上皮细胞黏附分子阳性的正常成体小鼠肝祖细胞向肝干细胞转化及HBx蛋白对肝干细胞稳定性的影响

EPCAM-positive normal hepatic progenitor cells transformation into liver stem cells and HBx-mediated effects on stability in adult mouse

目的观察上皮细胞黏附分子（EPCAM）阳性的正常成体小鼠肝祖细胞转化为多项分化潜能的肝干细胞，并探讨HBx蛋白对肝干细胞的影响。方法改良型两步灌流法消化肝组织，差异性离心法联合密度梯度离心法筛选肝祖细胞；在表皮生长因子和胶原蛋白作用下，观察肝祖细胞向肝干细胞转化，并经细胞免疫荧光法鉴定干细胞中CD133、EPCAM、CD49f及细胞角蛋白19表面标志物的表达情况。添加二甲基亚砜，过碘酸雪夫氏染色、细胞免疫荧光、流式细胞术检测干细胞向肝细胞和胆管上皮细胞分化情况。转染HBx蛋白后，PCR检测细胞转染效率，并观察HBx转染后干细胞形态学差异性及其增殖能力。两组间数据比较采用t检验。结果肝祖细胞存活率为78．67％±4．04％，在表皮生长因子和胶原蛋白作用下，铺路石样贴壁生长的肝祖细胞中部分细胞聚集成细胞球，具有干细胞样性质，高表达CD133、CD49f、细胞角蛋白19，低表达EPCAM。在二甲基亚砜作用下，能够向肝细胞和胆管上皮细胞分化。HBx转染后肝干细胞能维持干细胞形态，克隆能力增强。结论EPCAM阳性的正常成体小鼠肝祖细胞能够向肝干细胞转化，同时HBx蛋白对维持肝干细胞的稳定性、促进增殖有重要作用。

Objective To investigate the transformative potential of hepatic progenitor cells to differentiate into liver stem cells using a normal adult mouse system and to determine the effects of HBx protein in these liver stem cells＇ differentiation into hepatic cells. Methods Hepatic progenitor cells were obtained from mice by means of an optimized two-step digestion and perfusion method followed by joint differential centrifugation and density gradient centrifugation. Transformation of the hepatic progenitor cells into liver stem cells was observed by immunofluorescent detection of CD133, EPCAM, CD49f and CK19. Differentiation of the resultant liver stem cells into hepatic cells and bile duct epithelial cells was observed after DMSO addition by Periodic Acid-Schiff （PAS） staining followed by cell immunofluorescence and flow cytometry. To determine the effects of HBx on these liver stem cells＇ ability to differentiate into hepatic cells, cell transfection was used followed by observation of morphology and proliferation capacity. Results Cell viability of the isolated hepatic progenitor cells was 78.67±4.04%. Stimulation with EGF and collagen led to growth of some of the paving-stone shaped cells attached to the hepatic progenitor cells which had gathered into spherical clumps, as is the nature of stem cells. The liver stem cells showed high expression of CD133, CD49f and CK19, and low expression of EPCAM. Under the effect of DMSO, the liver stem cells differentiated into hepatocytes and bile duct epithelial cells. After HBx transfection, the liver stem cells maintained the characteristic shape of stem cells and showed enhanced proliferation. Conclusion EPCAM-positive adult hepatic progenitor cells can transform into liver stem cells. The HBx protein may play an important role in maintaining the stability of liver stem cells in the adult mouse.