脾虚内环境Oatp表达对肝癌CSC“干性”及PTEN/AKT信号通路影响研究

The environment of spleen deficiency is closely related to the occurrence and development of hepatocellular carcinoma (HCC). In previous studies, we found the expression of organic anion transporter (OATP)in spleen deficiency environment was one of the most important reasons for environmental changes in the body, further study found that abnormal expression of OATP in mice with spleen deficiency is closely related to the occurrence and development of HCC. Our preliminary experiments found that the expression of CD133 in HCC mice with spleen deficiency was significantly increased, which has been identified as cancer stem cell (CSC) of HCC. As we all know, the CSC is an important factor in the occurrence and development of tumor, we therefore hypothesized that the abnormal expression of OATP in spleen deficiency environment might be closely related to function of CSC in occurrence and development of HCC. So we hypothesized that the expression of OATP in spleen deficiency environment might be closely related to the characteristic of CSC of HCC. To confirm this hypothesis, we will build of hepatocellular carcinoma models in mice with spleen deficiency, the OATP expression changes in spleen deficiency being as the breakthrough point, the CD133+ CSC self renew differentiation ability, proliferation, migration invasion and tumorigenicity will be detected in HCC mice with spleen deficiency, and detection of hepatocellular carcinoma stem cells PTEN/AKT signal transduction pathway f CSC will also be detected in the same time. Fuether explore the molecular mechanism of OATP expression in spleen deficiency environmental on the characteristic of CSC, provide new therapeutic strategies for TCM in prevention and treatment of HCC, as well as promote improvement and development the theory of TCM in turbid transport and transformation.

脾虚内环境与肝癌发生发展关系密切。前期研究中，我们发现脾虚内环境有机阴离子转运肽(Oatp)表达是机体内环境变化的重要原因,进一步研究发现脾虚小鼠肝癌组织Oatp表达异常与肝癌发生发展关系密切。我们预实验发现脾虚小鼠肝癌组织肿瘤干细胞（CSC）CD133表达明显升高,而CSC是导致肿瘤发生发展的重要因素，我们推测脾虚内环境Oatp异常表达可能与CSC在肝癌发生发展所起作用密切，提出“脾虚内环境Oatp表达变化--肝癌CSC‘干性’相关”假说。为证实这一假说，我们将建立脾虚肝癌小鼠模型，以脾虚内环境Oatp表达变化为切入点,通过检测脾虚肝癌小鼠和对照肝癌小鼠CD133＋CSC的自我更新能力、增殖能力、迁移侵袭能力和成瘤能力等，同时检测肝癌CSC相关PTEN/AKT信号转导通路变化，探讨脾虚内环境Oatp表达变化对肝癌CSC“干性”影响的分子机制，促进中医藏象理论和湿浊转运理论的完善和发展。

目前对肝癌的研究大多侧重于消除“种子”（肿瘤细胞），却忽略了“土壤”（机体内环境）对肝癌发生发展的影响，本课题在中医脾胃理论指导下，开展了脾虚内环境对肝癌CSC“干性”影响的研究，发现：.①脾虚内环境对肝癌 CSC 及其“干性”维持促进了肝癌发展。脾虚内环境是否通过对肝癌 CSC 及其“干性”维持来促进肝癌发展，是本课题需要回答的科学问题。研究发现脾虚肝癌小鼠肿瘤体积明显大于肝癌组，成瘤率明显高于肝癌组小鼠，病理分级恶性度更高；与肝癌组相比，脾虚肝癌小鼠癌组织CD133 mRNA表达明显高于非脾虚肝癌小鼠，脾虚肝癌组癌组织CD133+肝癌细胞体外增殖、迁移侵袭和成瘤能力明显高于肝癌组，提示脾虚内环境可能通过维护肝癌CSC“干性”来促进肝癌发展，提示脾虚内环境对肝癌 CSC 及其“干性”维持促进了肝癌发展。.②脾虚内环境Oatp相关亚家族发生了变化，可能与肝癌CSCs“干性”维持相关。在原位移植肝癌模型中，发现脾虚状态下各脏腑组织Oatp相关亚家族呈现相应表达形式。在药物诱导性肝癌小鼠模型中，发现脾虚肝癌组小鼠肝组织中Oatp4a1及oatp2a1较肝癌组明显升高；发现脾虚肝癌组与肝癌组比较，肺组织中CD133表达明显升高，而肾组织中CD133表达降低；肺组织中oatp2a1表达下降，肾组织oatp2a1的表达升高，CD133表达趋势同oatp2a1在各脏器中的表达相反，提示脾虚状态下各脏腑组织Oatp相关亚家族可能存在相互协调或者制约机制，与肝癌CSCs“干性”维持相关，促进了肝癌的发展。.③脾虚内环境PTEN/AKT通路发生了变化，可能是促进肝癌发展的重要原因。在原位移植模型中，与肝癌组相比，脾虚肝癌组PTENmRNA表达降低，而AKTmRNA的表达则被上调。提示脾虚机体内环境发生变化且促进了肝癌的发展，其机制可能是脾虚内环境变化致PTEN/AKT信号通路失衡进而促进肝癌CSC 及其“干性”维持有关。. 以上研究提示脾虚机体内环境促进了肝癌的发展，其机制可能是脾虚内环境Oatp相关亚家族变化致PTEN/AKT信号通路失衡,进而促进肝癌 CSC “干性”维持，将为中医药治理脾虚内环境防治肝癌提供新的治疗策略，促进中医脾胃理论的完善和发展，具有重要的理论意义和临床应用价值。