基于骨髓干细胞转化肝癌干细胞研究"髓失生肝"的病因病机

Based on the Sheng-ji theory, we develop the liver viscera-state theory of "liver-governing-growth" and suggest that the "marrow-not-form-liver" is the key etiology and pathogenesis of liver cancer. Based on the results of preliminary work, this project will focus on the hypotheses of "marrow-form-liver/marrow-not-form-liver dynamic unbalance" and mainly discuss the transformation of bone marrow stem cell to liver cancer stem cell in the initiation and development of liver cancer . We mainly use the combination protocols of clinical sample (blood serum of cirrhosis and liver cancer), animal model and cell model and apply the techniques for system biology, including sorting and identifying the liver SP cancer cell， liver cancer histopathology detection，small animals living imaging real-time dynamic observation and quantitive proteomics, to clarify the biological basis for the etiology and pathogenesis of "marrow-not-form-liver" in the initiation and development of liver cancer from the aspects of the transformation of bone marrow stem cell to liver cancer stem cell. Meanwhile, we also discuss the effect and mechanism for the regulation of "nourishing-kidney-to-produce-marrow-and-form-liver" to "marrow-form-liver/marrow-not-form-liver dynamic unbalance" in the prevention for the transformation of bone marrow stem cell to liver cancer stem cell. This project will reveal the biological basis for the etiology and pathogenesis of "marrow-not-form-liver" in the development of liver cancer from the aspects of prevention and effect of Chinese medicine, in order to improve the clinical effect and realize the breakthrough and innovation of traditional Chinese medicine.

"髓失生肝"是我们继承中医"生机学说"，创新"肝主生发"肝藏象理论而提出的肝癌（HCC）发生发展新的病因病机认识。在前期工作的基础上，围绕慢性肝病HCC发生发展过程中存在基于骨髓干细胞转化肝癌干细胞的"髓生肝/髓失生肝"失衡机制的科学假说，采用动物与细胞模型结合临床研究（运用肝硬化、肝癌患者及含药血清）的两个实验方案，从组织、细胞和分子三个不同层次，主要运用骨髓来源肝癌SP细胞的分离、鉴定 、肝癌组织病理学检测 、小动物活体成像实时动态观察、定量蛋白质组学为主的系统生物学等技术，拟从骨髓干细胞转化肝癌干细胞角度揭示HCC发生发展过程中"髓失生肝"病因病机的生物学基础，同时进一步探讨"补肾生髓成肝"通过调控"髓生肝/髓失生肝"失衡而防止肝干细胞转化为肝癌干细胞的作用及机制，从中医药的防治效应揭示HCC发生发展过程中"髓失生肝"病因病机的生物学基础，提高临床疗效，推动中医药理论突破与创新。

干预肝癌微环境防治肝癌是肝癌三级预防的新进展，我们提出肝癌肝再生微环境的新认识和改善肝再生微环境防治肝癌发生发展的新策略。地五养肝胶囊能改善MSG-大鼠-肝再生模型紊乱的肝再生过程（“髓失生肝”），调控EMT /MET失衡，抑制肝纤维化组织中Hh信号通路的过度激活可能是其作用机制之一。 地五养肝胶囊能提高2-AAF/PH模型大鼠存活率的同时，抑制肝癌前病变，恢复肝脏组织结构和功能，其可能作用机制之一是抑制Wnt/β-catenin通路的过度激活可能是其抑制卵圆细胞过度增殖、异常分化的分子机制，调控2-AAF/PH大鼠模型多个肝再生相关细胞因子（TNF-α、IL-1、GRO/KC、VEGF、IFN-γ）的表达，使之更加趋向于正常组，从而通过改善肝再生微环境，防治肝癌前病变的发生发展。 地五养肝胶囊能抑制Solt-Farbe二步法肝癌大鼠模型肝癌发生发展的作用与索拉菲尼等效，其作用机制可能是抑制骨髓干细胞转化肝癌干细胞，促进肝脏细胞再生修复，抑制卵圆细胞过度增殖，调控EMT/MET失衡（抑制EMT、促进MET），影响JAK/STAT、Ras/Raf/Mek/Erk信号通路相关蛋白的表达，改善肝再生微环境而抑制肝癌发生发展。采用甲基化芯片技术检测及分析，发现地五养肝胶囊可通过改变其相关甲基化水平防治肝癌的发生发展。采用Arraystar大鼠长链非编码RNA芯片技术检测及分析，发现地五养肝胶囊可能通过影响关键lncRNA的表达水平，调节肝再生相关基因表达、调控细胞增殖分化相关信号通路关键信号分子的表达，实现调控肝再生、防治肝癌发生发展的生物学效应。采用骨髓干细胞与肝癌细胞共培养体系，初步揭示地五养肝胶囊对该培养体系中肝癌细胞的影响及其机制。以上实验研究揭示的地五养肝胶囊改善肝再生微环境防治肝癌发生发展的作用及其机制，为构建基于“补肾生髓成肝”的肝癌三级预防方案提供了实验依据，具有重要的科学意义和临床价值。