Decorin-ICAP-1调控Integrinβ1内化再循环在肺血管重建中的作用和机制研究

Pulmonary vascular remodeling (PVR) is the important pathophysiological foundation of hepatopulmonary syndrome (HPS) and other diseases. Our previous research have found that pulmonary artery smooth muscle cells (PASMCs) occurred cytoskeleton remodeling, which as a key upstream event before cells phenotype modulation and abnormal proliferation may play an important role in the PVR. It has been reported that the internalization and recycling of integrinβ1 could regulate the cells cytoskeleton remodeling; which was regulated by p-ICAP-1. And the phosphorylation activity of ICAP-1 was regulated by Decorin. Preliminary experiments found the up-regulating of integrinβ1 expression in HPS rat lung tissue, while HPS rat serum suppressed the protein expression level of Decorin in PASMCs. According to the results above, we hypothesised that the internalization and recycling of integrinβ1 by Decorin-ICAP-1 leads to cells cytoskeleton remodeling, which is the molecular mechanisms of PVR. This study intends to establish HPS rats models in vivo and in vitro, using WB, qPCR, Laser scanning confocal and other methods to observe PASMCs cytoskeleton remodeling; Capture-ELISA and flow cytometry techniques to detect the internalization and recycling of integrinβ1 ; using transfection of specific decorin lentivirus and siRNA to regulate the expression of decorin, by which to research the regulatory role of the decorin mediated the internalization and recycling of integrinβ1. The results of this study may provide a new target for the prevention and treatment of diseases such as HPS.

肺血管重建（PVR）是肝肺综合征（HPS）等疾病的重要病理生理基础。前期研究发现肺动脉平滑肌细胞（PASMCs）骨架重塑可能是其表型转换及增殖的上游关键事件。既往报道，整合素β1内化再循环调控细胞骨架重塑；这一过程受磷酸化ICAP-1调控，而ICAP-1磷酸化活性可能受Decorin调节。预实验发现，HPS大鼠肺微血管整合素β1表达上调；HPS血清刺激下PASMCs中Decorin表达下调。由此推测，Decorin-ICAP-1调控的整合素β1内化再循环，进而调节PASMCs骨架重塑可能是PVR的重要分子机制。课题拟建立HPS模型，采用WB、qPCR、激光共聚焦等方法，观察细胞骨架重塑；通过俘获酶联免疫蛋白示踪、流式细胞等技术，检测整合素β1内化再循环；采用慢病毒及siRNA干预Decorin表达，检测其对整合素β1内化再循环的调控效应；研究结果可能为HPS等疾病的防治提供新靶点。

肺血管重建（PVR）是肝肺综合征（HPS）等疾病的重要病理生理基础。前期研究发现肺动脉平滑肌细胞（PASMCs）骨架重塑可能是其表型转换及增殖的上游关键事件。本项目组在前期研究的基础上，发现Decorin参与磷酸化调节的整合素胞浆结构域关联蛋白-1（ICAP-1）的活性，进而在整合素β1（Integrinβ1）内化再循环调控细胞骨架重塑中发挥重要调控作用，这一分子机制具有重要研究价值。本项目建立了HPS大鼠模型和PASMCs模型，采用WB、qPCR、激光共聚焦、酶联免疫蛋白示踪、流式细胞、慢病毒及siRNA干扰等技术进行研究。研究结果：1、Talin1和ICAP-1竞争性结合Integrinβ1，调节其内化再循环；2、胞膜活化的Integrinβ1促进黏着斑复合物（FAC）的形成和激活；3、Integrinβ1的活化，进一步导致actomyosin的激活（包括：F-actin的聚合增加，Rho和MLC相关通路的活化）并最终导致PASMCs的异常迁移和增殖；4、ICAP-1受Smad泛素化相关因子1（Smurf1）泛素化调节，ICAP-1的泛素化降解导致对Integrinβ1的抑制减弱。而ICAP-1的磷酸化调节因子Decorin表达明显下调，调控Integrinβ1的活化并影响PASMCs的迁移和增殖；5、应用固相指数扩增反应（solidoid exponential amplification reaction, sEXPAR）-表面等离子共振（surface plasmon resonance, SPR）生物传感器检测技术，以及基因芯片技术分析HPS大鼠血清中miRNA的表达变化；发现miRNA-30a、miRNA-194和miRNA145-5p的表达改变可能通过调控F-actin聚合和actomyosin活化影响PASMCs的迁移和增殖。上述研究结果证实了Decorin-ICAP-1调控的整合素β1内化再循环，进而调节PASMCs骨架重塑，最终导致PASMCs的异常迁移和增殖，并发现miRNA在调节细胞骨架重塑中的作用，阐明了PVR的重要分子机制，为HPS相关PVR的防治提供新策略。