CX43影响诱导多能干细胞形成的机制研究

The creation of induced pluripotent stem cells (iPS cells) facilitate the generation of human patient-specific stem cell lines for modeling disease processes in vitro, providing a useful tool in studies of developmental biology, drug discovery and regenerative medicine. Although great progress has been made in the iPS cell field, reprogramming efficiency is low and the reprogramming mechanism remains unclear. The applicant has found that connexin 43 (CX43) was highly expressed in hiPS cells and it was the most dramatically unregulated connexin following reprogramming. Most importantly, the ectopic expression of CX43 significantly enhanced the reprogramming efficiency, whereas shRNA-mediated knockdown of endogenous CX43 expression greatly reduced the efficiency. But the mechanism of CX43 effecting the cell reprogramming efficiency is still unclear. CX43 is well known as a component of gap junctions is involved primarily in gap junction intercellular communication (GJIC); and some studies suggest that CX43 may also have a GJIC-independent function. In this study, we will induce chemical inhibitors or peptides blocking GJIC during the reprogramming process and observe the reprogramming efficiency. Real-time PCR, Western blot and co-immunoprecipitation will be performed to find out whether CX43 effecting the cell reprogramming by playing a direct role in a signaling pathway as a signaling molecule. We aim to found out the function of CX43 play on cell reprogramming and the mechanism of cell reprogramming.

诱导多能干细胞(iPSCs) 是体外研究疾病发病机制和药物筛选的良好模型，但如何提高其形成效率及阐明细胞重编程机制仍是未解决的问题。申请人前期研究发现在人iPSCs形成过程中缝隙连接蛋白43（connexin 43, CX43）的表达量随时间显著增强；过表达CX43提高人iPSCs的形成效率而干扰CX43抑制其形成（Hum Mol Genet，2013）。但CX43影响iPSCs形成的机制尚未明确，我们推测CX43可能通过参与形成缝隙连接调节胞内代谢物运输，或者直接参与胞内信号通路调控影响iPSCs形成。本课题拟在细胞重编程过程中抑制缝隙连接胞间通讯观察iPSCs形成效率的变化，并利用荧光定量PCR、免疫印迹、免疫共沉淀等方法探讨CX43是否作为信号分子直接参与胞内信号通路的调控，以期阐明CX43影响iPSCs形成的分子机理，为进一步揭示细胞重编程的机制提供科学依据。

本课题前期发现缝隙连接蛋白43（connexin43，CX43）在细胞重编程过程中表达逐渐上调，通过过表达CX43能促进诱导多能干细胞（Induced Pluripotent Stem Cells, iPSCs）的形成效率，研究进一步探讨CX43影响iPSCs形成的分子机理，为深入揭示细胞重编程的机制、提高细胞重编程效率提供科学依据。影响iPSCs形成效率可以通过调节细胞增殖来实现，按照项目的实施，我们首先通过CCK8检测发现过表达及干扰CX43不影响iPSCs形成过程中细胞的增殖，这可能不是CX43促进iPSCs形成的主要途径。在iPSCs形成过程，促使细胞发生间质上皮转化（Mesenchymal epithelial transition, MET）也是一个重要的促进因素。随着CX43变化上皮及间质标记物的变化研究，我们发现CX43的改变引起上皮标记物E-Cadherin、E-Pcam、Cldn3，间质标记物N-Cadherin以及Vimentin的表达变化，证实其可能通过参与细胞重编程过程中来促进iPSCs的形成。以往的研究表明，CX43除了作为形成细胞缝隙连接的结构蛋白以外，还可能以信号分子的形式直接参与胞内信号通路的调控。进一步研究发现，CX43对E-Cadherin的调节并不通过其上游调控的转录因子SP1来实现，而是通过其上游的转录因子Snail及Slug来实现。同时CX43对iPSCs形成的影响，还可能通过在细胞重编程过程中添加系列提高CX43表达的药物来实现，其中番茄红素可促进iPSCs的形成效率，这可能是更安全有效的促进iPSCs形成的方式。.此外我们还发现作为 CX 家族的另一重要成员，CX45 在重编程过程中表达量增加了 9 倍（远高于 CX43），而 iPSCs 在分化阶段 CX45 表达逐渐下降，随后发现过表达CX45能够提高iPSCs的形成效率，而干扰CX45几乎抑制了iPSCs的形成，而其作用方式主要是通过促进细胞的增殖来实现。这些结果为进一步阐明细胞重编程机制，提高iPSCs的形成效率提供了研究参考。