层粘连蛋白对肝脏卵圆细胞的调控作用及其机制研究

Hepatic oval cell is adult liver stem cell or progenitor cell. Stem cell therapy has promise as a new approach for treating life-threatening liver disease. Recently, attention has focused on elucidating the responses and mechanisms involved in the activation and expansion of hepatic oval cells. The microenvironment is highly regulated within an organ and plays an important role in maintaining stem cell division under signaling modulation. Laminin protein is a very important compartment involved in hepatic oval cell microenvironment. It is documented that laminin has a role in regulating oval cell differentiation and proliferation, suggesting a potential role of laminin to be a novel matrix protein supporting oval cell expansion in vitro or cell transplantation in vivo. However, the mechanism still needs elucidation. Most importantly, the most active laminin subtype in oval cell microenvironment is not identified. Based on our previously research, we plan to set a serious in vitro and in vivo experiments, employing rat hepatic oval cell as an object of the study. The aims of this project are to identify the most active subtype of laminin in oval cell regulation, to investigate the mechanism of Laminin-Integrin signal pathway in oval cell proliferation, apoptosis and differentiation, to establish a Laminin based extracellular matrix supporting system for oval cell culture and transplantation. This study will provide valuable data for further understanding the regulation mechanisms of hepatic progenitor cell, and may help the application of hepatic stem cell transplantation to liver disease treatment. The scientific significance is important.

肝脏卵圆细胞是肝成体干细胞，具有应用于干细胞移植治疗肝脏疾病的重大前景，其分子调控机制研究是关键所在。Laminin是卵圆细胞微环境的重要成分。研究发现Laminin可以调控卵圆细胞分化并支持其增殖，可能作为卵圆细胞体外增殖、体内移植的重要基质蛋白。但其作用机制尚不明了，且具有活性的Laminin亚型种类也尚不明确。基于我们的研究基础，本项目拟以大鼠肝卵圆细胞为研究对象，通过体内及体外实验明确对卵圆细胞具有调控作用的Laminin具体亚型种类，明了Laminin-Integrin信号通路对卵圆细胞增殖、凋亡及分化的调控作用并探讨其分子机制，并初步观察以Laminin为基础的细胞外基质支持体系对卵圆细胞移植的辅助作用。本项目将加深对卵圆细胞分子调控机制的认识，在卵圆细胞移植治疗肝脏疾病等方面具有重要的科学意义及潜在的应用价值。

肝脏成体干细胞也被称为卵圆细胞，可能作为肝细胞来源治疗肝脏疾病。微环境的调控对卵圆细胞的生物学行为起着重要作用。层粘连蛋白（Laminin）由3条多肽链α、β和γ组成，共存在15种亚型，是细胞外基质最主要的组成部分之一。目前关于Laminin在卵圆细胞调控作用方面的认识尚不十分明确。.本研究初步确定在卵圆细胞扩增过程中发挥作用的Laminin-Integrin亚型；再通过体外研究进一步观察所确定有活性的Laminin亚型对卵圆细胞的作用，并探讨其分子机制；最后观察这些Laminin亚型对移植卵圆细胞存活生长的作用。.我们在大鼠卵圆细胞增殖模型中检测Laminin各亚基mRNA及蛋白的表达变化，发现α2，α4，α5，β2，γ1亚基在肝切除术后4小时mRNA表达量达到峰值，随后逐渐降低。蛋白印迹实验发现α5，β2，γ1亚基在术后上调。由此我们推测最具活性的Laminin亚型可能是Laminin 521。体外实验中我们进一步验证了对卵圆细胞最具活性的亚型是Laminin 521。Laminin 521能显著提高细胞的延展性，促进细胞骨架的重排；明显加快卵圆细胞的运动速率，增加迁移距离。.卵圆细胞能表达Laminin的受体整合素多种亚基。抑制了整合素α6和β1后，可使细胞迁移得到明显抑制。Laminin 521可使整合素下游的Src、FAK及Paxillin蛋白发生磷酸化，且这种磷酸化可被整合素α6β1的阻断抗体抑制。而抑制FAK或阻断FAK磷酸化可显著抑制Laminin 521对卵圆细胞的迁移，说明Src、FAK、Paxillin参了与Laminin 521对卵圆细胞的迁移。同时，我们还发现Laminin 521可显著增加Akt磷酸化水平，但这种磷酸化水平通过抑制FAK并不能完全逆转，说明Laminin 521促进卵圆细胞迁移还可能存在其他非整合素途径。另外，抑制Akt可显著抑制Laminin 521对卵圆细胞的迁移作用但并不能影响卵圆细胞rac1/cdc42蛋白的表达。说明Akt与rac1/cdc42并不是上下游的关系，而是平行的非依存性关系。Laminin 521能促进卵圆细胞rac1/cdc42蛋白的表达上调，这种上调可以通过抑制FAK而阻断；且Laminin 521可增加Rac1的激活，证实被FAK直接激活的rac1/cdc42参与Laminin 521对卵圆细胞