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Volume 11,Issue 2,2004 Table of Contents
2004, 11(2):79-83.
DOI: 10.3872/j.issn.1007-385X.2004.2.001
Abstract:
Objective：To investigate the anti-tumor effects of adoptively transferred splenocytes induced by gene immunization with ehCGβ tumor vaccine. Methods：Sp2/0 cells were transfected with the plasmids containing ehCGβ or HBV-preS2/S and the postive clones were screened by G418. BALB/c mice were immunized with plasmid TR421-hCGβ or mock DNA three times. Two weeks after immunization, spleen cells from the immunized mice were harvested, and then analyze the CTL activity against Sp2/0-ehCGβ cells. The splenocytes derived from the immunized mice were adoptively transferred to the normal mice, which were subsequently given injections i.p. of Sp2/0-ehCGβ and β Sp2/0-preS2/S respectively. Results：Splenocytes derived from the mice immunized with TR421-hCGβ exhibited a strong specfic cytotoxic activity against Sp2/0-ehCGβ cells, The average tumor weight between test and control group is statistically significant (P<0.05). Only two mice out of six had tumors in Sp2/0-ehCGβadoptively transferred group, while all mice had tumors in control group. However, all mice challenged with Sp2/0-preS2/S formed tumors, and the average tumor weight was not obviously varied between the two groups. Conclusion：ehCGβ gene vaccine could induce specific cytotoxic splenocytes against ehCGβ and adoptive transfer of the splenocytes showed the anti-tumor activity.
Objective：To investigate the anti-tumor effects of adoptively transferred splenocytes induced by gene immunization with ehCGβ tumor vaccine. Methods：Sp2/0 cells were transfected with the plasmids containing ehCGβ or HBV-preS2/S and the postive clones were screened by G418. BALB/c mice were immunized with plasmid TR421-hCGβ or mock DNA three times. Two weeks after immunization, spleen cells from the immunized mice were harvested, and then analyze the CTL activity against Sp2/0-ehCGβ cells. The splenocytes derived from the immunized mice were adoptively transferred to the normal mice, which were subsequently given injections i.p. of Sp2/0-ehCGβ and β Sp2/0-preS2/S respectively. Results：Splenocytes derived from the mice immunized with TR421-hCGβ exhibited a strong specfic cytotoxic activity against Sp2/0-ehCGβ cells, The average tumor weight between test and control group is statistically significant (P<0.05). Only two mice out of six had tumors in Sp2/0-ehCGβadoptively transferred group, while all mice had tumors in control group. However, all mice challenged with Sp2/0-preS2/S formed tumors, and the average tumor weight was not obviously varied between the two groups. Conclusion：ehCGβ gene vaccine could induce specific cytotoxic splenocytes against ehCGβ and adoptive transfer of the splenocytes showed the anti-tumor activity.
2004, 11(2):84-88.
DOI: 10.3872/j.issn.1007-385X.2004.2.002
Abstract:
Objective：We utilized genotype well-defined 17 strains of BXD RI strains mice to determine if genetic loci underlie the dendritic cell response to the breast tumor cell line TS/A. Dendritic cells were assayed for tumor antigen processing, presenting function and IL-12 induction.Methods：T cells isolated from BXD strain 9 (BXD9, H-2d) or BXD strain 2 (H-2b) of mice previously immunized with breast tumor TS/A cells were stimulated in vitro with dendritic cells isolated from MHC matched BXD RI mice fed with TS/A tumor antigens for 3 days. Analysis of Dendritic phagocytosis apoptotic tumor cells.IL-12 induction of Dendritic cells isolated from BXD mice. Dendritic cells inhibit tumor cell growth and induce IFN-γ produced by T cells. Flow Cytometric Analysis of DC surface antigen expression. Dendritic cells Quantitative genetic mapping with MapManagerQTX and WebQTL. Results：Proliferation determined by MTT indicated that the ability of dendritic cells to stimulate T-cell proliferation greatly depended on BXD RI strain that was used as a stimulator. Furthermore, the phagocytosis capability of dendritic cells to tumor cells and induction of IL-12 in vitro were correlated (P<0.01) with the T-cell proliferative response stimulated by dendritic cells. Quantitative genetic mapping identified two loci that were associated with the processing of tumor antigens by dendrtic cells and the cytotoxicity response to the tumor cells.Conclusions：Processing and presenting of tumor Ag of Dendritic cells is variable and BXD strain dependent. Induction of the CD80 and CD54 Co-stimulation molecular is greatly affected by co-cultured tumor cells and BXD strain dependent. IL-12 induction and stimulation of tumor primed T cells are also BXD strain dependent. Inhibition of tumor growth by co-cultured DC were co-related with the induction of IL-12 and co-stimulation molecular induction and QTL mapped to Chromosome 6 and 13, respectively.
Objective：We utilized genotype well-defined 17 strains of BXD RI strains mice to determine if genetic loci underlie the dendritic cell response to the breast tumor cell line TS/A. Dendritic cells were assayed for tumor antigen processing, presenting function and IL-12 induction.Methods：T cells isolated from BXD strain 9 (BXD9, H-2d) or BXD strain 2 (H-2b) of mice previously immunized with breast tumor TS/A cells were stimulated in vitro with dendritic cells isolated from MHC matched BXD RI mice fed with TS/A tumor antigens for 3 days. Analysis of Dendritic phagocytosis apoptotic tumor cells.IL-12 induction of Dendritic cells isolated from BXD mice. Dendritic cells inhibit tumor cell growth and induce IFN-γ produced by T cells. Flow Cytometric Analysis of DC surface antigen expression. Dendritic cells Quantitative genetic mapping with MapManagerQTX and WebQTL. Results：Proliferation determined by MTT indicated that the ability of dendritic cells to stimulate T-cell proliferation greatly depended on BXD RI strain that was used as a stimulator. Furthermore, the phagocytosis capability of dendritic cells to tumor cells and induction of IL-12 in vitro were correlated (P<0.01) with the T-cell proliferative response stimulated by dendritic cells. Quantitative genetic mapping identified two loci that were associated with the processing of tumor antigens by dendrtic cells and the cytotoxicity response to the tumor cells.Conclusions：Processing and presenting of tumor Ag of Dendritic cells is variable and BXD strain dependent. Induction of the CD80 and CD54 Co-stimulation molecular is greatly affected by co-cultured tumor cells and BXD strain dependent. IL-12 induction and stimulation of tumor primed T cells are also BXD strain dependent. Inhibition of tumor growth by co-cultured DC were co-related with the induction of IL-12 and co-stimulation molecular induction and QTL mapped to Chromosome 6 and 13, respectively.
2004, 11(2):89-91.
DOI: 10.3872/j.issn.1007-385X.2004.2.003
Abstract:
Objective:To probe the potent effects of dendritic cells (DCs)-based vaccine by enhancing the mutual action between DCs and T lymphocytes. Methods: Mouse bone marrow DCs transduced with human Macrophage Inflammatory Protein-1 beta (MIP-1β) gene by adenovirus vector (MIP-1β-DC) were pulsed with murine CT26 colorectal adenocarcinoma cell antigen, and used to vaccinate syngeneic mice.Results: Immunization with CT26 cell antigen-pulsed MIP-1β-DC induced specific CTL against CT26 cells and induced protective antitumor immunity, which rendered the immunized mice resistant completely to CT26 tumor challenge. The result of the study on antitumor immunity mechanism showed that the antitumor response depended on both CD8+ T cells and CD4+ T cells ,which played important roles,while NK cells were not necessary.Conclusions:MIP-1β gene modified DCs are more potent in induction of antitumor immunity through the preferential chemotaxis of DCs on T cells. Vaccination with tumor antigen-pulsed MIP-1β-DC may be a novel approach to immunotherapy of cancer.
Objective:To probe the potent effects of dendritic cells (DCs)-based vaccine by enhancing the mutual action between DCs and T lymphocytes. Methods: Mouse bone marrow DCs transduced with human Macrophage Inflammatory Protein-1 beta (MIP-1β) gene by adenovirus vector (MIP-1β-DC) were pulsed with murine CT26 colorectal adenocarcinoma cell antigen, and used to vaccinate syngeneic mice.Results: Immunization with CT26 cell antigen-pulsed MIP-1β-DC induced specific CTL against CT26 cells and induced protective antitumor immunity, which rendered the immunized mice resistant completely to CT26 tumor challenge. The result of the study on antitumor immunity mechanism showed that the antitumor response depended on both CD8+ T cells and CD4+ T cells ,which played important roles,while NK cells were not necessary.Conclusions:MIP-1β gene modified DCs are more potent in induction of antitumor immunity through the preferential chemotaxis of DCs on T cells. Vaccination with tumor antigen-pulsed MIP-1β-DC may be a novel approach to immunotherapy of cancer.
2004, 11(2):92-95.
DOI: 10.3872/j.issn.1007-385X.2004.2.004
Abstract:
Objective: To study the effect on cytotoxicity and drug resistance by blocking ERCC1 gene expression in ovarian cancer cell lines. Methods: ERCC1 antisense expression vector was constructed and transfected into human ovarian cancer cell lines. Northern blotting and Western blotting were used to detect the RNA and protein expression level in the cells. Luciferase assay system was used to reveal the host cell reactivation function. MTT assay was used to study the effect on cytotoxicity and drug resistance in the cell lines.Results: After transfection of the antisense ERCC1, Northern and Western blotting indicated that the RNA and protein expression level of ERCC1 was obviously decreased. Luciferease assay showed reduced DNA-damage repair capacity as assessed by host cell reactivation. MTT assay also showed decreased drug resistance to cisplatin in the lines. Conclusion: Transfection of antisense ERCC1 may enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines.
Objective: To study the effect on cytotoxicity and drug resistance by blocking ERCC1 gene expression in ovarian cancer cell lines. Methods: ERCC1 antisense expression vector was constructed and transfected into human ovarian cancer cell lines. Northern blotting and Western blotting were used to detect the RNA and protein expression level in the cells. Luciferase assay system was used to reveal the host cell reactivation function. MTT assay was used to study the effect on cytotoxicity and drug resistance in the cell lines.Results: After transfection of the antisense ERCC1, Northern and Western blotting indicated that the RNA and protein expression level of ERCC1 was obviously decreased. Luciferease assay showed reduced DNA-damage repair capacity as assessed by host cell reactivation. MTT assay also showed decreased drug resistance to cisplatin in the lines. Conclusion: Transfection of antisense ERCC1 may enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines.
2004, 11(2):96-99.
DOI: 10.3872/j.issn.1007-385X.2004.2.005
Abstract:
Objective: To investigate the inhibitory effects and mechanism of a novel anti-human DR5(death receptor 5 of TRAIL) monoclonal antibody to glioma cell lines U343.Methods:DR5 protein was tested quantitative through FCM; DR5 mRNA was observed through RT-PCR and distribution was tested by immunocytochemistry. Inhibitory effects and inducing apoptosis of anti human DR5 monoclonal antibody to U343 were analysed by MTT, DNA Ladder,FCM. Results: The expression of death receptor 5 (DR5) was certificated in U343,DR5 appeared to be located in intracellular perinuclear compartment. Inhibitory effects of anti-human DR5 monoclonal antibody on U343 were achieved by 3 μg/ml at 4 hours and the mechanism was associated with apoptosis. Conclusion: Apoptosis of glioma cell lines U343 can be induced by anti-human DR5 monoclonal antibody, and targeted on DR will provide new way to treating cancer.
Objective: To investigate the inhibitory effects and mechanism of a novel anti-human DR5(death receptor 5 of TRAIL) monoclonal antibody to glioma cell lines U343.Methods:DR5 protein was tested quantitative through FCM; DR5 mRNA was observed through RT-PCR and distribution was tested by immunocytochemistry. Inhibitory effects and inducing apoptosis of anti human DR5 monoclonal antibody to U343 were analysed by MTT, DNA Ladder,FCM. Results: The expression of death receptor 5 (DR5) was certificated in U343,DR5 appeared to be located in intracellular perinuclear compartment. Inhibitory effects of anti-human DR5 monoclonal antibody on U343 were achieved by 3 μg/ml at 4 hours and the mechanism was associated with apoptosis. Conclusion: Apoptosis of glioma cell lines U343 can be induced by anti-human DR5 monoclonal antibody, and targeted on DR will provide new way to treating cancer.
2004, 11(2):100-104.
DOI: 10.3872/j.issn.1007-385X.2004.2.006
Abstract:
Objective:To verify that COX-2 promoter can drive its downstream genes specifically in COX-2-positive ovarian cancer cells; Moreover, comparing with CMV promoter, analyze the transcript efficiency of COX 2 promoter. Methods:Contructing the recombinant plasmids named COX-2-BAX and CMV-Luc. After transient transfection liposome-mediated with the plasmids COX-2-Luc and CMV-Luc, respectively, the expression of Luciferase reporter gene was measured in COX-2-positive ovarian cancer cell line-SKOV3 and COX-2-negative colon cancer cell line-SW480. SKOV-3 and SW480 were transfected with COX-2-BAX and CMV-BAX in the same way, respectively. The apoptosis rates were measured through flow ytometry.Results:The recombinant plasmids named COX-2-BAX and CMV-Luc were constructed successfully. The expression efficiency of reporter gene was 1554±86.5 in SKOV3 and 53.7±10.9 in SW480 after 24 hours transfected with phPES2, 9851.7±129.5 in SKOV3 and 8831.0±167.3 in SW480 after 24 hours transfected with CMV-Luc in the same way. The apoptosis rate was 10.4% in SKOV3 and 3.7% in SW480 after transfected with COX-2-BAX, 21.7%in SKOV3 and 25.6% in SW480 after 36 hours transfected with pcDNA3-BAX in the same way.Conclusions:COX-2 promoter can drive its downstream genes specifically in COX-2-positive ovarian cancer cell lines, but its expression efficiency wasmarkedly lower than CMV promoter's. With proper modification, COX-2 promoter is expected to be useful in gene therapy of ovarian cancers.
Objective:To verify that COX-2 promoter can drive its downstream genes specifically in COX-2-positive ovarian cancer cells; Moreover, comparing with CMV promoter, analyze the transcript efficiency of COX 2 promoter. Methods:Contructing the recombinant plasmids named COX-2-BAX and CMV-Luc. After transient transfection liposome-mediated with the plasmids COX-2-Luc and CMV-Luc, respectively, the expression of Luciferase reporter gene was measured in COX-2-positive ovarian cancer cell line-SKOV3 and COX-2-negative colon cancer cell line-SW480. SKOV-3 and SW480 were transfected with COX-2-BAX and CMV-BAX in the same way, respectively. The apoptosis rates were measured through flow ytometry.Results:The recombinant plasmids named COX-2-BAX and CMV-Luc were constructed successfully. The expression efficiency of reporter gene was 1554±86.5 in SKOV3 and 53.7±10.9 in SW480 after 24 hours transfected with phPES2, 9851.7±129.5 in SKOV3 and 8831.0±167.3 in SW480 after 24 hours transfected with CMV-Luc in the same way. The apoptosis rate was 10.4% in SKOV3 and 3.7% in SW480 after transfected with COX-2-BAX, 21.7%in SKOV3 and 25.6% in SW480 after 36 hours transfected with pcDNA3-BAX in the same way.Conclusions:COX-2 promoter can drive its downstream genes specifically in COX-2-positive ovarian cancer cell lines, but its expression efficiency wasmarkedly lower than CMV promoter's. With proper modification, COX-2 promoter is expected to be useful in gene therapy of ovarian cancers.
2004, 11(2):105-109.
DOI: 10.3872/j.issn.1007-385X.2004.2.007
Abstract:
Objective:To explore the variation of the proteins of erbB-2, p53 and MDR1 in repeated treatment by anticancer drugs and the effects on biotherapy. Methods:The human lung cancer cell line (LPET-a-1) was repeatedly treated by anticancer drugs—doxorubicin, etoposide, cisplatin and combination of the three drugs, and every drug was given in two concentrations. erbB-2, p53 and MDR1 were tested by flow cytometry. The percentage of cells expressing these proteins and the mean quantity of them were acquired. We could calculate the number of cells expressing each protein, the mean quantity of each protein and the total one after each treatment by different drugs in different concentration. Results:The levels of every protein decreased along with the time of cell culture. In high-dose groups, almost every item of erbB-2 and MDR1 was downregulated along with the times of treatment. In low-dose groups, there was not a rule of the item's variation and some of them increased. There was not a rule of p53 variation, and the expression of it in drug groups was higher than that in control at the third drug treatment.Conclusion:After treated by anticancer drugs, especially in low-dose, the expressions of erbB-2, p53 and MDR1 increased at different level. These data suggested that the patients suffering from cancer should be given adequate biotherapy after chemotherapy in clinical practice.
Objective:To explore the variation of the proteins of erbB-2, p53 and MDR1 in repeated treatment by anticancer drugs and the effects on biotherapy. Methods:The human lung cancer cell line (LPET-a-1) was repeatedly treated by anticancer drugs—doxorubicin, etoposide, cisplatin and combination of the three drugs, and every drug was given in two concentrations. erbB-2, p53 and MDR1 were tested by flow cytometry. The percentage of cells expressing these proteins and the mean quantity of them were acquired. We could calculate the number of cells expressing each protein, the mean quantity of each protein and the total one after each treatment by different drugs in different concentration. Results:The levels of every protein decreased along with the time of cell culture. In high-dose groups, almost every item of erbB-2 and MDR1 was downregulated along with the times of treatment. In low-dose groups, there was not a rule of the item's variation and some of them increased. There was not a rule of p53 variation, and the expression of it in drug groups was higher than that in control at the third drug treatment.Conclusion:After treated by anticancer drugs, especially in low-dose, the expressions of erbB-2, p53 and MDR1 increased at different level. These data suggested that the patients suffering from cancer should be given adequate biotherapy after chemotherapy in clinical practice.
LIU Feng
,
LOU Guo-liang
,
LI Yong-mei
,
CHEN Li
,
HUANG Zheng-xia
,
YANG Hong-bin
,
ZHU Yang
,
WANG Liang-hua
,
Qiu Yi
,
JIAO Bing-hua
2004, 11(2):110-113.
DOI: 10.3872/j.issn.1007-385X.2004.2.008
Abstract:
Objective：To obtain alloantigen-specific hyporesponsivness via depletion of alloreactive cells from haematopoietic stem cell grafts.Methods：BALB/c mouse bone marrow-derived dendritic cells genetically engineered to express FasL were cultured with C57BL/6 mouse spleen lymphocytes, and MLR was performed to evaluate the alloantigen-specific hyporesponsivness.Results：FasL-transfected DC induced T cell apoptosis and inhibited alloantigen-specific hyporesponsiveness in vitro.Conclusion：DC transfected with FasL gene may prevent graft-versus-host disease by selective removal of alloreactive cells from haematopoietic stem cell grafts
Objective：To obtain alloantigen-specific hyporesponsivness via depletion of alloreactive cells from haematopoietic stem cell grafts.Methods：BALB/c mouse bone marrow-derived dendritic cells genetically engineered to express FasL were cultured with C57BL/6 mouse spleen lymphocytes, and MLR was performed to evaluate the alloantigen-specific hyporesponsivness.Results：FasL-transfected DC induced T cell apoptosis and inhibited alloantigen-specific hyporesponsiveness in vitro.Conclusion：DC transfected with FasL gene may prevent graft-versus-host disease by selective removal of alloreactive cells from haematopoietic stem cell grafts
CHEN Jie
,
QIAN Qi-jun
,
DA Wan-ming
,
OU Ying-xian
,
XUE Hui-bin
,
CUI Zhen-fu
,
SU Chang-qing
,
LI Lin-fang
,
JIANG Li-hua
,
WU Hong-ping
2004, 11(2):114-118.
DOI: 10.3872/j.issn.1007-385X.2004.2.009
Abstract:
Objectives: To construct the replication-competent adenovirus CNHK200--mIFN-γ and the replication-deficient adenovirus AdEasy-mIFN-γ, and to compare their expression of mIFN-γ and the replicative activities of CNHK200-mIFN-γ，ONYX-015 and wide-type adenovirus (Ad5) in normal and cancer cells. Their antitumor activities were also observed.Methods:The replicative activities of viruses in cells were measured by viral replication assay; The replication of CNHK200-mIFN-γ in Hep3B cells was observed under the electron microscopy; The expression of mIFN-γ in cancer cells was detected by ELISA. CPE assay was used to detect viral antitumor activity.Results: Both CNHK200-mIFN-γ and ONYX-015 replicated only in cancer cells, and CNHK200-mIFN-γ replicated more potential than ONYX-015; The infection of CNHK200-mIFN-γ led to an obvious expression of mIFN-γ, whereas the expression of mIFN-γ mediated by AdEasy-mIFN-γ could not be detected at all. CNHK200-mIFN-γ and ONYX-015 could kill cancer cells but spare normal cells. Conclusions: The insertion of mIFN-γ causes no alterations on the tumor-specific replication of the original virus. CNHK200-mIFN-γ can selectively replicate and effectively express mIFN-γ in tumor cells, and specifically kill cancer cells, suggesting a splendid future as a new anticancer agent.
Objectives: To construct the replication-competent adenovirus CNHK200--mIFN-γ and the replication-deficient adenovirus AdEasy-mIFN-γ, and to compare their expression of mIFN-γ and the replicative activities of CNHK200-mIFN-γ，ONYX-015 and wide-type adenovirus (Ad5) in normal and cancer cells. Their antitumor activities were also observed.Methods:The replicative activities of viruses in cells were measured by viral replication assay; The replication of CNHK200-mIFN-γ in Hep3B cells was observed under the electron microscopy; The expression of mIFN-γ in cancer cells was detected by ELISA. CPE assay was used to detect viral antitumor activity.Results: Both CNHK200-mIFN-γ and ONYX-015 replicated only in cancer cells, and CNHK200-mIFN-γ replicated more potential than ONYX-015; The infection of CNHK200-mIFN-γ led to an obvious expression of mIFN-γ, whereas the expression of mIFN-γ mediated by AdEasy-mIFN-γ could not be detected at all. CNHK200-mIFN-γ and ONYX-015 could kill cancer cells but spare normal cells. Conclusions: The insertion of mIFN-γ causes no alterations on the tumor-specific replication of the original virus. CNHK200-mIFN-γ can selectively replicate and effectively express mIFN-γ in tumor cells, and specifically kill cancer cells, suggesting a splendid future as a new anticancer agent.
2004, 11(2):119-123.
DOI: 10.3872/j.issn.1007-385X.2004.2.010
Abstract:
Objective:To evaluate the gene therapeutic efficiency of TRAIL combined with DOX on human colon cancer cell line RKO. Methods:Human colon cancer cell line RKO was transfected with adenovirus-mediated TRAIL gene Ad/GT-TRAIL and TRAIL/DOX combination. Cell growth and apoptosis were measured by MTT method and flow cytometry. TRAIL gene expression before and after combination with DOX was measured by RT-PCR.Results: The cell line RKO was slightly suppressed by mediate adenovirus and significantly suppressed by TRAIL gene. The suppression percentages were 11.9% and 50.1% respectively. Combined with DOX, the suppression and the apoptosis of cell line RKO could be significantly enhanced by TRAIL gene. The suppression and the apoptic percentage reached 60.3% and 49.0%respectively. RT-PCR showed the expression of TRAIL gene was not enhanced in the RKO cells exposed to the DOX/TRAIL combination.Conclusion: TRAIL gene was able to induce apoptosis and suppress the growth of human colon cancer cell line RKO. Combined with DOX, the suppression and apoptosis could be significantly enhanced. But this result came out not by increasing the expression of TRAIL gene.
Objective:To evaluate the gene therapeutic efficiency of TRAIL combined with DOX on human colon cancer cell line RKO. Methods:Human colon cancer cell line RKO was transfected with adenovirus-mediated TRAIL gene Ad/GT-TRAIL and TRAIL/DOX combination. Cell growth and apoptosis were measured by MTT method and flow cytometry. TRAIL gene expression before and after combination with DOX was measured by RT-PCR.Results: The cell line RKO was slightly suppressed by mediate adenovirus and significantly suppressed by TRAIL gene. The suppression percentages were 11.9% and 50.1% respectively. Combined with DOX, the suppression and the apoptosis of cell line RKO could be significantly enhanced by TRAIL gene. The suppression and the apoptic percentage reached 60.3% and 49.0%respectively. RT-PCR showed the expression of TRAIL gene was not enhanced in the RKO cells exposed to the DOX/TRAIL combination.Conclusion: TRAIL gene was able to induce apoptosis and suppress the growth of human colon cancer cell line RKO. Combined with DOX, the suppression and apoptosis could be significantly enhanced. But this result came out not by increasing the expression of TRAIL gene.
WEN Jian-yan
,
ZHANG Ling
,
WAGN Yun
,
MAO Hai-ting
,
WEN Pei-e
,
CUI Shu-ling
,
LI Deng-hua
,
GU Hong-tao
2004, 11(2):124-128.
DOI: 10.3872/j.issn.1007-385X.2004.2.011
Abstract:
Objective: To explore the mechanism of HLA-G antisense oligodeoxynucleotides (ASODN) on reversion of tumour immune evasion and enhancement of killer activity of immune effector cells. Methods: The expression of HLA-G in JEG-3 cell line was studied by RT-PCR and flow cytometry. MTT assay was used in our studies to detect the change of NK and CD3AK killer activity after ASODN treatment. The effect of IFN-γ secreted by CD3AK after coculture of tumour-lymphocytes was explored by ELISA.Results： HLA-G ASODN could obviously inhibit the expression of HLA-G mRNA and protein. The supernatant of JEG-3 treated by ASODN could reverse the inhibiton of soluble HLA-G molecules to NK killer activity. On the other hand, HLA-G ASODN also could reverse the inhibition of HLA-G to the production of IFN-γ secreted by CD3AK and enhanced the proliferation of CD3AK. Meanwhile, it also improved the susceptibility of JEG-3 to CD3AK.Conclusion:HLA-G ASODN could inhibits the expression of HLA-G mRNA and protein in tumour cells and partly restore the cytotoxicity of NK and CD3AK cells, and thereby partly reverse immune evasion of tumour cells caused by souble HLA-G molecules.
Objective: To explore the mechanism of HLA-G antisense oligodeoxynucleotides (ASODN) on reversion of tumour immune evasion and enhancement of killer activity of immune effector cells. Methods: The expression of HLA-G in JEG-3 cell line was studied by RT-PCR and flow cytometry. MTT assay was used in our studies to detect the change of NK and CD3AK killer activity after ASODN treatment. The effect of IFN-γ secreted by CD3AK after coculture of tumour-lymphocytes was explored by ELISA.Results： HLA-G ASODN could obviously inhibit the expression of HLA-G mRNA and protein. The supernatant of JEG-3 treated by ASODN could reverse the inhibiton of soluble HLA-G molecules to NK killer activity. On the other hand, HLA-G ASODN also could reverse the inhibition of HLA-G to the production of IFN-γ secreted by CD3AK and enhanced the proliferation of CD3AK. Meanwhile, it also improved the susceptibility of JEG-3 to CD3AK.Conclusion:HLA-G ASODN could inhibits the expression of HLA-G mRNA and protein in tumour cells and partly restore the cytotoxicity of NK and CD3AK cells, and thereby partly reverse immune evasion of tumour cells caused by souble HLA-G molecules.
GE Lin-fu
,
JIANG Guo-sheng
,
LIU Xi-min
,
HUANG Ning
,
LIU Chuan-fang
,
TANG Tian-hua
,
DONG Zhen-jun
,
MA Huan-wen
,
ZHANG Yu-kun
,
KONG Fan-sheng
,
GUO Peng
2004, 11(2):129-132.
DOI: 10.3872/j.issn.1007-385X.2004.2.012
Abstract:
Objective:To observe graft-versus-host disease (GVHD) reaction in activated half-matched mixed bone marrow transplantation (BMT). Methods: Mice 615 with acute radiation disease were used as recipient mice model, with 615C57BL/6 hybrid F1 mice being half-matched donor mice. Half-matched murine bone marrow and spleen cells mixed with certain percent of syngeneic spleen cells were activated in vitro with IL-2 and underwent mixed half-matched BMT to observe their death rate, hematopoietic recovery of myelocytic lineage, spleen node count, mixed lymphocyte reaction in vivo, chimerism and pathological changes, to compared with GVHD reactions of various transplant models. Results:GVHD was relatively severe in both activated and non-actived half-matched BMT groups, showing that activated half-matched mixed 3∶1 BMT group after activation could not alleviate GVHD, whereas the activated half-matched mixed 1∶1 BMT group or 2∶1 group with high percentage of syngeneic spleen cells could lessen the extent of GVHD, as manifested by the quick recovery of peripheral blood WBC count and bone marrow GM-CFU formation, by the low of mixed lymphocyte reaction in vivo as well as by the increase of chimeric rate. Conclusions:Half-matched mixed BMT after activation could lower the GVHD reaction, which was relative to the ratio of syngeneic to allogeneic spleen cells.
Objective:To observe graft-versus-host disease (GVHD) reaction in activated half-matched mixed bone marrow transplantation (BMT). Methods: Mice 615 with acute radiation disease were used as recipient mice model, with 615C57BL/6 hybrid F1 mice being half-matched donor mice. Half-matched murine bone marrow and spleen cells mixed with certain percent of syngeneic spleen cells were activated in vitro with IL-2 and underwent mixed half-matched BMT to observe their death rate, hematopoietic recovery of myelocytic lineage, spleen node count, mixed lymphocyte reaction in vivo, chimerism and pathological changes, to compared with GVHD reactions of various transplant models. Results:GVHD was relatively severe in both activated and non-actived half-matched BMT groups, showing that activated half-matched mixed 3∶1 BMT group after activation could not alleviate GVHD, whereas the activated half-matched mixed 1∶1 BMT group or 2∶1 group with high percentage of syngeneic spleen cells could lessen the extent of GVHD, as manifested by the quick recovery of peripheral blood WBC count and bone marrow GM-CFU formation, by the low of mixed lymphocyte reaction in vivo as well as by the increase of chimeric rate. Conclusions:Half-matched mixed BMT after activation could lower the GVHD reaction, which was relative to the ratio of syngeneic to allogeneic spleen cells.
2004, 11(2):133-137.
DOI: 10.3872/j.issn.1007-385X.2004.2.013
Abstract:
Objective: To generate a new kind of cytokine MIP-2γ/GM-CSF fusion protein by genetic engineering technology and investigate its biological bifunction. Methods: The eukaryotic expressing vector for MIP-2γ/GM-CSF fusion protein was constructed by primer design and step by step cloing. MIP-2γ gene and GM-CSF gene were linked via a linker sequence（Gly4Ser）3. After transient expression， the cell supernatants of 293-T cells containing the aimed protein were obtained. The hematopoietic and chemotactic activities of the fusion protein were investigated in vitro.Results: MIP-2γ/GM-CSF fusion gene vector was confirmed by restriction analysis and sequencing analysis. The molecular weight of the protein was about 24.9 kD. MIP-2γ/GM-CSF fusion protein could stimulate the proliferation of TF-1 cells like GM-CSF. The specific activity of the fusion protein was 2.2×107IU/mg. The fusion protein was found to be effective in stimulating bone marrow cell colony-formation and chemoattracting immature dendritic cells and CD3+T cells. Conclusion: MIP-2γ/GM-CSF fusion protein exhibits bifunctions which including the promotion of hematopoiesis and chemoattration of immune cells, suggesting that the fusion protein can be used to regulate hematopoiesis and antitumor immune response as a new kind of cytokine.
Objective: To generate a new kind of cytokine MIP-2γ/GM-CSF fusion protein by genetic engineering technology and investigate its biological bifunction. Methods: The eukaryotic expressing vector for MIP-2γ/GM-CSF fusion protein was constructed by primer design and step by step cloing. MIP-2γ gene and GM-CSF gene were linked via a linker sequence（Gly4Ser）3. After transient expression， the cell supernatants of 293-T cells containing the aimed protein were obtained. The hematopoietic and chemotactic activities of the fusion protein were investigated in vitro.Results: MIP-2γ/GM-CSF fusion gene vector was confirmed by restriction analysis and sequencing analysis. The molecular weight of the protein was about 24.9 kD. MIP-2γ/GM-CSF fusion protein could stimulate the proliferation of TF-1 cells like GM-CSF. The specific activity of the fusion protein was 2.2×107IU/mg. The fusion protein was found to be effective in stimulating bone marrow cell colony-formation and chemoattracting immature dendritic cells and CD3+T cells. Conclusion: MIP-2γ/GM-CSF fusion protein exhibits bifunctions which including the promotion of hematopoiesis and chemoattration of immune cells, suggesting that the fusion protein can be used to regulate hematopoiesis and antitumor immune response as a new kind of cytokine.
2004, 11(2):138-141.
DOI: 10.3872/j.issn.1007-385X.2004.2.014
Abstract:
Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector (pShuttle 2-BMP-2) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCeⅠ and I-CeuⅠ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2), which was finally linearized with PacⅠ and transfered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also identified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method descinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.
Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector (pShuttle 2-BMP-2) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCeⅠ and I-CeuⅠ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2), which was finally linearized with PacⅠ and transfered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also identified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method descinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.
联系方式
- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
- 国内定价： ￥20元/册
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.