“补肾生髓成肝”对大鼠肝干细胞诱导分化肝细胞及调控机理的影响

Project team in the Chinese medicine "liver advocate germinal" Innovation Guided by basic theory put forward "Nourishing Kidney to Produce Marrow and Form Liver" theory and the treatment principle of chronic liver disease, the early group application "Zuogui Pill" as the representative of "Nourishing Kidney to Produce Marrow and Form Liver" method to promote the formation of bone marrow were studied in liver cells, mechanism expression profiles of genes may be complete regeneration factor and liver tissue by influencing the regulation of liver. The research group of "Application of soothing liver and invigorating spleen and "Yiqi Jiedu" can also induce oval cell into hepatocyte cells differentiation. This study in the early stage of the innovation of theory and experiment foundation, further the use of RT-PCR technology, Western blot technology, gene expression microarray technology research to "Diwuyanggan capsule" as the representative of "Nourishing Kidney to Produce Marrow and Form Liver” method on rat hepatic oval cells and bone marrow mesenchymal stem cells induced to differentiate into liver cells, hepatic stem cells (oval cells) signal pathway related gene of hepatocyte differentiation possibility and liver regeneration regulation mechanism. To clarify the "Nourishing Kidney to Produce Marrow and Form Liver" has important significance for the mechanism of liver cell regeneration, prevention and treatment of chronic liver disease liver "lay the foundation for further experimental theory of" Nourishing Kidney to Produce Marrow and Form Liver, provides the thinking way of TCM treatment for clinical prevention and treatment of chronic liver disease.

课题团队在中医“肝主生发”基础理论指导下创新性提出了“补肾生髓成肝”的理论和慢性肝病的治疗法则，前期课题组应用”左归丸“为代表的”补肾生髓成肝“法促进骨髓形成肝细胞进行了研究，机制可能是通过影响肝再生调控因子和肝组织基因表达谱完成的。课题组应用的”疏肝健脾法“和”益气解毒法“也能诱导卵圆细胞向肝细胞分化。本研究拟在前期理论创新和实验基础上，进一步运用RT-PCR技术、Western blot技术、基因组表达谱芯片技术研究以”地五养肝方"为代表的“补肾生髓成肝”法对大鼠肝卵圆细胞和骨髓间充质干细胞的诱导分化为肝细胞、肝干细胞（卵圆细胞）的可能性和肝再生中肝细胞分化相关基因信号通路调控机制。对阐明“补肾生髓成肝”对肝细胞再生机制有重要意义，为“补肾生髓成肝”防治慢性肝脏病症进一步奠定实验理论基础，为临床防治慢性肝病提供中医治疗思路。

课题团队在中医“肝主生发”基础理论指导下创新性提出了“补肾生髓成肝”的理论和应用于慢性肝病的治疗法则，前期应用“补肾生髓成肝“法促进骨髓形成肝细胞进行了研究，对防治肝癌、慢性乙肝也进行了相关的基础和临床研究。本研究首先采用HPLC-MS/MS技术对“补肾生髓成肝”法的地五养肝方（胶囊）药物成分进行分析，确认地五养肝含药血清中有丰富的色谱峰，表明含有多种成分，通过高效液相色谱法检测到的“补肾生髓成肝”法含药血清样品中的潜在活性化合物主要含有11种成分和多种分析成分，包括姜黄素、梓醇、槲皮素等，而正常血清样本不含地五养肝（胶囊）相关成分；研究证实了“补肾生髓成肝”法代表方地五养肝（胶囊）可以通过调节Bax/Bcl-2信号通路，调节ALT、AST、Bax、Bcl-2的水平，对四氯化碳诱导的体内、外模型的急性肝脏损伤具有治疗作用（肝保护作用）；研究还通过微阵列分析阐明“补肾生髓成肝”法代表方地五养肝（胶囊）抑制2-乙酰氨基芴（2-AAF）/部分肝切除（PH）大鼠肝癌进展的潜在机制。结果表明，模型组激活了趋化因子信号通路、PI3K-Akt信号通路、癌细胞蛋白多糖信号通路、癌细胞肌动蛋白骨架调节通路，“补肾生髓成肝”法组则被抑制。此外，设置Fold change＞4，模型组/正常组和“补肾生髓成肝”法/模型组大鼠共有54个重叠的差异表达基因。其中2-AAF/PH大鼠有48个基因上调，3个基因下调。地五养肝胶囊处理可逆转这些基因的表达趋势；网络药理学分析初步提示HIF-1α/VEGF通路可能是地五养肝（胶囊）治疗2-AAF-PH大鼠模型（卵圆细胞增生模型）的抑癌机制之一，研究正在进行中。转录组学分析进一步揭示了肝癌的致病途径，而且也揭示了“补肾生髓成肝”法的抑癌机制。研究证实“补肾生髓成肝”法代表方地五养肝(胶囊)可能是一种潜在的肝癌、癌前病变的治疗药物。研究为“补肾生髓成肝”防治慢性肝脏病症进一步奠定实验理论基础，为临床防治癌前病变、肝癌提供中医治疗思路。