Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for develo...Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.展开更多
Objective: To study the correlation of the methylation of the promoters of hMLH1 and hMSH2 with microsatellite instability (MSI) in the tissues of gastric carcinomas. Methods: A total of 68 sporadic cases of gastric c...Objective: To study the correlation of the methylation of the promoters of hMLH1 and hMSH2 with microsatellite instability (MSI) in the tissues of gastric carcinomas. Methods: A total of 68 sporadic cases of gastric carcinoma were studied. Ten specimens of normal gastric mucosa served as control. Methylation of hMLHl and hMSH2 was observed with methylation-specific PCR, and MSI analyzed with PCR-based techniques. Results: No methylation of hMLHl and hMSH2 was found in 10 specimens of normal gastric mucosa. Methylation of hMLHl was detected in 11 cases (16. 2%) of gastric cancers and MSI in at least one locus was found in 17 cases (25%) of the 68 with aid of 5 microsatellite markers, in which eight were MSI-H (≥2loci showed instability) nine MSI-L (only one locus showed instability), and fifty-one were MSS (no instability at any marker). The frequency of methylation was significantly high in MSI-H (87. 5%) than in MSI-L (11.1%) and MSS (5. 9%). CP<0. 01 - 0. 001) but there was no difference of methylation frequency between the cases with MSI-L and those with MSS. Conclusion: Methylation of hMLHl promoter is involved to the MSI pathway but not to the loss of heterozygosity (LOH) pathway in gastric carcinogenesis.展开更多
Objective To investigate effect of let-7a gene over-expression on apoptosis of gastric cancer cell lines SGC-7901. Methods The stable let-7a gene over-expressing SGC-7901 cells,SGC-7901/let-7a cells,were established u...Objective To investigate effect of let-7a gene over-expression on apoptosis of gastric cancer cell lines SGC-7901. Methods The stable let-7a gene over-expressing SGC-7901 cells,SGC-7901/let-7a cells,were established using shRNA lentiviral vector methods. Real-time RT-PCR analysis was used to evaluate the expression level of let-7a mRNA. Cells apoptosis was assessed by flow cytometry. Results RT-PCR analysis revealed let-7a expression in SGC-7901/let-7a cells was significantly increased. Cellular apoptosis assay showed that over-expression of let-7a could increase apoptosis of SGC-7901 cells(P=0.002). Conclusion Up-regulating let-7a expression promoted apoptosis in SGC-7901 cells.展开更多
Objective:To study the combined therapeutic effects of cytotoxic agent and differentiation-inducer on human gas tric carcinoma celll line SGC-7901 in vitro. Methods: The combined therapeutic effects of all-trans retin...Objective:To study the combined therapeutic effects of cytotoxic agent and differentiation-inducer on human gas tric carcinoma celll line SGC-7901 in vitro. Methods: The combined therapeutic effects of all-trans retinoic acid (ATRA), interferon a (IFNa)and fluorouracil (5-Fu) on gastric carcinoma cell line ax 7901 were observed when one of the 3, the combination of any 2 of the 3 and combination of all the 3 were administered respectively. The morphological and functional changes of gastric carcinoma cells were studied with MTT assay, flow cytometry, image analysis and determination of CEA content in the culture medium of the cells. Results: The cytostatic rate was increased as shown by the decrease of the rate of colony formation of the cells on culture disc when one agent, the combination of 2 agents and the combination of the 3 were administered progressively. The cells were relatively accumulated in the phase of G0/G1 and synthesis of DNA in he cells was inhibited.The malignant phenotype of the cells disappeared gradually while the characteristics of matUre cells were in creased. Meanwhile, CEA Level in the culture medium was decreased progressively. Apoptosis of the cells was oborved and a large amount of apoptotic apoptotic were found. Conclusion: The administration of the 3 agents in combination result in signif icant inhibition on proliferation, inducing of differention and promotion of apoptosis of gastric caxcinoma cells. The combina tion of cytotoxic agent and differention-inducer exerts significant inhibition on gastric carcinoma cells in vitro.展开更多
The effects of two antisense oligodeoxynucleotides on the expression of c-Ha-ras proto-oncogene and the growth of human gastric carcinoma cell lines were observed. Synthetic 15-mer directed at the region of the transl...The effects of two antisense oligodeoxynucleotides on the expression of c-Ha-ras proto-oncogene and the growth of human gastric carcinoma cell lines were observed. Synthetic 15-mer directed at the region of the translational initiation site of c-Ha-ras proto-oncogene (ASO-r) greatly inhibited the proliferation (55. 61%,P<0. 05) and DNA synthesis (76. 79%,P<0. 05) of MGc-803 cell line. It also inhibited the proliferation (62. 02%,P<0. 05) and DNA synthesis (76. 78%, P<0. 05) of SGc-7901 cell line. A reduction in intracellular P21 ras protein levels in MGc-803 cell line was observed 6 h after the treatment with ASO-r and maintained over 12 h. Another synthetic 15-mer targeted against the initiation codon and downstream 4 codons of c-myc proto-oncogene (ASOm) inhibited only DNA synthesis of MGc-803 cell line (71. 37%, P<0. 05). The control 15-mer did not inhibit the expression of P21 protein and proliferation of these cell lines. These experiments seemed to provide evidence that ASO-r could be effective in inhibiting the expression of c-Ha-ras proto-oncogene and controlling the growth of human gastric carcinoma cells,and that the over-expression of c-Ha-ras proto-oncogene might mainly be associated with the malignant proliferation of human gastric carcinoma cells.展开更多
DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcin...DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcinomas with liver metastases were anueploid(75%) as compared to 13 out of 45(28.8%) of cases without liver metastases(P<0.01);the one gastric carcinoma with adrenal metastasis was also anueploid.DNA ploidy was not related to ovarian or lymph node metastases.Another interesting finding was that all of 3 gastric carcinomas with liver metastases which showed a diploid DNA pattern,expressed p53 protein, while all of 3 carcinomas with liver metastases but no p53 protein expression were anueploid.The expression of p53 protein was not related to ovarian metastases.The results suggested that an anueploid DNA pattern and the expression of p53 protein are both objective markers valuable in predicting high risk potential of metastases to the liver,and that the combined detection of these markers can be a most useful method in the follow-up of Patients with gastric carcinoma in detecting those at high risk of developing metastases following surgical resection.Also the poorer prognosis of Patients with gastric carcinoma showing an anueploid DNA pattern may be related to the development of distant organ metastases through the blood vascular system.Furthermore,the clone of gastric carcinoma cells which accumulate p53protein or show an anueploid DNA pattern may have a causative role in the development of liver(&.adrenal) metastases.展开更多
Objective:To study the effects of recombinant antisense c-myc adenovirus (rAS-c-myc-Ad) on SGG 7901 human gastric carcinoma cell line in for and in nude mice. Methods:The effects of rAS-c-myc-Ad and LacZ-Ad on SGG 790...Objective:To study the effects of recombinant antisense c-myc adenovirus (rAS-c-myc-Ad) on SGG 7901 human gastric carcinoma cell line in for and in nude mice. Methods:The effects of rAS-c-myc-Ad and LacZ-Ad on SGG 7901 gastric carcinoma cells were observed with X-galstaining, MTT, DNA gradient degradation test, TUNEL, flow cytometry, PCR and western blot. The therapeutic effects of rAS-c-myc-Ad on the implanted ax 7901 cells in nude mice were also ob served.Results: rAS-c-myc-Ad significantly inhibited the growth of SGG 7901 cells and induced their apoptosis. After the treatment of rAS-c-myc-Ad, the prolifetion rate of the cells was decreased by 44’ l% in de and SGC 7901 cells failed to form caxcinoma ther they were implanted into nude mice. Injection of rAS-c-myc-Ad into the carcinoma subcutaneously implanted to the nude mice significantly inhibited the growth of the implanted carcinoma with an inhibition rate of 68. 9%. Conclusion: rAS-c- myc- Ad significantly inhibits the growth of SGG 7901 human gastric carcinoma cells in vitro and in nude展开更多
Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation...Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza-CdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.展开更多
Objective： To compare laparoscopic gastrectomy and conventional surgery on the dissemination and seeding of tumor cells. Methods：Intraoperative peritoneal lavage cytologic examination was performed in 65 patients wi...Objective： To compare laparoscopic gastrectomy and conventional surgery on the dissemination and seeding of tumor cells. Methods：Intraoperative peritoneal lavage cytologic examination was performed in 65 patients with gastric cancer, during laparoscopic gastrectomy （n = 34） and conventional surgery （n = 31）. Cytology was examined twice, immediately after opening the peritoneal cavity and just before closing the abdomen. Saline was poured into the peritoneal cavity, and 100 ml fluid was retrieved after irrigation. Laparoscopic instruments were lavaged after surgery with 100 ml saline. Carbon dioxide （COz） was derived through the trocar side orifice after pneumoperitoneum during laparoscopic gastrectomy and filtered through 100 ml saline. Cytologic examination of the filtrate was performed after the filtration process. Results： The incidence of positive cytology during laparoscopic surgery was 32.26% in the preoperative lavage and 22.58% in the postoperative lavage. The incidence of positive cytology during conventional surgery was 41.18% before lavage and 26.47% after lavage. Only one positive cytology was detected in the CO2 filtrate gas. The incidence of positive cytology in the lavage of the instruments during laparoscopic surgery was 6.45 %. Conclusion： During gastric laparoscopic surgery, CO2 pneumoperitoneum does not affect tumor cell dissemination and seeding. In this study, laparoscopic techniques used in gastric cancer surgery were not associated with a higher risk for intraperitoneal dissemination of cancer cells than the conventional surgery.展开更多
基金This work was supported by the National Natural Sciences Founda- tion of China(NSFC, No. 39800057, No. 30200338) the National "863" High-tech Project Foundation (No. 102-10-01 -06) +1 种基金National Distinguished Youth Program of NSFC(No. 39525020) This wor
文摘Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
基金Supported by National Natural Science Foundation of China (No. 30070043) , and "the Tenth five-year Plan"Scientific Research Foundation of Chinese PLA (No. 01Z075)
文摘Objective: To study the correlation of the methylation of the promoters of hMLH1 and hMSH2 with microsatellite instability (MSI) in the tissues of gastric carcinomas. Methods: A total of 68 sporadic cases of gastric carcinoma were studied. Ten specimens of normal gastric mucosa served as control. Methylation of hMLHl and hMSH2 was observed with methylation-specific PCR, and MSI analyzed with PCR-based techniques. Results: No methylation of hMLHl and hMSH2 was found in 10 specimens of normal gastric mucosa. Methylation of hMLHl was detected in 11 cases (16. 2%) of gastric cancers and MSI in at least one locus was found in 17 cases (25%) of the 68 with aid of 5 microsatellite markers, in which eight were MSI-H (≥2loci showed instability) nine MSI-L (only one locus showed instability), and fifty-one were MSS (no instability at any marker). The frequency of methylation was significantly high in MSI-H (87. 5%) than in MSI-L (11.1%) and MSS (5. 9%). CP<0. 01 - 0. 001) but there was no difference of methylation frequency between the cases with MSI-L and those with MSS. Conclusion: Methylation of hMLHl promoter is involved to the MSI pathway but not to the loss of heterozygosity (LOH) pathway in gastric carcinogenesis.
文摘Objective To investigate effect of let-7a gene over-expression on apoptosis of gastric cancer cell lines SGC-7901. Methods The stable let-7a gene over-expressing SGC-7901 cells,SGC-7901/let-7a cells,were established using shRNA lentiviral vector methods. Real-time RT-PCR analysis was used to evaluate the expression level of let-7a mRNA. Cells apoptosis was assessed by flow cytometry. Results RT-PCR analysis revealed let-7a expression in SGC-7901/let-7a cells was significantly increased. Cellular apoptosis assay showed that over-expression of let-7a could increase apoptosis of SGC-7901 cells(P=0.002). Conclusion Up-regulating let-7a expression promoted apoptosis in SGC-7901 cells.
文摘Objective:To study the combined therapeutic effects of cytotoxic agent and differentiation-inducer on human gas tric carcinoma celll line SGC-7901 in vitro. Methods: The combined therapeutic effects of all-trans retinoic acid (ATRA), interferon a (IFNa)and fluorouracil (5-Fu) on gastric carcinoma cell line ax 7901 were observed when one of the 3, the combination of any 2 of the 3 and combination of all the 3 were administered respectively. The morphological and functional changes of gastric carcinoma cells were studied with MTT assay, flow cytometry, image analysis and determination of CEA content in the culture medium of the cells. Results: The cytostatic rate was increased as shown by the decrease of the rate of colony formation of the cells on culture disc when one agent, the combination of 2 agents and the combination of the 3 were administered progressively. The cells were relatively accumulated in the phase of G0/G1 and synthesis of DNA in he cells was inhibited.The malignant phenotype of the cells disappeared gradually while the characteristics of matUre cells were in creased. Meanwhile, CEA Level in the culture medium was decreased progressively. Apoptosis of the cells was oborved and a large amount of apoptotic apoptotic were found. Conclusion: The administration of the 3 agents in combination result in signif icant inhibition on proliferation, inducing of differention and promotion of apoptosis of gastric caxcinoma cells. The combina tion of cytotoxic agent and differention-inducer exerts significant inhibition on gastric carcinoma cells in vitro.
文摘The effects of two antisense oligodeoxynucleotides on the expression of c-Ha-ras proto-oncogene and the growth of human gastric carcinoma cell lines were observed. Synthetic 15-mer directed at the region of the translational initiation site of c-Ha-ras proto-oncogene (ASO-r) greatly inhibited the proliferation (55. 61%,P<0. 05) and DNA synthesis (76. 79%,P<0. 05) of MGc-803 cell line. It also inhibited the proliferation (62. 02%,P<0. 05) and DNA synthesis (76. 78%, P<0. 05) of SGc-7901 cell line. A reduction in intracellular P21 ras protein levels in MGc-803 cell line was observed 6 h after the treatment with ASO-r and maintained over 12 h. Another synthetic 15-mer targeted against the initiation codon and downstream 4 codons of c-myc proto-oncogene (ASOm) inhibited only DNA synthesis of MGc-803 cell line (71. 37%, P<0. 05). The control 15-mer did not inhibit the expression of P21 protein and proliferation of these cell lines. These experiments seemed to provide evidence that ASO-r could be effective in inhibiting the expression of c-Ha-ras proto-oncogene and controlling the growth of human gastric carcinoma cells,and that the over-expression of c-Ha-ras proto-oncogene might mainly be associated with the malignant proliferation of human gastric carcinoma cells.
文摘DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcinomas with liver metastases were anueploid(75%) as compared to 13 out of 45(28.8%) of cases without liver metastases(P<0.01);the one gastric carcinoma with adrenal metastasis was also anueploid.DNA ploidy was not related to ovarian or lymph node metastases.Another interesting finding was that all of 3 gastric carcinomas with liver metastases which showed a diploid DNA pattern,expressed p53 protein, while all of 3 carcinomas with liver metastases but no p53 protein expression were anueploid.The expression of p53 protein was not related to ovarian metastases.The results suggested that an anueploid DNA pattern and the expression of p53 protein are both objective markers valuable in predicting high risk potential of metastases to the liver,and that the combined detection of these markers can be a most useful method in the follow-up of Patients with gastric carcinoma in detecting those at high risk of developing metastases following surgical resection.Also the poorer prognosis of Patients with gastric carcinoma showing an anueploid DNA pattern may be related to the development of distant organ metastases through the blood vascular system.Furthermore,the clone of gastric carcinoma cells which accumulate p53protein or show an anueploid DNA pattern may have a causative role in the development of liver(&.adrenal) metastases.
基金Supported by National 863 High Science and Technology Foundation of China, No.Z20-01-02
文摘Objective:To study the effects of recombinant antisense c-myc adenovirus (rAS-c-myc-Ad) on SGG 7901 human gastric carcinoma cell line in for and in nude mice. Methods:The effects of rAS-c-myc-Ad and LacZ-Ad on SGG 7901 gastric carcinoma cells were observed with X-galstaining, MTT, DNA gradient degradation test, TUNEL, flow cytometry, PCR and western blot. The therapeutic effects of rAS-c-myc-Ad on the implanted ax 7901 cells in nude mice were also ob served.Results: rAS-c-myc-Ad significantly inhibited the growth of SGG 7901 cells and induced their apoptosis. After the treatment of rAS-c-myc-Ad, the prolifetion rate of the cells was decreased by 44’ l% in de and SGC 7901 cells failed to form caxcinoma ther they were implanted into nude mice. Injection of rAS-c-myc-Ad into the carcinoma subcutaneously implanted to the nude mice significantly inhibited the growth of the implanted carcinoma with an inhibition rate of 68. 9%. Conclusion: rAS-c- myc- Ad significantly inhibits the growth of SGG 7901 human gastric carcinoma cells in vitro and in nude
文摘Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza-CdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.
文摘Objective： To compare laparoscopic gastrectomy and conventional surgery on the dissemination and seeding of tumor cells. Methods：Intraoperative peritoneal lavage cytologic examination was performed in 65 patients with gastric cancer, during laparoscopic gastrectomy （n = 34） and conventional surgery （n = 31）. Cytology was examined twice, immediately after opening the peritoneal cavity and just before closing the abdomen. Saline was poured into the peritoneal cavity, and 100 ml fluid was retrieved after irrigation. Laparoscopic instruments were lavaged after surgery with 100 ml saline. Carbon dioxide （COz） was derived through the trocar side orifice after pneumoperitoneum during laparoscopic gastrectomy and filtered through 100 ml saline. Cytologic examination of the filtrate was performed after the filtration process. Results： The incidence of positive cytology during laparoscopic surgery was 32.26% in the preoperative lavage and 22.58% in the postoperative lavage. The incidence of positive cytology during conventional surgery was 41.18% before lavage and 26.47% after lavage. Only one positive cytology was detected in the CO2 filtrate gas. The incidence of positive cytology in the lavage of the instruments during laparoscopic surgery was 6.45 %. Conclusion： During gastric laparoscopic surgery, CO2 pneumoperitoneum does not affect tumor cell dissemination and seeding. In this study, laparoscopic techniques used in gastric cancer surgery were not associated with a higher risk for intraperitoneal dissemination of cancer cells than the conventional surgery.
