人骨髓间质干细胞对肝星状细胞的体外调控

背景:目前肝纤维化尚没有公认的特效疗法,近年来骨髓间质干细胞应用于肝纤维化的治疗已取得一定效果,但具体机制仍不明确.目的:体外探讨人骨髓间质干细胞对肝星状细胞的调控作用.设计、时间及地点:细胞学体外观察,于2008-06/12在中山大学干细胞与组织工程研究中心、中山大学第三附属医院中心实验室完成.材料:骨髓来源于青年健康志愿者髂骨,入肝星状细胞购自中山大学实验动物中心,人正常肝细胞系L-02购自中山大学实验动物中心.方法:将分离提纯的人骨髓间质干细胞与肝星状细胞在β孔Transwell板上建立上下共培养体系.接种密度均为2×104cells/well.另设L-02代替人骨髓间质干细胞作为阴性对照,单纯培养的肝星状细胞为空白对照,培养72 h.主要观察指标:肝星状细胞形态及免疫细胞化学染色结果,流式细胞仪检测肝星状细胞凋亡情况,Western blot法检测肝星状细胞活化标志α-肌动蛋白的表达.结果:倒置显微镜下活化的肝星状细胞呈扁平状,胞浆内缺乏脂肪滴,α-肌动蛋白位于肝星状细胞胞质内,呈高张力纤维状分布.与L-02+肝星状细胞组、单纯肝星状细胞组比较,骨髓间质干细胞+肝星状细胞共培养组的肝星状细胞凋亡率明显升高(P<0.05),α-肌动蛋白表达水平明显下调.结论:人骨髓间质干细胞可抑制肝星状细胞活化,促进其凋亡,这可能是骨髓间质干细胞抗肝纤维化的作用机制.

In vitro regulation effect of human bone marrow mesenchymal stem cells on hepatic stellate cells

BACKGROUND: There is no accepted treatment for liver fibrosis recently. Bone marrow meaenchymal stern cells (BMSCs) used in the treatment of liver fibrosis has been reported as an effectively treatment, but the mechanism is unclear.OBJECTIVE: To study the regulation of hepatic stellate cells mediated by human BMSCs in vitro.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Center for Stem Cells and Tissue Engineering of Sun Yat-sen University and the Central Laboratory of Third Affiliated Hospital of Sun Yat-sen University from June to December 2008.MATERIALS: Human bone marrow masenchymal stem cells were collected from normal youth volunteers; Human hepatic stellate cells and normal liver call line L-O2 were supplied by the Animal Experimental Center of Sun Yat-sen University.METHODS: The purified human BMSCs and hepatic stellate calls were set up in Transwell co-culture system. The incubation density was 2×104cells/well. L-O2 was set up instead of human BMSCs as negative control. Hepatic stellate cells cultured alone served as blank control group. The culture was performed for 72 hours.MAIN OUTCOME MEASURES: Morphology of hepatic stellate cells and results of immunocytochemical staining. Apoptosis of hepatic stellte calls was determined by flow cytometry. Western blot were used to assay the expression of α-actin.RESULTS: Activated hepatic stellate cells presented fiat and thin shape under an inverted microscope. Fat drop was lack in cytoplasm, a -actin located in hepatic stellate calls, with the presence of high tension fibers. Compared with the L-O2 + hepatic stellate cell and hepatic stellate call groups, the apoptotic rate of hepatic stellate cells was significantly increased in the BMSC + hepatic stellate cell group (P < 0.05). α -actin expression was significantly down-regulated.CONCLUSION: Human BMSCs can inhibit activation of hepatic stellate ceils and promote them apoptosis, which may be the anti-hepatic fibrosis mechanism of BMSCs.