基于14-3-3蛋白调控骨髓干细胞转化肝细胞研究“髓生肝”的生物学基础

According to our previous research of 14-3-3 proteins up-regulated expression in the process of bone marrow stem cells transformed hepatocyte-like cells and the research status that the scientific connotation of "the marrow produces the liver" was not fully reveal, we put forward a scientific hypothesis that the biological foundation of "the marrow produce the liver" is the liver regeneration repair mechanism of which 14-3-3 proteins possibly regulate bone marrow stem cells transforming into hepatocyte through binding TAZ to influence Wnt/PI3K/Akt/MAPK signaling pathways network. This project contrastively observes 14-3-3 proteins over-expression, silence-expression and normal-expression influencing the process of bone marrow stem cell transforming into hepatocyte through Lentivirus transfection technique and in vitro culture system of bone marrow mesenchymal stem cell differentiated into hepatocyte. Using molecular biological technique, we would reveal the mechanism of bone marrow stem cell transform into hepatocyte which affect by 14-3-3 proteins and relevant signal pathways, and the intervention effect of Zuogui Pill and its mechanism. This research would help us to find some new therapeutic targets of liver regeneration regulation, to further enrich the scientific connotation of visceral manifestation theory "the liver governing growth" (the physiological mechanism of "the marrow produces the liver", the etiology and pathogenesis of "the marrow produces the liver abnormally" and the therapeutic principle and method of “nourishing kidney to produce marrow and form liver”), to establish the experimental and theoretical basis of improving the clinical curative effect, and to promote innovation and development of traditional Chinese medicine theory.

根据我们前期骨髓干细胞转化肝细胞的过程中14-3-3 蛋白表达规律的发现和“髓生肝”的科学内涵尚未完全揭示的研究现状，我们提出 14-3-3蛋白可能通过其结合蛋白TAZ参与Wnt/PI3K-Akt/MAPK信号通路网络调控骨髓干细胞转化肝细胞的肝再生修复机制是“髓生肝”生物学基础的科学假说。本课题研究采用慢病毒转染技术结合骨髓干细胞体外诱导为肝细胞的培养体系，观察14-3-3 蛋白过表达、沉默表达和正常表达对骨髓干细胞转化肝细胞的影响及左归丸调控14-3-3蛋白表达并促进骨髓干细胞转化肝细胞的作用，揭示14-3-3蛋白影响骨髓干细胞转化肝细胞的调控机制及左归丸的作用机制，明确新的调控肝再生的治疗靶点，进一步丰富“肝主生发”藏象理论体系（“髓生肝”的生理机制、“髓失生肝”的病因病机、“补肾生髓成肝”的治则治法）的科学内涵，为提高临床疗效奠定实验及理论基础，推动中医药理论的创新与发展。

根据我们前期骨髓干细胞转化肝细胞的过程中14-3-3 蛋白表达规律的发现和“髓生肝”的科学内涵尚未完全揭示的研究现状，我们提出 14-3-3蛋白可能通过其结合蛋白TAZ参与Wnt/PI3K-Akt/MAPK信号通路网络调控骨髓干细胞转化肝细胞的肝再生修复机制是“髓生肝”生物学基础的科学假说。本课题分别采用体外诱导模型和体内卵圆细胞增殖模型探讨14-3-3家族及其相关下游靶基因在骨髓细胞转化肝细胞过程中的表达规律，以及体现“补肾生髓成肝”治疗法则的地五养肝胶囊对这一过程的干预。结果提示采用肝细胞诱导培养基处理MSC细胞1d后AFP基因表达上调、5d时表达显著增加，Alb基因表达规律与AFP相同；14-3-3家族及相应信号通路靶基因也随着诱导时间延长而变化，14-3-3蛋白及其下游靶基因可能是地五养肝胶囊影响骨髓干细胞转化肝细胞的调控机制，有望成为调控肝再生的治疗靶点。