卵巢癌肿瘤微环境中间充质干细胞通过c-Met／HGF信号转导通路调控卵巢癌增生的分子机制研究

Ovarian cancer has become the highest mortality disease in gynecological cancer,because of its inconspicuous initial symptoms and rapidly development and metastases ability.Previous Flow cytometry and Western Blot showed that c-Met was overexpressed in ovarian cancer cells, and stem cells (MSC) promote the proliferation of cancer cells abviously,which is related to prognosis. It is speculated that the c-Met / HGF signaling pathway plays an important role in the development and progression of ovarian cancer. To further establish ovarian cancer /MSC co-culture, on the behave of HGF and c-Met inhibitor ,use ELISA to analyse cytokines and chemokines, Western blot and animal model to test the changes of downstream tyrosine kinase domain Tyr1349 and p44/p42MAP kinase Thr202/Tyr204 phosphorylation, c-Met siRNA knock-down to simulate function loss and gain experiment, microarray and compare KEGG、 GO database to screen key factors ,RT-PCR and proteome protein chip array to validate previous data, so that we can elucidate the molecular mechanism of ovarian cancer hyperplasia and also provide scientific basis for early diagnosis and targeted therapy.

卵巢癌初期症状不明显且发展迅速易多发性转移，极易错过最佳治疗时期成为妇科肿瘤中死亡率最高的恶性肿瘤。前期流式细胞术及Western Blot显示c-Met在卵巢癌细胞上过表达，干细胞（MSC）促进癌细胞异常增生且与预后相关。推测c-Met／HGF信号通路在卵巢癌的发生发展过程中起到促进作用。拟进一步建立MSC卵巢癌共培养，ELISA分析多种细胞因子、Western blot及小鼠模型检测HGF和c-Met抑制剂作用前后下游酪氨酸激酶域Tyr1349和p44/p42MAP激酶Thr202/Tyr204磷酸化变化，c-Met siRNA knock-down功能缺失及获得实验、microarray基因检测及KEGG、GO数据库对比分析筛选关键因子，RT-PCR、蛋白组学蛋白质芯片阵列法等进行验证，阐明卵巢癌异常增生分子机制并提供早期诊断和靶点治疗的科学依据。

本课题从新生儿脐带中分离人类间充质干细胞进行原代培养，与肿瘤细胞共培养建立肿瘤微环境模型，发现共培养过程中肿瘤细胞的细胞增殖能力得到显著提高，并且在与干细胞相互作用过程中肿瘤细胞获得了MSC特有的干细胞表面标记物CD90表达，部分CD73及CD105表达。同时分析得出卵巢癌细胞与干细胞通过多种信号传导途径如直接接触通路 Notch signaling、nanotube、cell-to-cell 以及间接接触通路细胞因子、趋化因子、exosome 交换等发生相互作用的同时。采用GFP、cherry荧光蛋白转染MSC和肿瘤细胞后在共培养过程中我们发现一种融合细胞即hybrids细胞，可以同时显影GFP和cherry荧光。c-Met/HGF 信号转导通路在卵巢癌细胞和干细胞 MSC 上同时发生的表达变化情况，并采取酪氨酸激酶抑制剂Foretinib进行信号通路的阻断，进一步观察下 游信号传导通路及细胞生物活性的改变，例如细胞周期改变、特性细胞蛋白表 达及基因表达的变化，发现foretinib可以明显抑制肿瘤细胞的生长及c-Met/HGF下游信号通路磷酸化。进一步阐明卵巢癌肿瘤微环境中间充质干细胞通过 c-Met /HGF 信号转导通路调控卵巢癌增生的分子生物学机制，Foretinib目前是肺癌的一线化疗药物，亦在卵巢肿瘤的临床早期靶向治疗提供相当可观的应用前景。