6):429-434.
DOI: 10.3872/j.issn.1007-385X.2006.6.006
Abstract:
To investigate the relationship between coxsackie adenovirus receptor (CAR) expression and the efficiency of adenovirus gene transfer, in an effort to provide evidence for the clinical application of adenovirus-related bio-preparations. Methods: The esophageal cancer cell lines (KYSE510, KYSE150, EC9706), cervical cancer cell line (HeLa), ovary cancer cell line (SKOV3), hepatoma cell line (HepG2), and lung cancer cell line (A549) were infected with Ad-GFP (100 MOI, 200 MOI) labeled by immunofluorescence; 48 h later we measured the Ad-GFP transduction efficiencies in the above cell lines by flow cytometric analysis. CAR expression levels in vitro were assayed by Western blotting. HeLa (3×106), A549 (3×106), SKOV3 (3×106), and EC9706 (2×106) cells were implanted subcutaneously into nude mice. When the tumor sizes reached 5-7 mm in diameter, the xenografts were injected with Ad-GFP (1×109PFU) twice with a 48 h interval. After another 48 h the mice were killed and the GFP expression were observed by fluorescence microscopy to determine the transduction efficiency. CAR expression was detected by immunohistochemistry in the xenograft tissues. Results: We found that Ad transduction efficiencies varied greatly in different tissues, even among the cells of the same tissue origin. The transduction efficiencies in A549 (92.67%), HeLa (89.31%), HepG2 (84.98%) and KYSE150 (74.59%) were higher than those in SKOV3 (30.06%), KYSE510 (27.40%), and EC9706 (1893%). Consistently, GFP expression was much higher in exografts sections resulting from HeLa and A549 than those from SKOV3 and EC9706. Increased CAR expression was predictive for more efficient gene transfer in vitro and in vivo. Conclusion: CAR expression level is closely correlated to Ad transduction efficiency, which suggests that measurement of CAR expression in tumor tissues is useful for individualized adenovirus based gene therapy in clinic.
To investigate the relationship between coxsackie adenovirus receptor (CAR) expression and the efficiency of adenovirus gene transfer, in an effort to provide evidence for the clinical application of adenovirus-related bio-preparations. Methods: The esophageal cancer cell lines (KYSE510, KYSE150, EC9706), cervical cancer cell line (HeLa), ovary cancer cell line (SKOV3), hepatoma cell line (HepG2), and lung cancer cell line (A549) were infected with Ad-GFP (100 MOI, 200 MOI) labeled by immunofluorescence; 48 h later we measured the Ad-GFP transduction efficiencies in the above cell lines by flow cytometric analysis. CAR expression levels in vitro were assayed by Western blotting. HeLa (3×106), A549 (3×106), SKOV3 (3×106), and EC9706 (2×106) cells were implanted subcutaneously into nude mice. When the tumor sizes reached 5-7 mm in diameter, the xenografts were injected with Ad-GFP (1×109PFU) twice with a 48 h interval. After another 48 h the mice were killed and the GFP expression were observed by fluorescence microscopy to determine the transduction efficiency. CAR expression was detected by immunohistochemistry in the xenograft tissues. Results: We found that Ad transduction efficiencies varied greatly in different tissues, even among the cells of the same tissue origin. The transduction efficiencies in A549 (92.67%), HeLa (89.31%), HepG2 (84.98%) and KYSE150 (74.59%) were higher than those in SKOV3 (30.06%), KYSE510 (27.40%), and EC9706 (1893%). Consistently, GFP expression was much higher in exografts sections resulting from HeLa and A549 than those from SKOV3 and EC9706. Increased CAR expression was predictive for more efficient gene transfer in vitro and in vivo. Conclusion: CAR expression level is closely correlated to Ad transduction efficiency, which suggests that measurement of CAR expression in tumor tissues is useful for individualized adenovirus based gene therapy in clinic.
2006, 13(6):435-441.
DOI: 10.3872/j.issn.1007-385X.2006.6.007
Abstract:
To construct recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin 1 mutant and to study their anti-oophoroma effect in vitro and in vivo. Methods: The genes encoding attenuated Shiga-like toxin 1 mutant were amplified by overlap PCR and then cloned into eukaryotic expression plasmid pcDNA31. The recombinant plasmids were transfected into SKOV3 cells and RT-PCR was used determine the expression of Stx l mRNA in SKOV3 cells. The influence of attenuated Stx 1 on the cell cycle of SKOV3 cells and the mechanism by which Stx 1 induces apoptosis of SKOV3 cells were observed. Then Stx 1 with different cyotoxicites (after packed by liposome) were intratumorally injected into immunodeficient mice harboring SKOV3 to assess their anti-oophoroma effect in vivo. Results: It was showed that the genes encoding attenuated Stx 1 mutant were successfully cloned into pcDNA3.1 and their mRNA expression in transfected SKOV3 cells was verified by RT-PCR. In vitro study showed that the constructed plasmid arrested SKOV3cells at G 2 /M stage. The post-transfection death of SKOV3 cells was mainly through necrosis as assessed by flow cytometry. In vivo study showed that the constructed plasmid had a significant anti-oophoroma effect on the growth of SKOV3 tumor in immunodefficient mice. Conclusion: Two sequences encoding attenuated Stx 1 mutant have been successfully constructed and expressed in eukaryotic expression system, which have been confirmed to have obvious anti-oophoroma effect in vitro and in vivo .
To construct recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin 1 mutant and to study their anti-oophoroma effect in vitro and in vivo. Methods: The genes encoding attenuated Shiga-like toxin 1 mutant were amplified by overlap PCR and then cloned into eukaryotic expression plasmid pcDNA31. The recombinant plasmids were transfected into SKOV3 cells and RT-PCR was used determine the expression of Stx l mRNA in SKOV3 cells. The influence of attenuated Stx 1 on the cell cycle of SKOV3 cells and the mechanism by which Stx 1 induces apoptosis of SKOV3 cells were observed. Then Stx 1 with different cyotoxicites (after packed by liposome) were intratumorally injected into immunodeficient mice harboring SKOV3 to assess their anti-oophoroma effect in vivo. Results: It was showed that the genes encoding attenuated Stx 1 mutant were successfully cloned into pcDNA3.1 and their mRNA expression in transfected SKOV3 cells was verified by RT-PCR. In vitro study showed that the constructed plasmid arrested SKOV3cells at G 2 /M stage. The post-transfection death of SKOV3 cells was mainly through necrosis as assessed by flow cytometry. In vivo study showed that the constructed plasmid had a significant anti-oophoroma effect on the growth of SKOV3 tumor in immunodefficient mice. Conclusion: Two sequences encoding attenuated Stx 1 mutant have been successfully constructed and expressed in eukaryotic expression system, which have been confirmed to have obvious anti-oophoroma effect in vitro and in vivo .
2006, 13(6):442-446.
DOI: 10.3872/j.issn.1007-385X.2006.6.008
Abstract:
To evaluate the efficacy of 3LL/GM-CSF tumor vaccine combined with pacilitaxel chemotherapy in treatment of mice bearing transplanted Lewis lung carcinoma. Methods: The tumor vaccine 3LL/GM-CSF was prepared by infecting Lewis lung carcinoma cell line 3LL with adenovirus encoding GM-GSF. Mice model of Lewis lung carcinoma was established by subcutaneous injection of 2×104 3LL cells into C57BL/6（H-2b）mice. The sensitivity of Lewis lung carcinoma cell line-3LL to the treatment of pacilitaxel was detected in vivo and in vitro. The mice tumor model was first treated with pacilitaxel chemotherapy and then with 3LL/GM-CSF, or first with 3LL/GM-CSF and then with pacilitaxel. Tumor growth and the long-term survival of mice were observed after treatment. The immune memory and cytotoxicity against target cells were studied in the mice. Results: Pacilitaxel at 100 nmol/L killed 32.10% 3LL cells after 24 hour in vitro; but pacilitaxel at 5-25 mg/kg only had a poor effect on growth of 3LL cells in vivo. The tumors rebated in 70% of mice treated with pacilitaxel chemotherapy and 3LL/GM-CSF vaccination successively, and the survival of these mice was obviously longer than that of pure pacilitaxel chemotherapy group (70.0 days vs 27.5 days). The killing rate of 3LL/GM-CSF after pacilitaxel chemotherapy was 41.35% on day 3. Meanwhile, the survival mice could resist the re-attack of 3LL cells (2×104). We also noticed that first treatment with 3LL/GM-CSF and then pacilitaxel chemotherapy had no effect on tumors. Conclusion: Application of tumor vaccine shortly after pacilitaxel chemotherapy can induce specific immune responses and prolong the survival of experimental mice, which provide a basis for future clinical practice.
To evaluate the efficacy of 3LL/GM-CSF tumor vaccine combined with pacilitaxel chemotherapy in treatment of mice bearing transplanted Lewis lung carcinoma. Methods: The tumor vaccine 3LL/GM-CSF was prepared by infecting Lewis lung carcinoma cell line 3LL with adenovirus encoding GM-GSF. Mice model of Lewis lung carcinoma was established by subcutaneous injection of 2×104 3LL cells into C57BL/6（H-2b）mice. The sensitivity of Lewis lung carcinoma cell line-3LL to the treatment of pacilitaxel was detected in vivo and in vitro. The mice tumor model was first treated with pacilitaxel chemotherapy and then with 3LL/GM-CSF, or first with 3LL/GM-CSF and then with pacilitaxel. Tumor growth and the long-term survival of mice were observed after treatment. The immune memory and cytotoxicity against target cells were studied in the mice. Results: Pacilitaxel at 100 nmol/L killed 32.10% 3LL cells after 24 hour in vitro; but pacilitaxel at 5-25 mg/kg only had a poor effect on growth of 3LL cells in vivo. The tumors rebated in 70% of mice treated with pacilitaxel chemotherapy and 3LL/GM-CSF vaccination successively, and the survival of these mice was obviously longer than that of pure pacilitaxel chemotherapy group (70.0 days vs 27.5 days). The killing rate of 3LL/GM-CSF after pacilitaxel chemotherapy was 41.35% on day 3. Meanwhile, the survival mice could resist the re-attack of 3LL cells (2×104). We also noticed that first treatment with 3LL/GM-CSF and then pacilitaxel chemotherapy had no effect on tumors. Conclusion: Application of tumor vaccine shortly after pacilitaxel chemotherapy can induce specific immune responses and prolong the survival of experimental mice, which provide a basis for future clinical practice.
2006, 13(6):447-451.
DOI: 10.3872/j.issn.1007-385X.2006.6.009
Abstract:
To investigate the expression of protein kinase B(PKB)and hypoxia inducible factor 1α(HIF-1α) in gastric cancer and its clinical significance. Methods: RT-PCR was used to detect the expression of PKB1,PKB2,PKB3, and HIF-1α mRNA in 26 patients with gastric cancer tissue samples and the corresponding para-cancer tissues. The expression of pPKB and HIF-1α in 64 gastric carcinomas and 26 para-cancer tissue samples were evaluated by immunohistochemical methods. Results: (1) PKB1, PKB2, and PKB3 mRNA were positive in both gastric cancer and the corresponding adjacent normal tissues, with no significant difference ( P ＞0.05). The expression of HIF-1α mRNA in cancerous tissues was significantly higher than that in para-cancer tissues (1.36±0.14 vs 0.54±0.18,P＜0.05). (2)The expression levels of pPKB and HIF-1α proteins in cancerous tissues were higher than those in para-cancer tissues (χ2=26936,15.950, P＜0.05). (3) The expression of pPKB and HIF-1α were significantly associated with TNM stage, invasion depth, lymph node metastasis, and distant metastases (P＜0.05）. Conclusion: The abnormal expression of PKB is at the translation or post-translation level. PKB and HIF-1α are overexpressed in gastric cancer tissues, which is correlatedwith cell proliferation, apoptosis, and metastasis of gastric cancer.
To investigate the expression of protein kinase B(PKB)and hypoxia inducible factor 1α(HIF-1α) in gastric cancer and its clinical significance. Methods: RT-PCR was used to detect the expression of PKB1,PKB2,PKB3, and HIF-1α mRNA in 26 patients with gastric cancer tissue samples and the corresponding para-cancer tissues. The expression of pPKB and HIF-1α in 64 gastric carcinomas and 26 para-cancer tissue samples were evaluated by immunohistochemical methods. Results: (1) PKB1, PKB2, and PKB3 mRNA were positive in both gastric cancer and the corresponding adjacent normal tissues, with no significant difference ( P ＞0.05). The expression of HIF-1α mRNA in cancerous tissues was significantly higher than that in para-cancer tissues (1.36±0.14 vs 0.54±0.18,P＜0.05). (2)The expression levels of pPKB and HIF-1α proteins in cancerous tissues were higher than those in para-cancer tissues (χ2=26936,15.950, P＜0.05). (3) The expression of pPKB and HIF-1α were significantly associated with TNM stage, invasion depth, lymph node metastasis, and distant metastases (P＜0.05）. Conclusion: The abnormal expression of PKB is at the translation or post-translation level. PKB and HIF-1α are overexpressed in gastric cancer tissues, which is correlatedwith cell proliferation, apoptosis, and metastasis of gastric cancer.
2006, 13(6):452-456.
DOI: 10.3872/j.issn.1007-385X.2006.6.010
Abstract:
To investigate the effect of rTFAR19 protein on the expression of hTERT mRNA and telomerase activity in human nasopharyngeal carcinoma CNE-1 cells, so as to understand the mechanism by which rTFAR19 protein promotes the apoptosis of CNE-1 cells. Methods: Different concentrations of rTFAR19 protein were co-cultured with human CNE-1 cells. The apoptosis of CNE-1 cells was analyzed by 7-AAD and Annexin V-PE-labeled flow cytometry (FACS) assay. The expression of hTERT mRNA was detected by RT-PCR and was compared between the 2 groups. The changes of telomerase activity after TFAR19 treatment were examined by TRAP-sliver staining. Results: rTFAR19 protein incorporated into human nasopharyngeal carcinoma CNE-1 cells without a carrier. When the concentrations of rTFAR19 protein were 5mg/L,10 mg/L, 15 mg/L，20 mg/L, and 30 mg/L, the apoptosis rates of CNE-1 cells were 15.26％，2948％，37.11％，54.20％，and 72.36％, respecitively. The apoptosis rate of the positive control group (treated with stuanrosporin) and the negative control group (treated with PBS) were 98.37% and 13.40％, respectively. The expression of hTERT mRNA in CNE-1 cells was similar between the experimental group and the control group. The telomerase activity of CNE-1 cells was obviously decreased after cultured with rTFAR19 protein (>20 mg/L). Conclusion: High concentration of rTFAR19 protein can depress the telomerase activity of human nasopharyngeal carcinoma CNE-1 cells and subsequently accelerate the apoptosis of CNE-1 cells .
To investigate the effect of rTFAR19 protein on the expression of hTERT mRNA and telomerase activity in human nasopharyngeal carcinoma CNE-1 cells, so as to understand the mechanism by which rTFAR19 protein promotes the apoptosis of CNE-1 cells. Methods: Different concentrations of rTFAR19 protein were co-cultured with human CNE-1 cells. The apoptosis of CNE-1 cells was analyzed by 7-AAD and Annexin V-PE-labeled flow cytometry (FACS) assay. The expression of hTERT mRNA was detected by RT-PCR and was compared between the 2 groups. The changes of telomerase activity after TFAR19 treatment were examined by TRAP-sliver staining. Results: rTFAR19 protein incorporated into human nasopharyngeal carcinoma CNE-1 cells without a carrier. When the concentrations of rTFAR19 protein were 5mg/L,10 mg/L, 15 mg/L，20 mg/L, and 30 mg/L, the apoptosis rates of CNE-1 cells were 15.26％，2948％，37.11％，54.20％，and 72.36％, respecitively. The apoptosis rate of the positive control group (treated with stuanrosporin) and the negative control group (treated with PBS) were 98.37% and 13.40％, respectively. The expression of hTERT mRNA in CNE-1 cells was similar between the experimental group and the control group. The telomerase activity of CNE-1 cells was obviously decreased after cultured with rTFAR19 protein (>20 mg/L). Conclusion: High concentration of rTFAR19 protein can depress the telomerase activity of human nasopharyngeal carcinoma CNE-1 cells and subsequently accelerate the apoptosis of CNE-1 cells .
2006, 13(6):457-461.
DOI: 10.3872/j.issn.1007-385X.2006.6.011
Abstract:
To observe the effect of telomerase antisense DNA on apoptosis of hepatoma cells induced by arsenic trioxide, in an effort to look for a new anti-hepatic cancer agent with high efficiency, low cytotoxicity. Methods: We designed and synthesized a 20nt telomerase-antisense DNA targeting telomerase template and observed its influence on the telomerase activity of hepatoma cells. H-E staining, flow cytometry, and DNA agarose electrophoresis were used to study the preventive effect of telomerase-antisense DNA on hepatoma cells apoptosis induced by arsenic trioxide. Flow cytometry was used to detect the expression of Fas, Fas-L, and bcl-2. Results: Telomerase-antisense DNA (5 μmol/L) effectively inhibited the telomerase activity of hepatoma cells after 24 hours (P<0.01). When telomerase activity was inhibited by telomerase-antisense DNA, the hepatoma cells became more sensitive to arsenic trioxide induced apoptosis, which was induced through Fas and Fas-L pathway. Conclusion: Inhibition of telomerase activity can obviously increase the apoptosis rate of hepatoma cells induced by arsenic trioxide, therefore reducing the quantity of the latter in treating HCC, indicating a future for the combined application of both in clinical practice.
To observe the effect of telomerase antisense DNA on apoptosis of hepatoma cells induced by arsenic trioxide, in an effort to look for a new anti-hepatic cancer agent with high efficiency, low cytotoxicity. Methods: We designed and synthesized a 20nt telomerase-antisense DNA targeting telomerase template and observed its influence on the telomerase activity of hepatoma cells. H-E staining, flow cytometry, and DNA agarose electrophoresis were used to study the preventive effect of telomerase-antisense DNA on hepatoma cells apoptosis induced by arsenic trioxide. Flow cytometry was used to detect the expression of Fas, Fas-L, and bcl-2. Results: Telomerase-antisense DNA (5 μmol/L) effectively inhibited the telomerase activity of hepatoma cells after 24 hours (P<0.01). When telomerase activity was inhibited by telomerase-antisense DNA, the hepatoma cells became more sensitive to arsenic trioxide induced apoptosis, which was induced through Fas and Fas-L pathway. Conclusion: Inhibition of telomerase activity can obviously increase the apoptosis rate of hepatoma cells induced by arsenic trioxide, therefore reducing the quantity of the latter in treating HCC, indicating a future for the combined application of both in clinical practice.
2006, 13(6):462-468.
DOI: 10.3872/j.issn.1007-385X.2006.6.012
Abstract:
To construct the expressing vector of the extracellular domain of death receptor 5 (DR5), express it E.coli , identify the purified DR5 protein, and study its biological activity. Methods: The extracellular domain of DR5(eDR5) was assembled by overlapping PCR. The expression vector pET-22b(+)/ DR5 was constructed and transformed into E.coli BL21（DE3）. The expression of eDR5 protein was induced by IPTG and purified by Ni2+-affinity chromatographic column. The purity and specificities were detected by SDS-PAGE and ELISA, respectively. The blocking effects of purified eDR5 on FMU1.5-induced apoptosis of U343, U373 cells were observed. Results: The extracellular domain of DR5 was obtained by overlapping PCR. The eDR5 protein was expressed in both supernatants and inclusion bodies with a yield more than 30% of total bacterial proteins. The purity of eDR5 was more than 95% and the yield reached 9 mg/ml. The result of ELISA showed the purified protein was eDR5. Purified eDR5 partially blocked the apoptosis of U343 cells induced by FMU1.5 and TRAIL. Conclusion: The successful construction, expression, and purification of the extracellular domain of DR5 protein lays a foundation for further study of DR5 function.
To construct the expressing vector of the extracellular domain of death receptor 5 (DR5), express it E.coli , identify the purified DR5 protein, and study its biological activity. Methods: The extracellular domain of DR5(eDR5) was assembled by overlapping PCR. The expression vector pET-22b(+)/ DR5 was constructed and transformed into E.coli BL21（DE3）. The expression of eDR5 protein was induced by IPTG and purified by Ni2+-affinity chromatographic column. The purity and specificities were detected by SDS-PAGE and ELISA, respectively. The blocking effects of purified eDR5 on FMU1.5-induced apoptosis of U343, U373 cells were observed. Results: The extracellular domain of DR5 was obtained by overlapping PCR. The eDR5 protein was expressed in both supernatants and inclusion bodies with a yield more than 30% of total bacterial proteins. The purity of eDR5 was more than 95% and the yield reached 9 mg/ml. The result of ELISA showed the purified protein was eDR5. Purified eDR5 partially blocked the apoptosis of U343 cells induced by FMU1.5 and TRAIL. Conclusion: The successful construction, expression, and purification of the extracellular domain of DR5 protein lays a foundation for further study of DR5 function.
2006, 13(6):466-468.
DOI: 10.3872/j.issn.1007-385X.2006.6.013
Abstract:
目的： 探讨p53腺病毒（简称Ad-p53）注射液联合中药得力生及消癌平治疗小鼠腹水瘤的协同作用。 方法： 建立昆明小鼠H22肝癌腹水瘤模型，建模48 h后分组分别给予Ad-p53、中药得力生和消癌平或两药联用。用药1周后检测体重、腹围、腹水量、腹水瘤细胞计数，流式细胞仪测定瘤细胞凋亡百分率以及细胞周期情况。 结果： Ad-p53、得力生、消癌平单药治疗组，除体重以外的上述指标与对照组比较均有统计学差异（ P ＜0.05），Ad-p53+得力生和Ad-p53＋消癌平组的腹围增长率、腹水量、瘤细胞计数、晚期细胞凋亡率均明显少于各单药治疗组（ P ＜0.05）。 结论： 腹腔注射Ad-p53、得力生和消癌平均可抑制H22细胞生长并减少腹水形成，Ad-p53与中药得力生、消癌平联合腹腔内给药对治疗腹水瘤有协同作用。
目的： 探讨p53腺病毒（简称Ad-p53）注射液联合中药得力生及消癌平治疗小鼠腹水瘤的协同作用。 方法： 建立昆明小鼠H22肝癌腹水瘤模型，建模48 h后分组分别给予Ad-p53、中药得力生和消癌平或两药联用。用药1周后检测体重、腹围、腹水量、腹水瘤细胞计数，流式细胞仪测定瘤细胞凋亡百分率以及细胞周期情况。 结果： Ad-p53、得力生、消癌平单药治疗组，除体重以外的上述指标与对照组比较均有统计学差异（ P ＜0.05），Ad-p53+得力生和Ad-p53＋消癌平组的腹围增长率、腹水量、瘤细胞计数、晚期细胞凋亡率均明显少于各单药治疗组（ P ＜0.05）。 结论： 腹腔注射Ad-p53、得力生和消癌平均可抑制H22细胞生长并减少腹水形成，Ad-p53与中药得力生、消癌平联合腹腔内给药对治疗腹水瘤有协同作用。
14 Progesterone regulates expression of SSTR subtypes in human non-small cell lung cancer A549 cells
2006, 13(6):469-474.
DOI: 10.3872/j.issn.1007-385X.2006.6.014
Abstract:
目的：探讨人非小细胞肺癌细胞A549中孕酮对生长抑素受体（somatostatin recepter，SSTR）表达的调节。方法：免疫组化法测定A549细胞孕激素受体的表达, RT-PCR检测孕酮作用前后SSTR亚型mRNA的表达。结果： A549细胞表达孕激素受体,同时表达生长抑素受体亚型SSTR2、SSTR1、SSTR4和SSTR5 mRNA，不表达SSTR3 mRNA。不同浓度的孕酮作用A549细胞24 h后，SSTR各亚型mRNA表达普遍上调（P<0.05）；10 nmol/L组及1 000 nmol/L组细胞出现了SSTR3 mRNA的表达。延长10 nmol/L孕酮作用时间至48 h， SSTR3与SSTR5 mRNA表达继续上调（P<0.05）。结论：孕酮上调A549细胞SSTR各亚型mRNA的表达，尤其是SSTR3与SSTR5 mRNA；调节作用与孕酮的作用时间和浓度有关。
目的：探讨人非小细胞肺癌细胞A549中孕酮对生长抑素受体（somatostatin recepter，SSTR）表达的调节。方法：免疫组化法测定A549细胞孕激素受体的表达, RT-PCR检测孕酮作用前后SSTR亚型mRNA的表达。结果： A549细胞表达孕激素受体,同时表达生长抑素受体亚型SSTR2、SSTR1、SSTR4和SSTR5 mRNA，不表达SSTR3 mRNA。不同浓度的孕酮作用A549细胞24 h后，SSTR各亚型mRNA表达普遍上调（P<0.05）；10 nmol/L组及1 000 nmol/L组细胞出现了SSTR3 mRNA的表达。延长10 nmol/L孕酮作用时间至48 h， SSTR3与SSTR5 mRNA表达继续上调（P<0.05）。结论：孕酮上调A549细胞SSTR各亚型mRNA的表达，尤其是SSTR3与SSTR5 mRNA；调节作用与孕酮的作用时间和浓度有关。
2006, 13(6):471-474.
DOI: 10.3872/j.issn.1007-385X.2006.6.015
Abstract:
人35型（Ad35）和11型（Ad11）腺病毒同属于B 2 亚群，病毒受体均为CD46分子，Ad35和Ad11的中和抗体在不同地区不同人种的阳性率都比较低，且对肿瘤细胞、造血干细胞、树突状细胞等一些传统的5型和2型腺病毒嗜性较低的细胞有较高的感染效率，而且转移基因的表达水平较高，提示Ad11和Ad35是一类具有良好开发潜力的肿瘤基因治疗载体。目前，开发利用Ad35或Ad11主要有三种形式：嵌合型腺病毒载体、复制缺陷的35型或11型腺病毒载体和嵌合型的35型或11型“gutless”腺病毒载体。这些载体的体外疗效较好，但体内疗效不理想，原因主要是病毒颗粒在从注射部位到达靶组织会被多重屏障所拦截。因此，深入研究Ad35和Ad11感染细胞的过程和机制， 有助于将它们应用于包括肿瘤在内多种重大疾病的基因治疗。
人35型（Ad35）和11型（Ad11）腺病毒同属于B 2 亚群，病毒受体均为CD46分子，Ad35和Ad11的中和抗体在不同地区不同人种的阳性率都比较低，且对肿瘤细胞、造血干细胞、树突状细胞等一些传统的5型和2型腺病毒嗜性较低的细胞有较高的感染效率，而且转移基因的表达水平较高，提示Ad11和Ad35是一类具有良好开发潜力的肿瘤基因治疗载体。目前，开发利用Ad35或Ad11主要有三种形式：嵌合型腺病毒载体、复制缺陷的35型或11型腺病毒载体和嵌合型的35型或11型“gutless”腺病毒载体。这些载体的体外疗效较好，但体内疗效不理想，原因主要是病毒颗粒在从注射部位到达靶组织会被多重屏障所拦截。因此，深入研究Ad35和Ad11感染细胞的过程和机制， 有助于将它们应用于包括肿瘤在内多种重大疾病的基因治疗。
2006, 13(6):475-480.
DOI: 10.3872/j.issn.1007-385X.2006.6.016
Abstract:
NK细胞识别靶细胞时在接触表面形成大量的超分子集落，结构上与神经突触相似，称为NK细胞免疫突触。NK细胞抑制性受体和活化性受体分别与相应的配体结合形成抑制性免疫突触与活化性免疫突触。免疫突触的成熟是一个伴随着大量分子重新组合和隔离分布的动态过程。很多因素可以影响它的形成，其中肌动蛋白聚合抑制剂细胞松弛素C等可以明显抑制早期抑制性突触的形成，活化性突触中脂筏向细胞间接触位点极化能够为大量活化性信号提供一个锚定平台。免疫突触可以促进细胞间的接触、提供细胞间信号传递的平台、参与活化检查节点的形成。进一步对其结构、形成过程、信号整合等进行探讨将为提高NK细胞抗肿瘤和抗病毒治疗效果提供新的策略。
NK细胞识别靶细胞时在接触表面形成大量的超分子集落，结构上与神经突触相似，称为NK细胞免疫突触。NK细胞抑制性受体和活化性受体分别与相应的配体结合形成抑制性免疫突触与活化性免疫突触。免疫突触的成熟是一个伴随着大量分子重新组合和隔离分布的动态过程。很多因素可以影响它的形成，其中肌动蛋白聚合抑制剂细胞松弛素C等可以明显抑制早期抑制性突触的形成，活化性突触中脂筏向细胞间接触位点极化能够为大量活化性信号提供一个锚定平台。免疫突触可以促进细胞间的接触、提供细胞间信号传递的平台、参与活化检查节点的形成。进一步对其结构、形成过程、信号整合等进行探讨将为提高NK细胞抗肿瘤和抗病毒治疗效果提供新的策略。
2006, 13(6):478-480.
DOI: 10.3872/j.issn.1007-385X.2006.6.017
Abstract:
骨桥蛋白(osteopontin, OPN)是一种磷酸化酸性糖蛋白分子，人体的破骨细胞、巨噬细胞、T细胞、以及血管平滑肌细胞等均可分泌OPN，其基因定位于染色体4q13。OPN具有细胞黏连蛋白的功能，可以与CD44、整合素α v β 3 以及αvβ5结合，OPN还参与骨质重建、新血管生成和炎症反应；OPN在肿瘤转移中起协同作用。在卵巢癌的早期诊断和预后监测方面可作为CA125以及其他细胞因子等标志物的补充。检测方法可望更简便。在基因转录水平上抑制OPN及其主要受体可能成为治疗肿瘤的新药物靶点。
骨桥蛋白(osteopontin, OPN)是一种磷酸化酸性糖蛋白分子，人体的破骨细胞、巨噬细胞、T细胞、以及血管平滑肌细胞等均可分泌OPN，其基因定位于染色体4q13。OPN具有细胞黏连蛋白的功能，可以与CD44、整合素α v β 3 以及αvβ5结合，OPN还参与骨质重建、新血管生成和炎症反应；OPN在肿瘤转移中起协同作用。在卵巢癌的早期诊断和预后监测方面可作为CA125以及其他细胞因子等标志物的补充。检测方法可望更简便。在基因转录水平上抑制OPN及其主要受体可能成为治疗肿瘤的新药物靶点。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.