miR-9调控VSMC表型转化在脑血管支架植入术后再狭窄中的作用及机制研究

Restenosis after cerebrovascular stent implantation is one of the most important and difficult problem in prevention and treatment of cerebrovacular disease, and the key is phenotype switch of vascular smooth muscle cell ( VSMC ). Our previous study found that the phenotype switch of VSMC is closely related to TGFβ and miR-9 on the cerebrovascular restenosis of patient and rat, but the definite mechanism is unknown and is to be disclosed. According to the latest research and the foundation, We suppose that TGFβ can upregulate miR9, which regulate VSMC phenotype switch by targeting protein Myocd, and then trigger cerebrovascular restenosis after stent implantation. Using the method of luciferase system, this project aims that TGFβ and Myocd are respectively the upstream regulatory gene and the downstream target gene of miR-9. By means of qPCR ,Western blot and siRNA technology, We observe that whether TGFβ /SMAD signaling pathway upregulate miR9, inhibit the expression of Myocd, and promote the differentiation of VSMC phenotype. The purpose is to elucidate the specific mechanism of VSMC phenotype switch after the cerebrovascular stent implantation and the role of regulatory of TGFβ and miR-9, which can provide a new way to inhibit and prevent the clinical restenosis after cerebrovascular stent implantation.

脑血管支架植入术后再狭窄是脑血管病防治中的重点和难点。血管平滑肌细胞（VSMC）表型转化是防治的关键环节。我们前期在脑动脉再狭窄病人及大鼠模型中研究发现TGFβ和miR-9均增高且呈正相关，进一步体外预实验发现miR-9能下调Myocd基因并能促进VSMC表型转化，但具体机制尚不清楚。基于前期研究和文献报道，我们提出假设：TGFβ上调miR-9，后者通过靶蛋白Myocd调控VSMC表型转化，引发脑血管支架植入术后再狭窄。本项目应用荧光素酶报告基因、qPCR、WB和siRNA等技术，在细胞水平与动物模型明确TGFβ和Myocd分别是miR-9的上游调控基因和下游靶基因；观察TGFβ/SMAD信号通路是否可通过上调miRNA-9，抑制Myocd表达，从而促进VSMC表型转化。该研究旨在阐明脑血管支架植入术后miR-9促进VSMC表型转化的功能和分子机制，为支架植入术后再狭窄的防治提供理论依据。

脑血管支架植入术后再狭窄是脑血管疾病防治中的重点和难点之一，其中血管平滑肌细胞（VSMC）的表型转化是其关键环节。我们前期在脑动脉再狭窄病人及大鼠模型中研究发现TGFβ、miR-9均增高且有相关性，进一步的体外实验发现miR-9能下调Myocd并能引起VSMC的表型转化，但具体机制亟待进一步明确。本项目通过球囊损伤模型和TGFβ干预体外培养VSMC，采用免疫组化、流式细胞技术、迁移增殖实验、WB、PCR、EMSA等相关技术手段，证实在球囊损伤后，平滑肌细胞发生表型由合成型转化为分泌型，导致再狭窄发生。而提高miR-9水平，可以下调Myocd，促进PCNA、C-JUN阳性平滑肌细胞增多，ACTA2、SmMHC2阴性平滑肌细胞减少，从而抑制损伤后平滑肌细胞转化为分泌型，促进其转化为合成型，抑制再狭窄发生。本项目为脑动脉支架植入术后再狭窄的防治提供坚实的实验基础和理论数据。