AIM To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS B7 gene was transfected into mouse colon canc...AIM To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS B7 gene was transfected into mouse colon cancer cell line CMT93. The transfectants were selected in DMEM containing 800 mg/ L G418, and B7 molecules were detected by immunohistochemistry. Experiments in vivo include: ① 5×106 B7+ CMT93 cells were inoculated into the back of C57BL/ 6 mice subcutaneously to determine their tumorigenicity (n=4). As control, wild type CMT93 cells were inoculated the same as the experimental group (n=3). ② The mice primed by B7+ CMT93 cells whose tumors vanished were rechallenged with wild type CMT93 to observe the immune protection of these mice against the wild type CMT93 (n=4). Non-primed 4 native mice inoculated with wild type CMT93 were used as control. With in vitro cytotoxicity assay, the mice were immunized with B7+ CMT93 or the wild type CMT93 by intraperitoneal injection (n=4×2). The spleen cells and the abdominal cavity infiltrating lymphocytes were obtained and cultured for two days. Cytotoxicity of these cells against the B7 gene modified or wild type CMT93 was detected by MTT assay.RESULTS B7 high expression clones were obtained after the transfection of the B7 gene into CMT93 cells by electroporation. Immunohistochemistry results showed mainly membrance staining and partly cytoplasm staining in B7 gene transfected CMT93 cells. In vivo experiments: ① After the inoculation of the B7+ CMT93 cells into the back of C57BL/ 6 mice, they lost their tumorigenicity greatly (P＜0.01). All the small tumors growing in the early period in the experimental group vanished in one month, and the tumors in control group grew progressively. ② No tumors were found in all 4 mice primed by B7+ CMT93 cells after they were rechallenged with wild type CMT93. In the control group all mice had grown tumors (P＜0.05). In vitro cytotoxicity assay, the CTLs induced by B7+ CMT93 had a higher cytotoxity against the wild type CMT93 than that induced by wild type CMT93 (P＜0.05), and the cytotoxity of CTLs induced by B7+ CMT93 against B7+ CMT93 cells was higher than that against wild type CMT93 cells (P＜0.05).CONCLUSION The results suggest that the expression of costimulation B7 molecules by colorectal cancer cells can decrease their tumorigenicity greatly, and the B7 molecule can augment the activation of the CTLs against colorectal cancer, and it plays an important role in CTL effector function as well.展开更多
基金Supported by the National Natural Science Foundation of China,No.3950076.
文摘AIM To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS B7 gene was transfected into mouse colon cancer cell line CMT93. The transfectants were selected in DMEM containing 800 mg/ L G418, and B7 molecules were detected by immunohistochemistry. Experiments in vivo include: ① 5×106 B7+ CMT93 cells were inoculated into the back of C57BL/ 6 mice subcutaneously to determine their tumorigenicity (n=4). As control, wild type CMT93 cells were inoculated the same as the experimental group (n=3). ② The mice primed by B7+ CMT93 cells whose tumors vanished were rechallenged with wild type CMT93 to observe the immune protection of these mice against the wild type CMT93 (n=4). Non-primed 4 native mice inoculated with wild type CMT93 were used as control. With in vitro cytotoxicity assay, the mice were immunized with B7+ CMT93 or the wild type CMT93 by intraperitoneal injection (n=4×2). The spleen cells and the abdominal cavity infiltrating lymphocytes were obtained and cultured for two days. Cytotoxicity of these cells against the B7 gene modified or wild type CMT93 was detected by MTT assay.RESULTS B7 high expression clones were obtained after the transfection of the B7 gene into CMT93 cells by electroporation. Immunohistochemistry results showed mainly membrance staining and partly cytoplasm staining in B7 gene transfected CMT93 cells. In vivo experiments: ① After the inoculation of the B7+ CMT93 cells into the back of C57BL/ 6 mice, they lost their tumorigenicity greatly (P＜0.01). All the small tumors growing in the early period in the experimental group vanished in one month, and the tumors in control group grew progressively. ② No tumors were found in all 4 mice primed by B7+ CMT93 cells after they were rechallenged with wild type CMT93. In the control group all mice had grown tumors (P＜0.05). In vitro cytotoxicity assay, the CTLs induced by B7+ CMT93 had a higher cytotoxity against the wild type CMT93 than that induced by wild type CMT93 (P＜0.05), and the cytotoxity of CTLs induced by B7+ CMT93 against B7+ CMT93 cells was higher than that against wild type CMT93 cells (P＜0.05).CONCLUSION The results suggest that the expression of costimulation B7 molecules by colorectal cancer cells can decrease their tumorigenicity greatly, and the B7 molecule can augment the activation of the CTLs against colorectal cancer, and it plays an important role in CTL effector function as well.
