CREG在糖尿病性心肌病中的作用及机制研究

Diabetic cardiomyopathy (DCM) causes overt heart failure, which is the most common cause of death in patients with type 2 diabetes mellitus. Thus clarifying underlying mechanism and exploring effective target for treatment of DCM has become a hot topic in this field. Cellular repressor of E1A stimulated gene (CREG) is a small molecular glycoprotein locating in endosome-lysosome system, which is involved in maintenance of cellular homeostasis and protecting cells from pathological damages. Our preliminary data showed that CREG was highly expressed in cardiomyocytes under physiological condition, and over-expression of CREG alleviated cardiomyocyte damages induced by ischemia or inflammation. In addition, in a mouse model of DCM, we observed significant reduction of CREG in myocardium, suggesting that CREG might be a key factor involved in regulating onset and development of DCM. Therefore, the aim of this study is to elucidate the role and underlying mechanism of CREG in DCM. In this study, the cardiomyocyte specific CREG knock-out and CREG transgenic mice have been successfully established and will be used to prepare animal models and cell models of DCM to determine the role of CREG both in vivo and in vitro. Then by taking advantage of protein microarray assay, the molecular mechanism of CREG in regulating DCM will be elucidated, mainly focusing on the lysosome-autophagy pathway. Results from this study will provide strong theoretical support and novel therapeutic target for prevention and treatment of DCM.

糖尿病性心肌病（DCM）所致的心力衰竭是引起2型糖尿病患者死亡的主要原因。因此，阐明DCM发生机制并寻找有效治疗靶点，成为目前DCM防治研究的热点。E1A激活基因阻遏子（CREG）是定位于细胞内体-溶酶体系统的小分子糖蛋白。它能够参与细胞内稳态调控，对抗病理性细胞损伤。我们前期研究发现，CREG在正常心肌细胞中高表达，过表达CREG能够对抗缺血或炎症引起的心肌细胞损伤；而在DCM模型小鼠心肌中，CREG表达则显著降低，提示其可能是DCM发生的重要调控分子。因此，本研究选择CREG为靶向分子，通过心肌特异性敲除和过表达CREG的DCM小鼠模型和原代心肌细胞模型，从在体和细胞水平分别阐明CREG在DCM发生及防治中的作用；并尝试从溶酶体-自噬保护这一重要的细胞内稳态调控通路入手，通过蛋白质芯片技术，阐明CREG调控DCM的分子机制，为DCM防治提供新的理论依据和分子靶点。

项目背景：自噬功能障碍在糖尿病心肌病(DCM)的发生发展中起着重要作用。E1A激活基因阻遏子(CREG)是一种小分子分泌型糖蛋白，在多种细胞和组织中广泛表达，是一个重要的心肌保护因子。本研究拟利用CREG心肌特异性敲除小鼠和CREG转基因小鼠建立DCM动物模型，从在体和细胞水平阐明CREG在DCM发生中的作用和分子机制，争取为DCM的预防和治疗提供理论依据和治疗靶点。.主要完成了以下内容： .(1) 利用高脂喂养联合小剂量链脲佐菌素(HFD+STZ)腹腔注射方法建立2型DCM小鼠模型，定量PCR和western blot检测CREG基因mRNA和蛋白表达。结果发现，DCM后心肌组织CREG基因mRNA水平无明显变化，而CREG蛋白表达在2型DCM心脏组织中明显降低。.(2) 对CREG转基因小鼠和心肌特异性敲除小鼠建立DCM模型，评价建模24周各组心功能等情况。结果发现，CREG转基因小鼠心肌损伤及心肌肥厚减轻，而心肌特异性CREG敲除小鼠心肌损伤及心肌肥厚加重。.(3) 利用western blot、免疫荧光染色等方法检测CREG转基因小鼠和心肌特异性敲除小鼠DCM后心肌自噬及溶酶体指标表达。结果发现，CREG转基因可改善DCM后心肌自噬-溶酶体功能，而CREG心肌特异性敲除后可加重DCM后心肌自噬-溶酶体障碍。.(4) 提取小鼠原代心肌细胞(NMCMs)，给予棕榈酸PA刺激，western blot检测CREG蛋白表达。结果发现PA可抑制CREG蛋白表达。.(5) 建立CREG过表达和低表达的NMCMs，给予PA刺激后western blot、免疫荧光染色等方法检测自噬及溶酶体指标表达。结果发现，CREG过表达可改善PA导致心肌自噬-溶酶体功能，而CREG低表达可加重PA所致心肌自噬-溶酶体功能障碍。.(6) 利用CO-IP、定量PCR及western blot方法确定CREG与LAMP2的相互作用及上下游关系。结果发现，CREG与LAMP2之间存在相互作用，并且CREG作为LAMP2的上游分子，通过调控LAMP2表达影响了2型DCM心肌细胞的自噬-溶酶体功能。.科学意义：本研究从在体和细胞水平阐明CREG通过调控LAMP2影响心肌细胞自噬进而参与DCM发生发展，为深入阐明DCM的发病机制以及寻找DCM防治的新靶点提供理论依据。