基质素2网络结构稳定性对糖尿病肾小球硬化的作用研究

Glomerulosclerosis in diabetic nephropathy is caused by the accumulation of extracellular matrix proteins in the mesangial interstitial space, resulting in fibrosis. Matrilin2 is a widely distributed extracellular matrix protein that participates in the formation of both collagen-dependent and collagen-independent filamentous macromolecular network with adaptor functions which are involved in the development and homeostasis of network of extracellular matrix. There are two alternative splicing isoforms with unique domain of matrilin2 that have distinct roles in regulating oligomerization and proteolysis, both of which have an impact on the homeostasis of the matrilin filamentous network of the extracellular matrix. Quantitative analysis matrilin2 in glomerulus of normal and diabetic nephropathy mice might find the relationship between matrilin2 and glomerular sclerosis. In this project, we will investigate the distinct roles of the two alternative splicing isoforms of matrilin2 in the synthesis of major extracellular matrix protein in vitro using mouse mesangial cell. Identifying homeostasis of the matrilin2 filamentous network may provide new clues in understanding the mechanisms of glomerulosclerosis in diabetic nephropathy and in the development of specific treatments.

糖尿病肾小球硬化的主要病理改变是系膜间隙处细胞外基质过度聚集。细胞外非胶原基质网络结构的稳态取决于非胶原蛋白的合成与降解的动态平衡。基质素与细胞外基质中的胶原蛋白、蛋白多糖或其它非胶原蛋白分子形成细胞外基质网络。基质素2独特功能域的剪切影响该蛋白多聚体形成和酶切，从而影响细胞外基质网络中的稳定性。本研究应用分子生物学和形态学技术分析基质素2在正常和糖尿病肾病小鼠肾小球中的表达情况；通过体外观察两种剪接方式的基质素2及其酶切产物对肾系膜细胞合成和降解细胞外基质的影响，基质素2与其它基质蛋白的合成及特异性降解酶抑制剂干扰降解的相互关系，旨在阐明基质素2分子结构稳定性对自身和其它细胞外基质蛋白稳态的调节作用，从而为防治提供新的思路。

糖尿病肾病（Diabetic nephropathy, DN）是糖尿病患者最常见、最严重的的微血管并发症之一。其主要临床表现为肾小球系膜细胞外基质蛋白聚集而造成的系膜细胞凋亡，纤维组织增生，最终导致肾小球硬化。基质素2（matrilin-2）是细胞外基质的重要组成成分，参与细胞的迁移和再生，是维持细胞外基质稳态的重要因子。而基质素2对糖尿病肾小球硬化的肾小球系膜细胞的影响目前尚无报道。本研究旨在探讨基质素2在保护高糖和肿瘤坏死因子（TNF-α）在诱导小鼠肾小球系膜细胞（SV40-MES13）凋亡中的作用和分子机制。通过体外培养SV40-MES13细胞，过表达或敲降matrilin-2后给予高糖和TNF-α诱导凋亡，CCK-8、Hoechst-33258染色和流式细胞术检测细胞凋亡情况，western blot和RT-qPCR检测matrilin-2和JNK信号通路的的改变情况，免疫共沉淀和质粒转染以检测其具体分子机制。结果发现，高糖和TGF-β1处理上调SV40-MES13细胞matrilin-2的表达；过表达matrilin-2可逆转TNF-α对SV40-MES13细胞的凋亡诱导作用；JNK通路抑制剂可以减轻 matrilin-2 敲低诱导的 SV40 MES 13细胞凋亡；matrilin-2通过靶向TNFR/ASK1/MKK7途径中的ASK1来促进TNFα活化的JNK。本研究阐明了matrilin-2在保护TNF-α诱导的SV40-MES13细胞凋亡中的作用及其分子机制，为matrilin-2作为新的研究靶点在防治糖尿病肾病中的应用提供了新的思路。