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目的移植bcl-2转基因HepaRG细胞到小鼠体内,并联合使用Jo2抗体诱导鼠肝细胞凋亡策略,促进人鼠嵌合肝中人肝细胞的增殖。方法SCID鼠经脾移植2×106bcl-2转基因HepaRG细胞,移植后1d始腹腔注射0.2mg/kgJo2抗体,每周1次,持续10周为实验组;移植未转染bcl-2基因的HepaRG细胞,同样给予Jo2抗体腹腔注射为对照1组,移植未转染bcl-2基因的HepaRG细胞,未腹腔注射Jo2抗体为对照2组,建立人鼠嵌合肝动物模型。从2周开始,后4、8、12、16、20、24周,分别采用免疫组化、免疫荧光和逆转录聚合酶链反应(RT-PCR)检测不同时相点实验组和对照组嵌合肝中肝组织人清蛋白以及人清蛋白mRNA,并用酶联免疫法(ELISA)定量检测鼠血清人清蛋白的含量。结果实验组和对照组小鼠均能存活至24周,鼠肝组织和血清中均能检测到清蛋白的表达。实验组肝组织和血清人清蛋白的表达均可持续到24周,人血清清蛋白高峰值为95.32ng/mL,出现在16周;而对照1组肝组织和血清人清蛋白的表达可持续到20周,人血清清蛋白高峰值为42.37ng/mL,出现在12周;对照2组肝组织和血清人清蛋白的表达可持续到12周,人血清清蛋白高峰值为22.91ng/mL,出现在8周。结论利用bcl-2抗Fas凋亡特性,转染bcl-2基因的HepaRG细胞移植并联合小剂量Jo2抗体腹腔注射SCID鼠,有利于人鼠嵌合肝中人肝细胞的增殖和存活。
Objective To transfect bcl-2 transgenic HepaRG cells into mice and use Jo2 antibody in combination to induce rat hepatocyte apoptosis and promote the proliferation of human hepatocytes in human chimeric liver. Methods 2 × 106bcl-2 transgenic HepaRG cells were transplanted in the spleens of SCID mice. At 1d after transplantation, 0.2mg / kg of antibody was injected intraperitoneally once a week for 10 weeks. The HepaRG cells without transfection of bcl-2 gene Jo2 antibody was also given intraperitoneally to control group 1, HepaRG cells transfected with bcl-2 gene were transplanted, Jo2 antibody was not injected intraperitoneally into control group 2, and a human chimeric hepatic animal model was established. Immunohistochemistry, immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the mRNA expression levels at different time points from the beginning of 2 weeks, the 4th, 8th, 12th, 16th, Hepatic liver tissue of human albumin and human albumin mRNA, and enzyme-linked immunosorbent assay (ELISA) quantitative detection of human serum albumin content. Results Both experimental and control mice survived to 24 weeks, and albumin expression was detected in both liver and serum of mice. Liver tissue and serum albumin expression in the experimental group can be sustained until 24 weeks, the peak value of human serum albumin was 95.32ng / mL, appeared in 16 weeks; while the control group 1 liver tissue and serum human albumin expression is sustainable At 20 weeks, the peak value of human serum albumin was 42.37ng / mL, which appeared at 12 weeks. The expression of human albumin in control group 2 lasted for 12 weeks and the peak value of human serum albumin was 22.91ng / mL , Appeared in 8 weeks. Conclusion The anti-Fas apoptosis of bcl-2 transfected HepaRG cells transfected with bcl-2 gene combined with small doses of Jo2 antibody intraperitoneal injection of SCID mice is conducive to human liver chimeric liver cell proliferation and survival.