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Volume 9,Issue 2,2002 Table of Contents
2002, 9(2):77-81.
DOI: 10.3872/j.issn.1007-385X.2002.2.001
Abstract:
Objective: To evaluate the possibility of the anti-tumor immunity induction by co-transfecting the colorectal cancer cells with chemokine MCP-3 and B7 gene. Methods: Mouse MCP-3 and B7(B7.1) genes were transduced into mouse colorectal cancer CMT93 cells using Liposome. G418-ressistant clones were selected. MCP-3 and B7 mRNA expression was detected by RT-PCR. Chemotactic activity of MCP-3 was measured by chemotaxis assay. The tumoregericity of gene transfectants (CMT93/B7+MCP-3) was detected by in vivo experiments. The immune protection against rechallenged CMT93 in the mice primed by CMT93/B7+MCP-3 was observed, and the effect of CMT93/B7+MCP-3 as vaccine to treat established tumors in the early stage was detected by in vivo experiments as well. Results:CMT93/B7+MCP-3 expressed MCP-3 and B7 mRNA, but control groups not expressed. The secreted MCP-3 in the cell culture supernatant possesses the chemotatic activity. in vivo experiments showed that the tumorigenicity of CMT93/B7+MCP-3 was reduced significantly (P<0.01), All mice primed by CMT93/B7+MCP-3 got system immunity against the rechallenged CMT93 (P<0.05). And CMT93/B7+MCP-3 as vaccine retarded the growth of the tumors from the early stage of CMT93.Conclusion: The co-transfection of MCP-3 gene and B7 gene promotes the induction of anti-colorectal cancer immunity, and co-transfectant as vaccine is effective to treat the established tumors.
Objective: To evaluate the possibility of the anti-tumor immunity induction by co-transfecting the colorectal cancer cells with chemokine MCP-3 and B7 gene. Methods: Mouse MCP-3 and B7(B7.1) genes were transduced into mouse colorectal cancer CMT93 cells using Liposome. G418-ressistant clones were selected. MCP-3 and B7 mRNA expression was detected by RT-PCR. Chemotactic activity of MCP-3 was measured by chemotaxis assay. The tumoregericity of gene transfectants (CMT93/B7+MCP-3) was detected by in vivo experiments. The immune protection against rechallenged CMT93 in the mice primed by CMT93/B7+MCP-3 was observed, and the effect of CMT93/B7+MCP-3 as vaccine to treat established tumors in the early stage was detected by in vivo experiments as well. Results:CMT93/B7+MCP-3 expressed MCP-3 and B7 mRNA, but control groups not expressed. The secreted MCP-3 in the cell culture supernatant possesses the chemotatic activity. in vivo experiments showed that the tumorigenicity of CMT93/B7+MCP-3 was reduced significantly (P<0.01), All mice primed by CMT93/B7+MCP-3 got system immunity against the rechallenged CMT93 (P<0.05). And CMT93/B7+MCP-3 as vaccine retarded the growth of the tumors from the early stage of CMT93.Conclusion: The co-transfection of MCP-3 gene and B7 gene promotes the induction of anti-colorectal cancer immunity, and co-transfectant as vaccine is effective to treat the established tumors.
2002, 9(2):82-84.
DOI: 10.3872/j.issn.1007-385X.2002.2.002
Abstract:
Objective: To investigate the expression of MDR1 and GFP in the human hematopoietic cells mediated by adeno-associated virus. Methods:The GFP gene was transferred into the human hematopoietic cells by AAV vectors and created strong visible fluorescence by purely molecular biological means. Using adeno-associated virus vectors, we have transferred human mdr-1 gene into human hematopoietic cells and investigated the drug resistence of human hematopoietic cells modified with mdr-1 gene. PCR analysis confirmed that mdr1 cDNA had been successfully transferred into the human hematopoietic cells.An assay of MTT proved that the human hematopoietic cells modified by mdr1 gene had resistance to colchicine.Results: It was about 30% of the hematopoietic cells that expressed the green fluorescent proteins. The resistance of hematopoietic cells was increased parently when the cells were infected by the crude virus stocks. Conclusion: It is conducted that the AAV vector could successfully transfer the foreign gene into the human hematopoietic cells. The cells modified with mdr1 gene have increased the resistance to drugs.
Objective: To investigate the expression of MDR1 and GFP in the human hematopoietic cells mediated by adeno-associated virus. Methods:The GFP gene was transferred into the human hematopoietic cells by AAV vectors and created strong visible fluorescence by purely molecular biological means. Using adeno-associated virus vectors, we have transferred human mdr-1 gene into human hematopoietic cells and investigated the drug resistence of human hematopoietic cells modified with mdr-1 gene. PCR analysis confirmed that mdr1 cDNA had been successfully transferred into the human hematopoietic cells.An assay of MTT proved that the human hematopoietic cells modified by mdr1 gene had resistance to colchicine.Results: It was about 30% of the hematopoietic cells that expressed the green fluorescent proteins. The resistance of hematopoietic cells was increased parently when the cells were infected by the crude virus stocks. Conclusion: It is conducted that the AAV vector could successfully transfer the foreign gene into the human hematopoietic cells. The cells modified with mdr1 gene have increased the resistance to drugs.
2002, 9(2):85-89.
DOI: 10.3872/j.issn.1007-385X.2002.2.003
Abstract:
Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI)SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/TK,but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion:The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.
Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI)SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/TK,but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion:The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.
2002, 9(2):90-93.
DOI: 10.3872/j.issn.1007-385X.2002.2.004
Abstract:
Objective: To investigate the successive fluctuation of some Th1 type cytokines in the peripheral blood of tumor-bearing mice treated with tumor-derived heat shock protein 70(HSP70), explore the mechanism of HSP 70 in breaking through the tumor-immunity tolerance of the organism with tumor-burden and in inducing effective anti-tumor immune response, and provide valuable reference for tumor-derived HSP70 administration in treating human cancer. Methods:Cell culture, techniques for protein extraction and purification, SDS-PAGE, Western-blot, capillary electrophoresis, ELISA technique and animal experiment were applied. Results: HSP70 could result in apparent tumor-inhibitory effect and upregulate some Th1 type cytokines(IL-2, TNF-β, and IFN-γ) in the peripheral blood of the treated mice gradually to the frequency of HSP70 administration, and showed no reduction trend in two weeks after the final treatment. Statistically significant difference was observed contrasted with those of the control group (P<0.01).Conclusion: Tumor-derived HSP70 could dramatically upregulate the Th1 type cytokines of tumor-bearing mice to trigger and maintain effective immunological activity against tumor, which is an important aspect of its anti tumor activity.
Objective: To investigate the successive fluctuation of some Th1 type cytokines in the peripheral blood of tumor-bearing mice treated with tumor-derived heat shock protein 70(HSP70), explore the mechanism of HSP 70 in breaking through the tumor-immunity tolerance of the organism with tumor-burden and in inducing effective anti-tumor immune response, and provide valuable reference for tumor-derived HSP70 administration in treating human cancer. Methods:Cell culture, techniques for protein extraction and purification, SDS-PAGE, Western-blot, capillary electrophoresis, ELISA technique and animal experiment were applied. Results: HSP70 could result in apparent tumor-inhibitory effect and upregulate some Th1 type cytokines(IL-2, TNF-β, and IFN-γ) in the peripheral blood of the treated mice gradually to the frequency of HSP70 administration, and showed no reduction trend in two weeks after the final treatment. Statistically significant difference was observed contrasted with those of the control group (P<0.01).Conclusion: Tumor-derived HSP70 could dramatically upregulate the Th1 type cytokines of tumor-bearing mice to trigger and maintain effective immunological activity against tumor, which is an important aspect of its anti tumor activity.
GUO Cong-hui
,
TIAN Chang-sheng
,
ZHAO Dan
,
GAO Rui-juan
,
TAN Yan
,
JIANG Yan-fang
,
DI Qian
,
ZHAO Xue-jian
2002, 9(2):94-97.
DOI: 10.3872/j.issn.1007-385X.2002.2.005
Abstract:
Objective: To investigate the effect of purified recombinant secreted human prostate secretory protein of 94 amino acids (rsHPSP94) on the growth of androgen-independent human prostate cancer cells (PC-3) and its potential mechanism of action. Methods:Cell growth inhibition rate was determined by MTT and cell cycle distribution was analysed by Flow cytometry(FCM) .Results: rsHPSP94, in a dose- and time-dependent manner, inhibited the growth of PC-3. Flow cytometric analysis showed that rsHPSP94 arrested the cell cycle in the G1-phase and induced the cell apoptosis after treatment for 48 h. Conclusion: These results suggest that rsHPSP94 can inhibit the growth of PC-3 by arresting the cell cycle in the G1-phase.
Objective: To investigate the effect of purified recombinant secreted human prostate secretory protein of 94 amino acids (rsHPSP94) on the growth of androgen-independent human prostate cancer cells (PC-3) and its potential mechanism of action. Methods:Cell growth inhibition rate was determined by MTT and cell cycle distribution was analysed by Flow cytometry(FCM) .Results: rsHPSP94, in a dose- and time-dependent manner, inhibited the growth of PC-3. Flow cytometric analysis showed that rsHPSP94 arrested the cell cycle in the G1-phase and induced the cell apoptosis after treatment for 48 h. Conclusion: These results suggest that rsHPSP94 can inhibit the growth of PC-3 by arresting the cell cycle in the G1-phase.
2002, 9(2):98-100.
DOI: 10.3872/j.issn.1007-385X.2002.2.006
Abstract:
Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDfGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDfGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with 1mmol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDfGC and KDfGC+BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/DAla system on K652e cells.
Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDfGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDfGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with 1mmol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDfGC and KDfGC+BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/DAla system on K652e cells.
2002, 9(2):101-104.
DOI: 10.3872/j.issn.1007-385X.2002.2.007
Abstract:
Objective: To study the effect of inhibiting the expreesion of the first subtype of the monocarboxylate transporter(MCT1) gene on proliferation and apoptosis in tumor cells.Methods:MCT1 antisense gene expression vector was introduced into A549 cells with electroporation to inhibit the expression of MCT1 gene. The positive colonies were selected by G418. The integration of MCT1 antisense gene and A549 genomic DNA was confirmed by PCR and RT-PCR technique. The pHi, lactate content and ATP content were detected with spectrophotometry and bioluminescence method respectively. The conditions of cell apoptosis were measured with in situ- apoptosis assay kit. And cells transfected MCT1 antisense gene and A549 cells were inoculated in nude mouse subcutaneous to observe the growth of transplantation tumor. Results:Compared with A549 cells, the pHi of cells transfected MCT1 antisense gene was remarkably decreased (P<005), and lactate content was remarkably increased (P<0.001), ATP content was remarkably decreased (P<005). Apoptosis rate of cells transfected MCT1 antisense gene was remarkly higher than A549 cells(P<001). And the nude mouse transplantation tumors inoculated cells transfected MCT1 antisense gene were significantly lighter and smaller than that ones inoculated A549 cells(P<0.001). Conclusion: MCT1 gene plays an important role in proliferation and apoptosis of tumor cells.
Objective: To study the effect of inhibiting the expreesion of the first subtype of the monocarboxylate transporter(MCT1) gene on proliferation and apoptosis in tumor cells.Methods:MCT1 antisense gene expression vector was introduced into A549 cells with electroporation to inhibit the expression of MCT1 gene. The positive colonies were selected by G418. The integration of MCT1 antisense gene and A549 genomic DNA was confirmed by PCR and RT-PCR technique. The pHi, lactate content and ATP content were detected with spectrophotometry and bioluminescence method respectively. The conditions of cell apoptosis were measured with in situ- apoptosis assay kit. And cells transfected MCT1 antisense gene and A549 cells were inoculated in nude mouse subcutaneous to observe the growth of transplantation tumor. Results:Compared with A549 cells, the pHi of cells transfected MCT1 antisense gene was remarkably decreased (P<005), and lactate content was remarkably increased (P<0.001), ATP content was remarkably decreased (P<005). Apoptosis rate of cells transfected MCT1 antisense gene was remarkly higher than A549 cells(P<001). And the nude mouse transplantation tumors inoculated cells transfected MCT1 antisense gene were significantly lighter and smaller than that ones inoculated A549 cells(P<0.001). Conclusion: MCT1 gene plays an important role in proliferation and apoptosis of tumor cells.
2002, 9(2):105-109.
DOI: 10.3872/j.issn.1007-385X.2002.2.008
Abstract:
Objective: To explore the effects of tumor cells on the development and function of the mononuclear cell (MNC)-derived dendritic cells. Methods: The supernatants of cultured cell lines H7402 (human hepatocellular carcinoma), HCT-8 (human colon carcinoma) and HFCL (human bone marrow stromal cell) were collected and used as the conditionalculture medium (CCM) for MNC-derived DC culture. The dendritic cells were characterized by phenotypic analysis. We evaluated the capacity of phagocytizing dextran, and the capacity to stimulate allogeneic T cells of MNC-derived DC. Results: Phenotypic analysis showed that dendritic cells cultured in the presence of supernatants of tumor cells expressed lower levels of CD1a, MHC Ⅱ molecules(HLA-DR) and costimulatory molecules (CD86), all of which were significantly lower than those in control and HFCL groups(the negative control group). Also, they displayed a much lower capacity of phagocytizing dextran as shown by the FITC-dextran assay. Moreover, they were less efficient of inducing allogeneic T-cell proliferation in a mixed lymphocyte reaction. Conclusion: These data show that tumor cells negatively regulate the production and function of DC, which might be one of the underlying mechanisms that play a role in the immune escape of tumor cells.
Objective: To explore the effects of tumor cells on the development and function of the mononuclear cell (MNC)-derived dendritic cells. Methods: The supernatants of cultured cell lines H7402 (human hepatocellular carcinoma), HCT-8 (human colon carcinoma) and HFCL (human bone marrow stromal cell) were collected and used as the conditionalculture medium (CCM) for MNC-derived DC culture. The dendritic cells were characterized by phenotypic analysis. We evaluated the capacity of phagocytizing dextran, and the capacity to stimulate allogeneic T cells of MNC-derived DC. Results: Phenotypic analysis showed that dendritic cells cultured in the presence of supernatants of tumor cells expressed lower levels of CD1a, MHC Ⅱ molecules(HLA-DR) and costimulatory molecules (CD86), all of which were significantly lower than those in control and HFCL groups(the negative control group). Also, they displayed a much lower capacity of phagocytizing dextran as shown by the FITC-dextran assay. Moreover, they were less efficient of inducing allogeneic T-cell proliferation in a mixed lymphocyte reaction. Conclusion: These data show that tumor cells negatively regulate the production and function of DC, which might be one of the underlying mechanisms that play a role in the immune escape of tumor cells.
2002, 9(2):110-112.
DOI: 10.3872/j.issn.1007-385X.2002.2.009
Abstract:
Objective: To highly express rh-Endostatin from Pichia pastoris and purify it to homogeneity. Methods: Constructed Pichia pastoris X33/pLW202 was amplified and inoculated to ferment media. The supernatant of the strain was collected after induction. Through ultrafiltration,affinity and gel chromatography, rh-Endostatin with high purication was obtained. Results: 60mg/L rh-Endostatin was obstained from supernatant. HPLC showed its purity was above 98%. Conclusion: High level expression of secreted rh-Endostatin has been successfully achieved in Pichia pastoris expression system.
Objective: To highly express rh-Endostatin from Pichia pastoris and purify it to homogeneity. Methods: Constructed Pichia pastoris X33/pLW202 was amplified and inoculated to ferment media. The supernatant of the strain was collected after induction. Through ultrafiltration,affinity and gel chromatography, rh-Endostatin with high purication was obtained. Results: 60mg/L rh-Endostatin was obstained from supernatant. HPLC showed its purity was above 98%. Conclusion: High level expression of secreted rh-Endostatin has been successfully achieved in Pichia pastoris expression system.
2002, 9(2):113-116.
DOI: 10.3872/j.issn.1007-385X.2002.2.010
Abstract:
Objective: To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hTNF-α, investigate the antitumor effect of recombinant adenovirus. Methods: Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hTNF-α. Cell growth assay, colone formation test, flowcytometry assay and morphology were used to observe the effects on tumor cells. The hTNF-α gene, which was transduced into cancer cells mediated by recombinant adenovirus, was detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay. The intratumoral injection of rAd-LacZ and rAd-hTNF-α was carried out to evaluate their antitumor effects. Results:The titer of rAd reached 10 10 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd. Except the surface structure and ultrastructure of tumor cells infected with rAd had a light change, cell growth abillity assay, colone formation test, flow cytometry assay showed no significant difference compared with that of the control cells. The TNF-α gene expression at 24 h increased greatly. Antitumor study indicated that on the tumor-bearing mice treated with rAd the tumor grew slowly. Tumor volume was significantly smaller and survive time was prolonged than that of controls.Conclusion:There was no significantly changes occurred on tumoral cells after infected with recombinant adenovirus expressing hTNF-α. The intratumoral injection of rAd-LacZ and rAd-hTNF-α could inhibit the growth of solid tumor.
Objective: To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hTNF-α, investigate the antitumor effect of recombinant adenovirus. Methods: Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hTNF-α. Cell growth assay, colone formation test, flowcytometry assay and morphology were used to observe the effects on tumor cells. The hTNF-α gene, which was transduced into cancer cells mediated by recombinant adenovirus, was detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay. The intratumoral injection of rAd-LacZ and rAd-hTNF-α was carried out to evaluate their antitumor effects. Results:The titer of rAd reached 10 10 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd. Except the surface structure and ultrastructure of tumor cells infected with rAd had a light change, cell growth abillity assay, colone formation test, flow cytometry assay showed no significant difference compared with that of the control cells. The TNF-α gene expression at 24 h increased greatly. Antitumor study indicated that on the tumor-bearing mice treated with rAd the tumor grew slowly. Tumor volume was significantly smaller and survive time was prolonged than that of controls.Conclusion:There was no significantly changes occurred on tumoral cells after infected with recombinant adenovirus expressing hTNF-α. The intratumoral injection of rAd-LacZ and rAd-hTNF-α could inhibit the growth of solid tumor.
11 Cloning of Fab Gene of an Anti-Human Bladder Cancer Monoclonal Antibody and Its Expression in E.coli
2002, 9(2):117-121.
DOI: 10.3872/j.issn.1007-385X.2002.2.011
Abstract:
Objective:To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E.coli. Methods: Fd and κ genes of mAb BDI were cloned by RT-PCRand inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E.coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immunohistochemistry. Results: Fd and κ genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E.coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion:The Fab gene of an anti-human bladder cancer mAb was expressed in E.coli. The importance of the N-terminal sequence on antibody binding activity was suggested.
Objective:To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E.coli. Methods: Fd and κ genes of mAb BDI were cloned by RT-PCRand inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E.coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immunohistochemistry. Results: Fd and κ genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E.coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion:The Fab gene of an anti-human bladder cancer mAb was expressed in E.coli. The importance of the N-terminal sequence on antibody binding activity was suggested.
2002, 9(2):122-125.
DOI: 10.3872/j.issn.1007-385X.2002.2.012
Abstract:
Objective: To study the effect of angiogenesis inhibitor TNP-470 combination with 5-FU on liver metastasis of human colon cancer.Methods:Human colon cancer cell line, LOVO cells, were injected intrasplenically into BALB/c nude mice to produce diffuse liver metastases. Mice were randomly divied into four groups: TNP-470 treated group, 5-FU treated group, TNP-470+5-FU treated group and control group. Animals were sacrificed after 4 weeks, and their livers were processed for histological examination. Liver metastatic rate and tumor foci in liver were counted. Tumor microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined by immunohistochemistry SABC method with image analyse system. Results: TNP-470 in combination with 5-FU and TNP-470 alone display a significant inhibitory effect on liver metastasis compared to the control (P<0.01, P<0.01). The expression of VEGF in the liver metastatic tumors was clearly inhibited by TNP-470 in combination with 5-FU and 5-FU alone compared to the control (P<0.01, P<0.05). The expression of MVD was also inhibited by TNP-470 in combination with 5-FU or TNP-470 alone compared to the control (P<0.01, P<0.01). Conclusion:The angiogenesis inhibitor TNP-470 in combination with 5-FU has additive effect and inhibitory activity against liver metastasis of human colon cancer, therefore, might be a safe and effective anti-tumor strategy.
Objective: To study the effect of angiogenesis inhibitor TNP-470 combination with 5-FU on liver metastasis of human colon cancer.Methods:Human colon cancer cell line, LOVO cells, were injected intrasplenically into BALB/c nude mice to produce diffuse liver metastases. Mice were randomly divied into four groups: TNP-470 treated group, 5-FU treated group, TNP-470+5-FU treated group and control group. Animals were sacrificed after 4 weeks, and their livers were processed for histological examination. Liver metastatic rate and tumor foci in liver were counted. Tumor microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined by immunohistochemistry SABC method with image analyse system. Results: TNP-470 in combination with 5-FU and TNP-470 alone display a significant inhibitory effect on liver metastasis compared to the control (P<0.01, P<0.01). The expression of VEGF in the liver metastatic tumors was clearly inhibited by TNP-470 in combination with 5-FU and 5-FU alone compared to the control (P<0.01, P<0.05). The expression of MVD was also inhibited by TNP-470 in combination with 5-FU or TNP-470 alone compared to the control (P<0.01, P<0.01). Conclusion:The angiogenesis inhibitor TNP-470 in combination with 5-FU has additive effect and inhibitory activity against liver metastasis of human colon cancer, therefore, might be a safe and effective anti-tumor strategy.
CHEN Guo-you
,
LI Yong-jian
,
JIANG Ying-ming
,
ZHANG Yi
,
HUANG Xin
,
JIANG Bo
,
SHI Qun-ying
,
LU Lin
,
LU Jun-yan
2002, 9(2):126-128.
DOI: 10.3872/j.issn.1007-385X.2002.2.013
Abstract:
Objective: To set up an optimal method of fermentation and purification of recombinant human stem cell factor (rhSCF). Methods: The effect of culture medium on the expression of rhSCF and the contents of rhSCF after different induction time were observed. The optimal condition for purification of rhSCF was also studied by changing pH, concentration of protein and chromatography procedure. The bioactivity of rhSCF was determined by UT-7 proliferation.Results: The expression level of rhSCF increased significantly in M9 culture medium containing glycerol, casein hydrolysate, yeast extract, peptone, and trace element. The optimal induction time for rhSCF expression was 6 h, approximately 30% of total protein. The insoluble inclusion body of rhSCF was denatured by 8M urea. After refolding for 24 h, the protein was firstly purified by acid precipitation and the supernatant was applied to an Source 30S column. The disulfide-linked dimeric rhSCF was excluded by reverse phase chromatography. The buffer was converted to PBS by S200. The purity of final product was over 95% with the biological activity more than 6×10 5 U/mg. Conclusion: The optimal condition for rhSCF preparation was successfully established, which can be used for scale-up production in the future.
Objective: To set up an optimal method of fermentation and purification of recombinant human stem cell factor (rhSCF). Methods: The effect of culture medium on the expression of rhSCF and the contents of rhSCF after different induction time were observed. The optimal condition for purification of rhSCF was also studied by changing pH, concentration of protein and chromatography procedure. The bioactivity of rhSCF was determined by UT-7 proliferation.Results: The expression level of rhSCF increased significantly in M9 culture medium containing glycerol, casein hydrolysate, yeast extract, peptone, and trace element. The optimal induction time for rhSCF expression was 6 h, approximately 30% of total protein. The insoluble inclusion body of rhSCF was denatured by 8M urea. After refolding for 24 h, the protein was firstly purified by acid precipitation and the supernatant was applied to an Source 30S column. The disulfide-linked dimeric rhSCF was excluded by reverse phase chromatography. The buffer was converted to PBS by S200. The purity of final product was over 95% with the biological activity more than 6×10 5 U/mg. Conclusion: The optimal condition for rhSCF preparation was successfully established, which can be used for scale-up production in the future.
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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.