实时荧光定量RT-PCR检测肝卵圆细胞Alb和CK-19 mRNA的表达

Using real-time fluorescence quantitative RT-PCR to detect Alb and CK-19 mRNA in hepatic oval cells

目的建立Alb和CK-19 mRNA表达量检测的实时荧光定量聚合酶链式反应(RT-PCR)系统,检测Lewis大鼠肝卵圆细胞(HOC)及其分化细胞中Alb和CK-19 mRNA表达量并分析其意义。方法用实时荧光定量RT-PCR技术检测分别检测HOC、分化细胞、胆道及肝脏标本中Alb和CK-19 mRNA的表达情况,以上标本各20例。结果Alb mRNA在HOC(-1.820±0.614)中的表达水平明显低于其在肝脏(-1.139±0.588)中的表达水平(P<0.05);CK-19 mRNA在HOC(-3.852±0.974)中的表达水平明显低于在胆道(-1.739±0.704)、肝脏(-1.926±0.653)、分化细胞(-1.492±0.634)中的表达水平(P<0.05);HOC Alb mRNA表达水平高于CK-19mRNA表达水平(P<0.05),分化细胞Alb mRNA(-1.758±0.543)表达水平同ck-19 mRNA表达水平差异不明显(P>0.05)。结论使用实时荧光定量RT-PCR检测HOC Alb和CK-19 mRNA的表达克服了传统PCR只能定性不能定量的缺点,为研究Alb和CK-19 mRNA在HOC中的表达提供了相对定量的方法,成功地进行该项实验需要掌控好几个关键环节;HOC中Alb mRNA和CK-19 mRNA表达量低,经过诱导分化后两者表达量明显升高,证实HOC具有分化为肝细胞和胆管上皮细胞的双向分化特性,在本研究的诱导分化条件下HOC主要向胆管上皮细胞分化。

【Objective】Establishing a real-time fluorescence quantitative RT-PCR system to detect Alb and CK-19 mRNA in hepatic oval cells（HOC） and analysing respectively.【Method】Using real-time fluorescence quantitative RT-PCR to detect Alb and CK-19 mRNA in HOC,differentiated cells,biliary tissues and hepatic tissues.All specimens mentioned above were originated from Lewis rat and sample number in each group was 20.【Result】Between groups,the expression level of Alb mRNA in HOC（-1.820±0.614） was lower than that in hepatic tissues（-1.139±0.588）（P 0.05）,the expression level of CK-19 mRNA in HOC（-3.852±0.974） was lower than that in hepatic tissues（-1.926±0.653）,biliary tissues（-1.739±0.704） and differentiated cells（-1.492±0.634） respectively（P0.05）.Within individual group,the expression level of Alb mRNA was higher than CK-19 mRNA in HOC（P0.05）,while differentiated cells Alb mRNA（-1.758±0.543） level and CK-19 mRNA level show no difference（P0.05）.【Conclusion】 Compared with traditional PCR assay,real-time fluorescence quantitative RT-PCR offers a quantitative way to test Alb mRNA and CK-19 mRNA in HOC.For successfully handling it,there were some key steps shoud be controlled.Alb mRNA and CK-19 mRNA level in HOC showed low.But after inducing differentiation,Alb mRNA and CK-19 mRNA level obtained a higher level expression.In this experiment,HOC was proved to differentiate to two type cells：biliary epithelial cells and hepatic cells,but mainly differentiated to biliary epithelial cells.