AT2R-AngII轴介导的MSCs靶向归巢对血管损伤后新生内膜形成的影响与机制研究

Restenosis has become a common complication that severely influence the prognosis of vascular injury disease，there is no better solution. Our previous study found that: neointimal hyperplasia following balloon-induced injury can be effectively suppressed by transplant the AT2 gene modified bone marrow mesenchymal stem cells (MSCs) into the arterial injury localization, but the precisely mechanism of the MSCs targeted homing mediated by AT2R gene and the inhibition of neointimal hyperplasia is not clear.According to our previous study，we suspect that MSCs targeting homing can be enhanced by AngII through AT2R pathway. The role of AT1R activation in neointimal formation can be antagonisted by AT2R activation. In this study, the vitro co-culture model of vascular smooth muscle cells(VSMCs)，endothelial cells(ECs) and MSCs will be constructed at first. Quantitative PCR and western blot approach will be used to detect the change of cytokine secretion and biological behaviour which mediated by transfected AT2R gene in MSCs.The molecular mechanism of arterial injury recovery and the inhibition of neointimal formation which conducted by AT2R gene modified MSCs is to be discussed. Then, rat carotid injury model will be constructed . Tail vein will be used to injecting AT2R gene modified MSCs.Y chromosome in situ hybridization ,luciferase reporter gene assay and pathological detection are used to detect the aggregation of MSCs into arterial injury localization and their influence on neointimal formation. We hope to further clarify the role of AT2R and the molecule mechanism of the MSCs targeted homing mediated by AT2R gene in vascular injury repair. We believe the finding will supply experimental evidence to the prevention and cure of restenosis after vasular injury which mediated by stem cell.

再狭窄是严重制约血管损伤性疾病预后的瓶颈。我们前期研究显示转染AT2R的骨髓间充质干细胞(MSCs)可以抑制新生内膜形成，但AT2R对MSCs向血管损伤区的靶向归巢及其抑制新生内膜增生的确切机制尚不清楚。根据前期研究，我们推测AngII可以通过AT2R增强MSCs的靶向归巢，同时AT2R拮抗AT1R介导的新生内膜形成。本研究拟建立血管平滑肌/内皮细胞与MSCs共培养模型，通过定量PCR结合WB等方法观察AT2R在MSCs改变平滑肌/内皮细胞增殖能力及细胞因子分泌谱变化中的影响，明确AT2R修饰MSCs进而抑制平滑肌细胞迁移增殖的作用及分子机制。建立大鼠颈动脉损伤模型，通过尾静脉输注AT2R修饰MSCs,采用Y染色体探针、荧光素酶报告基因与病理检测等技术检测MSCs向动脉损伤区的聚集及对新生内膜形成的影响，阐明AT2R在MSCs修复血管损伤中的作用及分子机制,为干细胞防治再狭窄提供实验依据。

再狭窄是严重制约血管损伤性疾病预后的瓶颈。我们前期研究显示移植转染AT2R的骨髓间充质干细胞(BMSC)到损伤血管局部可以抑制新生内膜形成，本研究在前述研究基础上观察过表达AT2R的间充质干细胞外泌体对血管损伤修复的影响。实验首先采用缺氧模型在体外观察过表达AT2R的间充质干细胞外泌体对血管内皮细胞及平滑肌增殖、迁移及炎性因子产生的影响，结果显示过表达AT2R的间充质干细胞外泌体可以促进损伤的血管内皮细胞结构和功能修复，同时抑制损伤的血管平滑肌细胞由收缩型向合成型转变，抑制平滑肌细胞迁移、促进其凋亡增加；同时抑制损伤局部炎性因子的释放。上述研究结果表明，转染有AT2R基因的BMSC外泌体能对抗缺氧所致内皮细胞及平滑肌细胞损伤，促进其功能恢复。实验进一步采用球囊制作大鼠颈动脉损伤动物模型，观察过表达AT2R的间充质干细胞外泌体对在体血管损伤的治疗效应，结果显示含有荧光素酶-CD63的BMSC相关外泌体向血管损伤部位聚集，过表达AT2R基因的BMSC外泌体经尾静脉注射组中TNF-α、IL-1β、CTGF、TGF-β1蛋白的表达较对照组明显受到抑制，同时该组球囊损伤后大鼠颈动脉新生内膜增生得到有效抑制。研究结果证实：外源性过表达AT2R的间充质干细胞外泌体能定向向损伤部位募集归巢并发挥显著的抑炎效应、促进损伤血管修复，抑制新生内膜过度增生。本研究为治疗血管损伤后再狭窄提供了全新的临床思路。