AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of C...AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.展开更多
AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological character...AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.展开更多
基金the Natural Science Foundation of Anhui Province,No.99044312(WY) and No.9741006(LX)Natural Science Foundation of Anhui Educational Commission,No.JL-97-077(WY).
文摘AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.
基金Supported jby the Natural Science Foundation of Guangdong Province China,No.980180
文摘AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.
