【作者】 刘伟华；

【导师】 柳志红；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 背景肺动脉高压病因尚未完全明确,病理生理学改变复杂,治疗棘手。近年研究结果表明,他汀类药物对于肺动脉高压有改善作用,但是其具体机制尚未完全明确。小G蛋白是他汀类药物重要作用靶点。本研究旨在讨论辛伐他汀是否作用于小G蛋白家族成员而改善肺动脉高压。方法动物实验采用野百合碱腹腔注射构建大鼠肺动脉高压模型,并应用辛伐他汀进行干预。通过免疫组织化学法和Western blot测定小G蛋白家族成员蛋白表达位置及其水平变化,并确定血管内皮细胞凋亡及增殖改变,PCR测定小G蛋白家族成员的mRNA变化,从而确定辛伐他汀是否作用于小G蛋白而改善肺动脉高压。体外实验采用吡咯野百合碱刺激人肺动脉内皮细胞构建肺动脉高压模型,并应用辛伐他汀进行干预,同时应用甲羟戊酸、焦磷酸法呢酯和焦磷酸牛龙牛儿基牛龙牛儿酯进行刺激。观察细胞凋亡增生变化,并通过Western blot测定小G蛋白家族成员蛋白水平变化,PCR测定小G蛋白家族成员的mRNA变化,从而确定辛伐他汀是否作用于小G蛋白而改善肺动脉内皮细胞功能,并确定辛伐他汀作用的具体信号途径。结果动物实验结果表明,野百合碱组大鼠肺动脉压力明显增高,血管重构明显,凋亡增殖标志物caspase-3和PCNA蛋白表达及mRNA水平均明显升高,小G蛋白家族成员RhoA/ROCK1, Rab1, Rac1的蛋白表达明显增高,而且集中表达于血管壁,上述小G蛋白家族成员的mRNA水平增高。辛伐他汀干预后,大鼠肺动脉压力明显下降,血管重构改善,caspase-3、PCNA及小G蛋白家族成员的蛋白表达及mRNA水平也明显降低。体外研究表明,吡咯野百合碱刺激后,肺动脉内皮细胞增殖明显,凋亡增殖标志物caspase-3口PCNA的蛋白表达和mRNA水平均明显升高,小G蛋白家族成员RhoA/ROCK1, Rab1, Rac1的蛋白表达和nRNA水平增高。在吡咯野百合碱+辛伐他汀组,小G蛋白家族成员蛋白表达和mRNA水平均较吡咯野百合碱组下降,但同时给予甲羟戊酸、焦磷酸法呢酯和焦磷酸牛龙牛儿基牛龙牛儿酯后,观察到RhoA蛋白及mRNA升高,ROCK1的mRNA水平升高,Rabl蛋白及mRNA升高,Rac1蛋白表达未见改变,但其mRNA升高。结论动物实验结果表明辛伐他汀可以明显改善大鼠肺动脉内皮细胞增生及凋亡,延缓肺血管重构,有效改善肺动脉高压的进展。辛伐他汀通过抑制小G蛋白家族成员RhoA/ROCK1,Rabl,Rac1的表达而发挥作用。体外研究结果表明,辛伐他汀改善人肺动脉内皮细胞的增生及凋亡,并可有效抑制小G蛋白家族成员的基因激活及蛋白表达。辛伐他汀主要通过抑制甲羟戊酸、焦磷酸法呢酯和焦磷酸牛龙牛儿基牛龙牛儿酯改善RhoA/ROCK1基因及蛋白过度表达,并抑制和Rab1蛋白合成,但辛伐他汀对于Rac1的抑制作用途径未能明确。更多还原

【Abstract】 Background The etiology and pathophysiology of pulmonary arterial hypertension are not fully understood. Recent researches have proved that the statins could improve pulmonary arterial hypertension. But the mechanisms of statins in pulmonary arterial hypertension are not fully clear. Small G proteins are important pharmacological targets of statins. The research aimed to whether simvastatin improved pulmonary arterial hypertension by regulating small G proteins.Methods We constructed the rat model of pulmonary arterial hypertension by intraperitoneal injection of monocrotaline, and gave simvastation to therapy. We determined the small G protein family members’expression by immunohistochemistry and Western blot, the mRNA level of small G proteins by PCR, and determined the apoptosis and proliferation of vascular endothelial cells.The cellular model of pulmonary arterial hypertension was constructed with human pulmonary arterial endothelial cells treated with monocrotaline pyrrole. And we separately treated with simvastatin, mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Then we observed the apoptosis and proliferation of vascular endothelial cells, determined the proteins and mRNA levels of small G proteins by Western blot and PCR.Results Animal experiment showed that the pulmonary arterial hypertension rats had increased mean pulmonary arterial pressure, obvious vascular remodeling, and increased protein and mRNA levels of caspase-3, proliferating cell nuclear antigen and small G proteins family members. Administration of simvastatin could improve these changes.In vitro, the monocrotaline pyrrole group had an obvious proliferation of vascular endothelial cells, and increased protein and mRNA levels of caspase-3, proliferating cell nuclear antigen and small G proteins family members. Administration of simvastatin could improve these changes. After treated with mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, we observed increased mRNA levels of RhoA, ROCK1, Rab1and Rac1, and increased protein levels of RhoA and Rab1.Conclusions Simvastatin could improve the apoptosis and proliferation of vascular endothelial cells, vascular remodeling and the development of pulmonary arterial hypertension. Inhibition of RhoA/ROCK1, Rab1and Rac1played great roles in these effects of simvastatin. Simvastatin decreased the RhoA/ROCKloverexpression, the protein synthesis of Rabl by inhibition of mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate.更多还原