细胞外基质蛋白him1/fbln6与心脏发育构建的遗传机理研究

The extracellular matrix protein (ECM) hemicentin1 or fibulin6 (hem1/fbln6) is one of the highly conserved proteins, mainly distributing in diverse epithelial cells and cells of blood vessels in uterus, eyes and many other tissues and organs. Despite the prevalence of fact, their molecular mechanism remains related to this gene largely unknown although ECM proteins have already been shown in animal models to play a non-substitutable role in cell differentiation and tissue/organ architecture. Our unpublished data shows that the protein distributes in intercalated disk of cardiomyocytes between two cells and haploinsufficiency was observed on traditional knockout. Base on these evidences, we intend to perform the following specific aims by using our newly conditional knockout hem1/fbln6 animal model. In Aim 1, we intend to analyze that the distribution pattern of the protein within mouse early development stage including the first 3 mitosis of embryonic cells and the first different cell lineages of Epiblast (EPI）, trophectoderm (TE）and primitive endoderm (PE）. Using mouse early embryo development, we intend to investigate the detailed information of the ECM protein during the formation of EPI, TE and PE. This differentiation process will enhance our understanding of human tissue and organ genesis in both normal development and pathological remodeling events. In Aim 2 and 3, using newly generated conditional knockout hem1/fbln6 mouse model crossing with cardiomyocyte-specific MHC-cre recombinase transgenic mouse, we intend to produce the cardiomyocyte-specific deletion mouse (Mc::hem1/fbln6-/-) . Then we plan to investigate the expression pattern of ECM hem1/fbln6 during heart development from cardiac crescent stage, tubular heart stage, looping heart stage and 4-chamber heart (forming) stage. Compared to normal development, any developing defects occurred in Mc::hem1/fbln6-/- mutant hearts and Tc::hem1/fbln6-/-mutant hearts would reveal the function of ECM hem1/fbln6 in cardiomyocyte during the maturation of heart. In Aim 4, once defect of the cardiomyocyte-specific deletion of hem1/fbln6 did not affect the growth of mice, we intend to apply exercise-stress to investigate the cardiac function performance of Mc::hem1/fbln6-/- mutant mice, Tc::hem1/fbln6-/-mutant mice and Tc-hem1/ fbln6-/-/Mc-hem1/fbln6-/-. The alternation of other potential related proteins will be investigated as well. .Therefore, the investigation of our proposal will enhance the understanding of ECM hem1/fibl6 function during the development of heart, which will provide our further investigation on molecular pathological mechanism on human heart developing disorder and other cardiac diseases.

细胞外基质蛋白hem1/fbln6具高度进化保守性，主要分布于各类上皮细胞和血管组织中；我们尚未发表的实验结果表明了该蛋白在心脏中分布于心肌细胞闰盘的分布特征；经典敲除方法获得的突变小鼠研究显示存在基因半量效应，表现出心脏功能的缺陷。另外我们报道了该蛋白基因缺失会导致细胞有丝分裂之胞质分裂的失败以致细胞多核的现象。因而，本申请中我们拟以近期制得的hem1/fbln6基因条件敲除小鼠为材料，特异性地在心脏心肌细胞中敲除该基因以探索细胞外基质蛋白hem1/fbln6在心脏发育由心管到四腔心以至最后构建形成成体心脏过程中的作用，项目的实施将揭示心脏发育构建对成体心脏生理功能的影响的机制，从而建立hem1/fibl6相关的“动（细胞分裂）”与“静（组织结构）”结合的组织构建模式；实验结果也将为心脏先天性遗传疾病机理的研究提供新的思路并为建立相应的诊疗策略打下分子遗传学基础。

细胞外基质蛋白hem1/fbln6具高度进化保守性，主要分布于各类上皮细胞和血管组织中，该蛋白在心脑血管中的分布特别是心脏中分布于心肌细胞闰盘的分布特征备受同行关注。基于我们首次报道的该蛋白基因缺失会导致细胞有丝分裂之胞质分裂的失败以致细胞多核的现象得到国际同行关注的前提下，本课题使用经典敲除方法，规避了极具争议的CRISPR编辑脱靶的重要缺陷，获得了hem1/fbln6基因修饰突变小鼠，并以此突变小鼠获得了重要的研究结果，值得一提的是该hem1/fbln6基因修饰小鼠完全具有本土(即本实验室)知识产权。以此模式小鼠获得的研究结果、关键数据及其科学意义来看，在心脏发育组织构建成构建形成成体心脏及心脑血管系统中，确证了hem1/fibl6及钙相关调节蛋白在“动（细胞分裂）”与“静（组织结构）”组织构建动态模式，我们的研究结果,很有可能在相应领域中进入国际先进行列，结果概述如下：（1）使用具有本土知识产权的hem1/fbln6基因突变模式小鼠，在锻炼干预下发现脑出血性中风现象，并且伴随眼底视网膜出血病理现象，表明ECM hem1/fbln6基因是脑中风发生的重要遗传因素。（2）突变模式小鼠在心脏发育和结构成熟及重建中，发现了兴趣基因的功能特性，以及血管和心肌细胞特异敲除表现出二级三级AV传导阻滞病理状态；同时，对突变模式小鼠的肠系膜分级动脉血管进行结构功能的分析，包括离体血管收缩能力及应力分析。（3）发现突变模式小鼠在心脏发育和结构成熟及重建中hem1/fbln6 基因的功能特性；与此同时，在上述特异性敲除的AV传导阻滞病理状是发育构建异常所致。（4）成体hem1/fbln6基因突变模式小鼠在外加压力如锻炼和pressure-overload的施压情况下，伴随心脏生理功能的失常的Col1a1、Col3a1和Tgfb表征的选择性纤维化，NppaAPDH、Nppb和Myh7表征的心肌细胞肥大以及施压时间逻辑关系下的组织结构和分子机制的异变。（5）突变模式小鼠在脑出血性中风中基因表达谱系细胞结构组分和生理生学过程与功能相关性研究。就以上研究结果，我们正在加紧与国际顶尖学校合作，努力把这些重要实验发表于重要专业杂志上。

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