人端粒酶逆转录酶诱导的猪肝细胞初步鉴定

Characterization of porcine hepatocytes transfected with human catalytic subunit of telomerase reverse transcriptase(hTERT)

目的观察应用四步灌流法分离后原代猪肝细胞的数量和活力,初步鉴定人端粒酶逆转录酶(hTERT)诱导的猪肝细胞的生物学特性。方法四步灌流法分离12只猪肝脏组织中的猪肝细胞,采用锥虫蓝拒染法测定其肝实质细胞数量和活力;将含hTERT真核表达的载体转染到原代猪肝细胞,经G418硫酸盐筛选,获得耐药性的猪肝细胞克隆并传代3代。观察原代猪肝细胞和hTERT诱导后的猪肝细胞的形态,检测两种猪肝细胞不同时期培养上清液中白蛋白、尿素氮的浓度。结果每只肝脏的肝细胞产量约为2.0×1010个,具有活力的肝细胞所占百分比为(95.5±3.2)%。原代猪肝细胞和hTERT诱导的猪肝细胞培养上清中分泌白蛋白和尿素氮在前3天无统计学差异(P>0.05),第4、5天差异有统计学意义(P<0.05)。结论四步灌流法分离获取的猪肝细胞产量高、功能活性好。hTERT诱导的猪肝细胞具有正常肝细胞的基本功能,可作为生物人工肝及细胞移植等的理想的细胞源。

Objective To assess the yield and vriability of porcine primary hepatocytes separated by a modified four-step collagenase method; and try to immortalize porcine hepatocytes by transfection of human telomerase reverse transcriptase （hTERT）gene. Methods Porcine primary hepatocytes were obtained from Chinese experimental miniature pigs using a modified four-step collagenase perfusion method, the yield and viability were determined by trypan blue exclusion test. The cells were transfected with hTERT cloned in a eukaryotic expression plasmid. The morphology of cultured porcine hepatocytes and transfected porcine hepatocytes was observed by optical microscope. The concentrations of albumin and BUN in the supernatant in the culture media were determined. Results The average yield of porcine hepatocytes was 2.0×10^10cells per liver with an average viability of （95.5±3.2）%. Two cloned porcine hepatocytes was obtained by G418 selection, and expanded by 3 passages. There were no significant differences in albumin secretion and urea synthesis in the first three days between the porcine primary hepatocytes and transfected porcine hepatocytes （P〉0.05）, but there were significant differences in the albumin secretion and urea synthesis 3 days later （P〈0.05）. Conclusion Porcine hepatocytes can be harvested with high yields using the modified four-step collagenase perfusion method. The transfected porcine hepatocytes maintain the properties of primary hepatocytes. This generation of new porcine hepatocytes may be applied for the development of bioartifical liver devices and hepatocytes transplantation.