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经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P<0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P<0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P<0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。
The classical transient receptor potential channel 6 (TRPC6) protein is the molecular basis of the receptor-manipulating Ca2 + channel (ROCC). This article aims to investigate the role of TRPC6 / ROCC in monocrotaline (MCT) -induced pulmonary hypertension in rats. Sprague-Dawley rats were randomly divided into normal control group (CON group) and MCT group. CON group was maintained for three weeks, while MCT group was administered intraperitoneally with 2% MCT at a dose of 60 mg / kg. MCT-induced chronic pulmonary hypertension Mouse model. The right ventricular systolic pressure (RVSP) and right ventricular mass index (RVMI) were measured, and the morphology of pulmonary arteries was observed by HE staining. The pulmonary artery reconstruction was analyzed. Semi-quantitative RT-PCR and Western blot were used to detect TRPC6 mRNA and protein expression in rat pulmonary arteries. In the vascular tension experiment, the contractile effect of isolated rat pulmonary rings was detected by ROCC and transmembrane DAG mimics 1-oleoyl-2-acetyl-sn-glycerol (OAG). Fluorescence probe Fluo3-AM was used to measure the cytoplasmic free Ca2 + concentration ([Ca2 +] i) induced by OAG in rat pulmonary arterial smooth muscle cells (PASMCs). The results showed that RVSP and RVMI in MCT group were significantly increased compared with CON group (P <0.01). Morphological observation showed that the smooth muscle layer of pulmonary arteriole was thickened and the lumen was reduced. The mRNA and protein expression of TRPC6 was not significant Variety. In the CON group, OAG almost did not cause contraction of the pulmonary artery rings, while in the MCT group, the pulmonary artery ring contraction response was significantly enhanced, the difference was significant (P <0.01). MCT also significantly increased the [Ca2 +] i increase in OAG-triggered PASMCs compared to the CON group (P <0.05). These results suggest that MCT pretreatment did not significantly enhance the expression of TRPC6mRNA and protein in pulmonary artery, but could promote the influx of Ca2 + and pulmonary arterial pressure in PPCMCs induced by TRPC6 / ROCC, induce pulmonary hypertension in rats and further induce pulmonary vascular And right ventricular remodeling.