肝星状细胞诱导卵圆细胞向成熟肝细胞分化的体外研究

目的 研究肝星状细胞(HSC)在体外培养的卵圆细胞向成熟肝细胞分化过程中所起的作用.方法 (1)将卵圆细胞分别与原代培养的HSC混合共培养(混合共培养组),采用Millicell小室不接触共培养(不接触共培养组),卵圆细胞单独培养作为对照组.在共培养后第7、14、21天观测,采用Western blot和荧光定量PCR检测肝细胞核因子4 α(HNF-4 α)、白蛋白和甲胎蛋白(AFP)、细胞角蛋白19(CK-19)的表达量;(2)电子透射显微镜观察卵圆细胞超微结构的变化;(3)过碘酸希夫染色检测各时间点卵圆细胞内糖原颗粒的含量;(4)酶联免疫吸附法检测各时间点卵圆细胞分泌白蛋白的含量.统计学处理采用重复测量资料的多因素方差分析和LSD-t检验.结果 (1)卵圆细胞第7、14、21天表达HNF-4 α和白蛋白的mRNA的量(相对与共培养之前的表达量的倍数):混合共培养组HNF-4 α分别为(1.9±0.2)倍、(10.7±1.2)倍、(12.0±1.3)倍;白蛋白mRNA的量分别为(5.7±1.6)倍、(110.7±13.7)倍、(173.6±22.3)倍;不接触共培养组HNF-4 α分别为(1.4±0.1)倍、(3.2±0.6)倍、(8.9±1.4)倍;白蛋白mRNA的量分别为(2.9±1.4)倍、(22.3±8.5)倍、(96.3±16.3)倍.共培养组(混合共培养与不接触共培养组)的表达量均高于对照组,LSD-t值分别为32.98,10.08;13.38,7.96; P值均<0.01,差异有统计学意义.第7、14、21天AFP、CK-19的表达量:混合共培养组AFP分别为(1.1±0.2)倍、(0.2±0.0)倍、(0.0±0.0)倍;CK-19分别为(0.2±0.1)倍、(0.0±0.0)倍、(0.0±0.0)倍;不接触共培养组AFP分别为(1.0±0.2)倍、(0.2±0.1)倍、(0.1±0.0)倍;CK-19分别为(0.6±0.1)倍、(0.1±0.0)倍,(0.0±0.0)倍.共培养组(混合共培养与不接触共培养组)的表达量均低于对照组.LSD-t值分别为37.99,34.50;13.59,22.46;P值均<0.01,差异有统计学意义.(2)卵圆细胞共培养后白蛋白分泌量明显增加:混合共培养组:14 d为(15.30±0.09)ng/ml、21 d(20.98±0.12)ng/ml,不接触共培养组14 d为(11.41±0.13)ng/ml,21 d为(15.12±0.17)ng/ml,混合共培养组高于不接触共培养组,LSD-t=251.94,P<0.01,差异有统计学意义.(3)随着共培养时间的延长,卵圆细胞内质网、线粒体和高尔基体等细胞器逐渐丰富,且细胞间形成毛细胆管结构.(4)过碘酸希夫染色显示共培养后卵圆细胞内出现大量红色糖原颗粒.结论 HSC可以诱导卵圆细胞向成熟肝细胞分化,HSC除了通过细胞因子等可溶性因子途径发挥作用外,与卵圆细胞的直接接触也有着重要作用.

Differentiation of hepatic oval cell into mature hepatocyte induced by hepatic stellate cells

Objective To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte. Methods The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by milllcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-41A, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit. Result (1) The relative level of HNF-4 α and albumin mRNA expression compared with pre-coculture: M-coculture:HNF-4 α: 1.9±0.2, 10.7±1.2, 12.0±1.3; albumin: 5.7±1.6, 110.7±13.7, 173.6±22.3. S-coculture: 1.4±0.1, 3.2±0.6, 8.9±1.4 times; albumin: 2.9±1.4, 22.3±8.5, 96.3±16.3. The relative level of HNF-4α and albumin mRNA expression in coculture group (M- and S-colculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P<0.01); and a higher level of HNF-4α and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P<0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1±0.2, 0.2±0.0, 0.0± 0.0; S-coculture group: AFP: 1.0±0.2, 0.2±0.1, 0.1±0.0; CK-19:0.6±0.1, 0.1±0.0, 0.0±0.0; control group: AFP: 1.0±0.1, 1.0±0.1, 1.1±0.1, CK-19:1.0±0.1, 1.1±0.1, 1.0±0.1. The rela-tive level of AFP and CK-19 mRNA expression in cocuiture group(M- and S-colculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P<0.01). (2) The albumin secretion was detected in M-coculture: 14day: (15.30±0.09) ng/ml, 21:(20.98±0.12) ng/ml; S-coculture: 14 day: (11.41±0.13) ng/ ml, 21day:(15.12±0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochon-drion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared be-tween oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups. Conclusion HSC can induce differentiation of oval cell into mature hepatocyte.