NF2/Mst1信号通路在调节心肌缺血再灌注损伤中的机制研究

The molecular mechanisms of myocardial remodeling after ischemia reperfusion (Ischemia/Reperfusion, I/R) injury has an important significance. In vivo, nerve fibroma protein 2 (NF2), as a scaffold protein, is ubiquitously expressed and responsible for the cell signal transduction. Mammalian sterile 20-like kinase 1 (Mst1), a highly conserved serine / threonine protein kinase, plays an important role in the regulation of myocardial hypertrophy and apoptosis in response to I/R injury. Our previous studies show that Rassf1A (a tumor suppressor) activates Mst1 and leads to cadiomyocytes apoptosis during heartpressure overload. However, the exact mechanism of Rassf1A/Mst1 signal pathway and its upstream regulatory factors remain unclear. We hypothesize that NF2 is a positive regulatory factor of Mst1 signal, and the NF2/Mst1 signal pathway plays an important role in the I/R process. We will induce a I/R model using nf2 gene knockout mice. Primary cardiomyocytes and cardiac fibroblasts will be isolated and cultured. The effect of NF2/Mst1 signal pathway on cadiomyocytes survival and function will be investigated by gene transfection, immunoprecipitation, co-immunofluorescence, immunohistochemistry, real-time quantitative Polymerase Chain Reaction（qRT-PCR), at the molecular, cellular and whole animal levels. In this way, we expect to find new strategies for myocardial injury treatment.

探讨缺血再灌注 (Ischemia/Reperfusion，I/R) 损伤后心肌重塑的分子机制具有重要意义。神经纤维瘤蛋白NF2是在体内普遍表达的支架蛋白，负责细胞内外信号转导；高度保守的蛋白激酶Mst1主要调控心肌肥大和凋亡。我们前期研究表明，在压力负荷过度的心脏中，Rassf1A（一种肿瘤抑制剂）可激活Mst1表达从而促进心肌细胞凋亡，但其上游的调节因子还研究甚少。我们预测NF2是积极的Mst1信号调节因子，NF2/Mst1信号通路在I/R损伤过程可能发挥重要作用。本研究旨在利用课题组建立的全身性和心肌组织特异性nf2基因敲除小鼠的优势，制备I/R损伤模型，分离培养原代心肌细胞和心肌成纤维细胞，利用基因转染、免疫共沉淀、免疫荧光、免疫组化、流式细胞术、心肌功能测定等方法，在分子、细胞和整体动物水平探讨NF2/Mst1信号通路对心肌细胞存活和功能的影响，有助于找到新的心肌损伤治疗策略。

探讨缺血再灌注(Ischemia/Reperfusion，I/R)损伤后心肌重塑的分子机制具有重要意义。神经纤维瘤蛋白NF2是在体内普遍表达的支架蛋白，负责细胞内外信号转导；高度保守的蛋白激酶Mst1主要调控心肌肥大和凋亡。我们前期研究表明，在压力负荷过度的心脏中，Rassf1A（一种肿瘤抑制剂）可激活Mst1表达从而促进心肌细胞凋亡，但其上游的调节因子还研究甚少。我们预测NF2是积极的Mst1信号调节因子，NF2/Mst1信号通路在I/R损伤过程可能发挥重要作用。本研究旨在利用课题组建立的全身性和心肌组织特异性nf2基因敲除小鼠的优势，制备I/R损伤模型，分离培养原代心肌细胞和心肌成纤维细胞，利用基因转染、免疫共沉淀、免疫荧光、免疫组化、流式细胞术、心肌功能测定等方法，在分子、细胞和整体动物水平探讨NF2/Mst1信号通路对心肌细胞存活和功能的影响，有助于找到新的心肌损伤治疗策略。