HGF/c-met轴调控系统的建立及其诱导间充质干细胞定向归巢和分化治疗肝衰竭

In many cases of serious injury to the liver, liver cells die faster than they regenerate, and liver failure is inevitable. Transplantation has been the only effective treatment for this situation, but it is limited by shortage of organs and high expense, so the mortality for these patients is extremely high. Autologous Bone Mesenchymal Stem Cells (BMSCs) transplantation into patients with liver failure is a new method of clinical treatment for liver failure. The efficacy is limited, however, by the number of BMSCs that can be isolated from the bone marrow, non-specific markers, and the uncertainty of hepatic homing and differentiation. In this research, we used the albumin promoter to regulate the expression of green fluorescent protein (GFP) in gene-modified BMSCs, and flow cytometry to screen for GFP-positive cells that have the potential ability to differentiate into hepatocyte-like cells derived from BMSCs; c-met expression was controlled simultaneously in these cells by the Tet-on system to establish the GFP (+) Tet-on-c-met BMSCs. We then studied whether or not the expression level of c-met is dependent on doxycycline concentration in vitro and in vivo. We further determined the capacity of GFP (+) Tet-on-c-met BMSCs homing along HGF concentrations to the liver and differentiation into hepatocyte-like cells under the control of doxycycline. In addition, we analyzed the dose-dependent relationship between the ability of hepatic homing and differentiation with the expression of HGF/c-met axis. This project attempted to systematically clarify that the HGF/c-met axis plays an important role in BMSCs hepatic homing and differentiation, and to provide new approaches to carry out stem cell transplantation for liver failure in the future.

肝脏受到严重打击时，肝细胞进行性死亡速度超过肝脏再生的代偿能力,肝衰竭的发生不可避免。肝衰竭患者除肝移植以外缺少有效治疗手段，病死率极高。自体BMSCs移植是肝衰竭临床治疗的新方法，但受限于骨髓分离BMSCs数过少、无特异性标记，以及细胞移植后肝脏归巢和肝细胞分化能力不确定性等问题严重影响了其疗效。本课题拟利用白蛋白启动子调控的GFP表达筛选出具有类肝细胞分化潜力的BMSCs，并通过Tet-on系统调控c-met在该群细胞表达，建立GFP（+）Tet-on-c-met BMSCs，然后研究该群细胞体内外c-met表达水平对多西环素诱导浓度的依赖性，最后分析BMSCs顺HGF浓度归巢于肝脏并进一步向类肝细胞分化的能力，以及该能力与HGF/c-met轴表达水平之间的量效关系。本研究试图系统地阐明HGF/c-met轴在BMSCs肝脏归巢和分化机制中的重要作用，并为今后肝衰竭干细胞移植提供新思路。

肝衰竭除肝移植以外缺少特效治疗手段，因肝移植费用昂贵且供体短缺导致肝衰竭病死率极高。自体BMSCs移植是近年来发展起来的一种肝衰竭临床治疗新方法，但由于骨髓分离BMSCs数量少且无特异性标记，以及细胞移植后肝脏归巢、向肝细胞分化能力不确定性等问题严重影响了该治疗方法的临床疗效。. 本研究成功构建了rtTA advanced、TRE-c-met/GFP慢病毒，并感染大鼠BMSCs，G418筛选后获得稳定转染细胞株，然后利用流式细胞仪筛选出GFP（+）Tet-on-c-met BMSCs细胞。体外实验证实该细胞在多西环素诱导下可高表达c-met蛋白，且HGF可使过表达c-met磷酸化。GFP（+）Tet-on-c-met BMSCs经多西环素联合细胞因子（HGF、EGF）诱导可体外增殖和分化为类肝细胞。将GFP（+）Tet-on-c-met BMSCs尾静脉注射肝衰竭大鼠模型体内，经多西环素诱导c-met表达后，活体成像系统显示该群细胞主要集聚在动物肝脏。GFP（+）Tet-on-c-met BMSCs移植联合多西环素诱导可以明显改善肝衰竭大鼠肝组织病理学改变，包括降低HAI评分以及肝细胞凋亡指数（P<0.05）。GFP（+）Tet-on-c-met BMSCs移植后联合多西环素诱导可显著提高肝衰竭大鼠的生存率（P<0.01），长期观察GFP（+）Tet-on-c-met BMSCs移植在无多西环素诱导的情况下无致瘤性。. 本研究在动物水平论证HGF/c-met轴在BMSCs肝脏归巢和分化机制中的重要作用，为今后BMSCs移植治疗肝衰竭临床研究奠定理论基础，同时将Tet-on调控原理与ex vivo基因治疗途径相结合建立的HGF/c-met轴调控系统，有利于今后利用多西环素人为控制外源基因在BMSCs的表达，也为基因修饰BMSCs移植的临床研究提供了一条新的思路。