基于Wnt／β-catenin通路对肝卵圆细胞增殖分化调控探讨山仙颗粒抗肝癌前病变恶性转变的机制研究

Hepatocarcinogenesis is a multi-step process in which cirrhosis , hepatic precancerous lesions progress into hepatocellular carcinoma. Recent studies have found that excessive proliferation and abnormal differentiation of hepatic oval cell (HOC) after liver injury are the key process of malignant transformation of hepatic precancerous lesions . Wnt／β-cateninsignaling pathway may play an important role in the regulation of proliferation and differentiation of HOC. However, it’s still unclear that how to work of Wnt／β-catenin signaling pathway in the process of malignant transformation of hepatic precancerous lesions . Therefore, we will discuss Wnt／β-catenin signaling pathway in promoting proliferation and differentiation of HOC by detecting the gene wnt-1,β-catenin,c-myc,Cyclin D1 and Ep-CAM in the regulation of cell proliferation and differentiation of Wnt／β-catenin signaling pathway in vivo and in vitro level respectively. This study provides a new way for the treatment of hepatic precancerous lesions. SXG is effective for treatment of malignancy. Previous studies have shown that SXG can inhibit hepatocellular carcinoma and hyperplasia of fibrosis surrounding hepatoma. This study is to explore further whether the SXG against malignant transformation of hepatic precancerous lesions by inhibiting excessive proliferation and abnormal differentiation of HOC and provides experimental basis for clinical treatment of hepatic precancerous lesions of SXG.

肝癌发生经历较长的过程，即肝硬化－肝癌前病变-肝癌。研究发现肝卵圆细胞（HOC)在肝损伤后过度增殖和异常分化是肝癌前病变发生恶性转变的重要环节,而Wnt/β-catenin 信号通路可能是调控HOC增殖和分化的关键步骤。但该通路在肝癌前病变恶性转化过程中是如何发挥作用的尚不完全清楚。因此本研究拟在在体和离体水平，通过Wnt信号通路中调节细胞增殖和分化的相关基因wnt-1、β-catenin、c-myc 、Cyclin D1、Ep-CAM等的检测，旨在探讨Wnt信号通路在HOC增殖和分化中的作用，为肝癌前病变防治提供新的思路。山仙颗粒是对我院治疗恶性肿瘤疗效确切的中成药，前期研究发现该药能够抑制肝癌且对肝癌周围的纤维化增生有明显抑制作用，本研究进一步探究该药是否通过Wnt／β-catenin信号通路抑制HOC的过度增殖和阻止其异常分化来防治恶性转变，为山仙颗粒临床治疗肝癌前病变提供实验依据。

肝癌发生经历较长的过程，即肝硬化－肝癌前病变-肝癌。肝硬化-肝癌的恶性演进过程中会经历一个较长的肝癌癌前病变时期。因此如能阻止或逆转肝癌前病变恶性转化就有可能阻断肝癌的发生。本研究以 Wnt／β-catenin 信号通路的受体和相关靶基因为切入点，采用免疫组化、Q-PCR、Western bolt等综合研究方法，研究山仙颗粒是否通过调节 Wnt／β-catenin 信号通路抑制肝卵圆细胞异常活化增殖，并对此信号的相关下游分子进行研究，以阐明山仙颗粒治疗肝癌前病变的分子机制。研究结果表明：（1）山仙颗粒可改善肝癌前疾病-肝硬化SD大鼠肝脏病变，降低变性、坏死的肝细胞数目，减少异型增生结节的数目和体积；（2）山仙颗粒降低肝癌前疾病-肝硬化SD大鼠血清ALT、AST、GGT含量，且具有剂量依赖性；（3）山仙颗粒可抑制肝癌前疾病-肝硬化SD大鼠肝脏纤维化程度，纤维间隔的数目和厚度明显降低；（4）山仙颗粒可下调肝癌前疾病-肝硬化SD大鼠肝脏组织中Wnt／β-catenin信号通路中wnt-3a、β-catenin、C-Myc mRNA的表达；（5）山仙颗粒可降低肝癌前疾病-肝硬化SD大鼠肝脏组织中Wnt／β-catenin信号通路相关蛋白wnt-3a、β-catenin、C-Myc的表达量；（6）山仙颗粒可下调2-AAF/PH肝癌前病变大鼠肝脏组织中肝卵圆细胞的过度增殖和活化，肝卵圆细胞标志物AFP、CD90的表达明显降低。因此我们得出以下结论：肝癌前疾病的发生发展与Wnt／β-catenin信号通路过度激活密切相关；山仙颗粒可能通过干预Wnt／β-catenin信号通路相关蛋白抑制肝卵圆细胞的过度增殖和活化，抑制制肝细胞异常增生及肝硬化形成，从而阻断肝癌前疾病的恶化。