转基因肝星状细胞株对肝细胞生长的支持作用

Sustaining effect of gene-transferring hepatic stellate cell strain CFSC/HGF on hepatocytes development

目的探讨转基因肝星状细胞株CFSC/HGF对大鼠肝细胞生长的支持作用.方法将大鼠原代肝细胞培养于稳定表达肝细胞生长因子(HGF)的肝星状细胞株CFSC/HGF所构建的饲养层上,连续动态观察肝细胞的形态、超微结构、白蛋白分泌、尿素合成以及吲哚氰绿摄取排泌功能的变化,同时与传统胶原贴壁培养的肝细胞进行比较,并通过半定量逆转录聚合酶链反应(RT-PCR)检测HGF受体c-Met表达的变化.结果共培养的肝细胞体外培养至7～10d时细胞增殖、白蛋白分泌及尿素合成达到峰值,以后逐渐下降,至35d时仍保持一定的存活和功能.与传统胶原上培养的肝细胞相比,其寿命、形态和功能的维持时间明显延长; RT-PCR结果显示,与CFSC/HGF饲养层细胞共培养1周后,肝细胞表面c-Met表达上调2.2 3倍.结论转基因肝星状细胞株CFSC/HGF对肝细胞较长时间内保持高活性、高密度的生长有显著的支持作用,CFSC/HGF诱导的肝细胞表面c-Met表达上调可能参与了该支持作用.

Objective To investigate the sustaining effects of gene-transferring hepatic stellate cell strain CFSC/ HGF on the development of hepatocytes. Methods A CFSC/HGF strain, espressing HGF steadily and effectively was established by recombined retroviral vector pMSCV-HGF infection. Morphology and ultra structure of hepatocytes, albumin and urea production, as well as ICG uptake and excretion were studied continuously following the hepatocytes cultured on the CFSC/HGF feeder layers. Parallel group of collagen-dependent hepatocytes culturing and hepatocytes culturing on CFSC were also conducted. Semi-quantitative RT-PCR analysis was made to evaluate the expression of HGFreceptor c-Met. Results The hepatocytes cocultured on CFSC/HGF feeder layers had a higher survival rate, and the functions of albumin secretion and urea syntheses and ICG uptake and excretion, were superior to the other two culture methods. The result of RT-PCR indicated that the c-Met expressed on the CFSC/HGF coculturing hepatocytes was up-regulated 2.23 times. Conclusion Gene-transferring hepatic stellate cell strain CFSC/HGF exhibited a remarkable sustaining effect on the hepatocytes development. The up-regulation of c-Met expressed on the surfaces of the hepatocytes induced by CFSC/HGF might play some part in this function.