鳖甲煎丸通过调控miRNA-140促进间充质干细胞抑制肝癌干细胞治疗肝癌的机制研究

High recurrence and metastasis of hepatocellular carcinoma (HCC) is a serious threat to patients with liver cancer survival, a large number of studies confirm that there are cancer stem cells in tumor tissue, this is the root of tumor growth, invasion, metastasis and recurrence, therefore, the tumor is a disease of stem cells is due to gene mutations can lead to more stem cells lose growth control and and stop a certain stage in the differentiation of infinite proliferation of abnormal tissue or organ.Mesenchymal stem cells and cancer stem cells have many similarities, mesenchymal stem cells through competition with cancer stem cell nutrition and inhibit the growth of tumor stem cells, based on the turtle shell decoct pill for stem cell migration homing prophase research foundation of differentiation and the miRNA - 140 in the regulatory effect of inhibiting tumor cell proliferation, combination of turtle shell decoct pill, the theoretical basis of the anti liver cancer metastasis, turtle shell decoct pill resistance to recurrence and metastasis of liver cancer and liver cancer stem cells.The project is to study as the breakthrough point, to choose between mesenchymal stem cells and cancer stem cells in a certain proportion of mixed culture, the turtle shell decoct pill can intervene in vitro and in vivo transplantation, MMT Huh7 colorimetric determination of BMSC and liver cancer stem cells proliferation activity, rt-pcr and Western blot method to detect cell MicRNA - 140, CD133, CD144, EPCAM, AFP expression, to explore the turtle shell decoct pill affect the mechanism of prevention and treatment of liver cancer stem cells, discover new method for treatment of liver cancer。

肝癌的高复发及转移的问题已经严重威胁肝癌患者生存, 大量研究证实,肿瘤组织中存在着肿瘤干细胞,这是肿瘤生长、侵袭、转移和复发的根源,故肿瘤是一种干细胞疾病，而间充质干细胞和肿瘤干细胞有很多相似之处，间充质干细胞可通过调控细胞周期等方式抑制肿瘤干细胞生长，基于以上理论及鳖甲煎丸对干细胞迁移归巢分化的前期研究，以及miRNA-140在抑制肿瘤细胞增殖的调控作用，结合鳖甲煎丸抗肝癌转移的事实，我们推测：鳖甲煎丸抗肝癌的复发、转移与肝癌干细胞的关系密切。本项目以此为研究切入点，选用间充质干细胞与肝癌干细胞按一定比例混合培养，经鳖甲煎丸进行体外干预及体内移植实验，MMT比色法测定BMSC及肝癌干细胞 Huh7增殖活性，RT-PCR 及Western blot法检测细胞MicRNA-140、CD133、CD144、EPCAM、AFP表达， 探究鳖甲煎丸影响干细胞防治肝癌的机制，挖掘新的治疗肝癌方法。

肝癌的高复发及转移的问题严重威胁肝癌患者生存。肿瘤组织中存在着肿瘤干细胞,这是肿瘤生长、侵袭、转移和复发的根源；间充质干细胞可通过调控细胞周期等方式抑制肿瘤干细胞生长；miRNA-140在抑制肿瘤细胞增殖的调控作用；鳖甲煎丸在临床实验中有抗肝癌转移功效。本项目以此为出发点，推测鳖甲煎丸抗肝癌的复发、转移与肝癌干细胞的关系密切。以鳖甲煎丸含药血清为干预方法，采用体内结合体外的验证模式。在体内实验中，通过观察对照组、鳖甲煎丸组、鳖甲煎丸+miR-140 inhibitor组、骨髓间充质干细胞组、骨髓间充质干细胞+miR-140 inhibitor组、骨髓间充质干细胞+鳖甲煎丸组、骨髓间充质干细胞+鳖甲煎丸+miR-140 inhibitor组的细胞增殖、凋亡、相关干细胞表面生物标志物的影响。从而验证鳖甲煎丸含药血清联合骨髓间充质干细胞对肝癌干细胞的生长增殖、CD24、CD133、EpCAM的表达有抑制作用；对肝癌干细胞的凋亡有促进作用。在体外实验中，通过使用肝癌干细胞在裸鼠上注射产生异位移植瘤，分为小鼠肝癌模型组、鳖甲煎丸组、鳖甲煎丸+miR-140 inhibitor组、骨髓间充质干细胞组、骨髓间充质干细胞+miR-140 inhibitor组、骨髓间充质干细胞+鳖甲煎丸组、骨髓间充质干细胞+鳖甲煎丸+miR-140 inhibitor组。观察各组小鼠的肝脏组织病理变化、癌组织细胞凋亡率、癌组织中细胞增殖抗原Ki67及肝癌干细胞表面标志物CD24、CD133 和 EpCAM的表达情况。发现鳖甲煎丸含药血清联合骨髓间充质干细胞以及miRNA-140对肝癌模型小鼠的肝脏病理有明显改善作用；对癌组织中细胞增殖抗原Ki67及CD24、CD133、EpCAM的表达有抑制作用；对肝癌组织中细胞的凋亡有促进作用。