基于Smad3/Smad7“平衡”的健脾化瘀法抑制肝癌细胞EMT的机制研究

In China hepatocellular carcinoma (HCC) is characterized by high incidence and poor prognosis. High invasiveness and potential to metastasis is one of the most important characteristics of HCC. The theory of “Jianpi Huayu Therapy” is rooted in “Synopsis of Golden Chamber”. “Jianpi Huayu Therapy”is one of the most important therapies on HCC. Our previous studies revealed that “Jianpi Huayu Therapy” reduced the metastasis and recurrence of HCC and improved the survival of HCC patients. However, most of the studies on the anticancer mechanisms of “Jianpi Huayu Therapy”aimed at inhibition of proliferation or induction of apoptosis. Recent studies found epithelial-mesenchymal transition (EMT) provides the basis for invasion and metastasis of HCC. Our previous study revealed the therapy inhibited the invasiveness and metastasis of HCC cell. Activation of TGF-β/Smads pathway and EMT was observed in HCC cell. Smad3 and Smad7 were control points of TGF-β/Smads pathway. So we presumed that inhibition of EMT via regulating Smad3/Smad7 balance in HCC was one of the most important anticancer mechanisms of “Jianpi Huayu Therapy”. In present research，①we employ Real-time PCR, Western blot, immunofluorescence, shRNA and gene overexpression technologies as investigative tools,to explore the effect of “Jianpi Huayu Therapy”drug serum on EMT of HCC cell line via regulating Smad3/Smad7 balance in vitro；②we will then establish human HCC cell lines nude mice orthotopic models to study the anti-tumous effect and the underlying mechanisms of“Jianpi Huayu Therapy”drug on HCC. The expected results will reveal the anticancer mechanism of “Jianpi Huayu Therapy” on HCC, and lead to a novel therapeutic strategy and theory basis for Chinese medicine treatment on HCC.

我国肝癌发病率高而预后差，易侵袭转移是其特点。健脾化瘀法理论根源于《金匮要略》，是中医治疗肝癌重要治法。健脾化瘀法中药可降低肝癌转移复发、延长生存期；既往探讨其抗癌机制集中于抑制肿瘤细胞增殖和诱导凋亡。我们结合前期研究Smad蛋白、上皮间质转化(EMT)的基础和最新进展“EMT是肝癌侵袭转移关键步骤”，提出假设：健脾化瘀法调控Smad3/Smad7“平衡”从而抑制肝癌细胞EMT是其治疗肝癌重要分子机制。项目拟：①体外采用实时定量PCR、免疫荧光、Western blot、基因沉默和超表达等技术，研究健脾化瘀法中药对Smad3/Smad7“平衡”调控及对肝癌细胞EMT分子表型、细胞形态和侵袭转移能力的影响；②体内借助肝癌动物模型探讨健脾化瘀法中药抗癌作用和其对Smad3/Smad7“平衡”及EMT影响的关系。结果有望揭示健脾化瘀法治疗肝癌分子机制，为中医药治疗肝癌提供新思路和理论依据。

我国肝癌发病率高而预后差，易侵袭转移是其特点。健脾化瘀法理论根源于《金匮要略》，是中医治疗肝癌重要治法。我们结合前期研究Smad蛋白、上皮间质转化(EMT) 的基础和最新进展“EMT是肝癌侵袭转移关键步骤”，提出科学假设，分别从体外采用实时定量PCR 、免疫荧光、Western blot、基因沉默和超表达等技术，研究健脾化瘀法中药对Smad3/Smad7 “平衡”调控及对肝癌细胞EMT分子表型、细胞形态和侵袭转移能力的影响；体内借助肝癌 动物模型探讨健脾化瘀法中药抗癌作用和其对Smad3/Smad7“平衡”及EMT影响的关系。结果显示健脾化瘀方药可提高Smad7 mRNA和蛋白质水平，降低磷酸化Smad3 (p-Smad3) 蛋白质水平，提高E-cadherin并降低N-cadherin和Vimentin的表达水平，抑制肝癌细胞侵袭和转移能力。结果有望揭示健脾化瘀法治疗肝癌分子机制，为中医药治疗肝癌提供新思路和理论依据。