低氧高二氧化碳对肺动脉平滑肌细胞TRPC1/4表达影响及其与细胞增殖、凋亡的关系

The Expression of TRPC1/4 and Its Relationship with Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells under Hypoxic and Hypercapnic Conditions

该文旨在研究低氧高二氧化碳时C型瞬时受体电位通道1(transient receptor potential channel 1,TRPC1)和TRPC4的表达变化与肺动脉平滑肌细胞增殖和凋亡的关系。经免疫荧光鉴定后随机分为5组:常氧对照组(N)、低氧高二氧化碳组(H)、溶剂二甲基亚砜(dimethyl sulfoxide,DMSO)对照组(D)、TRPC1/4抑制剂SKF-96365组(S)和TRPC1/4激动剂环匹阿尼酸(cyclopiazonic acid,CPA)组(C)。N组置于常氧培养箱(21%O2、5%CO2、37°C)中培养24 h,其余4组放于低氧高二氧化碳培养箱(5%O2、6%CO2、37°C)中培养24 h。采用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和蛋白质印迹法(Western blot)分别检测TRPC1和TRPC4的m RNA和蛋白质水平;CCK-8法检测各组细胞增殖情况;原位末端标记法(Td T-mediated d UTP nick-end labeling,TUNEL)检测各组细胞凋亡指数(apoptotic index,AI);Fura 2-AM法检测细胞内钙离子浓度[Ca2+]i。结果发现,H组TRPC1和TRPC4的m RNA和蛋白质水平以及细胞增殖和[Ca2+]i均高于N组(P<0.05),而凋亡指数低于N组(P<0.05)。与H组相比,S组TRPC1/4的m RNA和蛋白质水平以及细胞增殖和[Ca2+]i均降低(P<0.05),细胞凋亡增加(P<0.05);C组TRPC1的m RNA和蛋白质水平以及细胞增殖和[Ca2+]i增加(P<0.05),细胞凋亡率下降(P<0.05),但C组TRPC4的m RNA和蛋白表达变化无统计学意义(P>0.05)。以上结果说明,在低氧高二氧化碳情况下,TRPC1/4的m RNA和蛋白质水平以及[Ca2+]i均增高,细胞的增殖和凋亡与细胞内TRPC1的表达变化有关。

This work was aimed to study the relationship between TRPC1/4 and pulmonary artery smooth muscle cells（PASMCs） proliferation and apoptosis under hypoxic and hypercapnic conditions. Cellular purity was assessed by immunofluorescence staining for α-SMA under fluorescence microscopy. PASMCs were divided into 5 groups randomly： normoxic group（N）, hypoxic and hypercapnic group（H）, DMSO group（D）, TRPC1/4 inhibitor SKF96365 group（S） and TRPC1/4 activator CPA group（C）. N group was incubated under normoxia（5% CO2, 21% O2, 37 °C） for 24 h, and the others were incubated under hypoxic and hypercapnic（6% CO2, 5% O2, 37 °C） atmosphere for 24 h. TRPC1/4 m RNA levels were detected by reverse transcription polymerase chain reaction（RTPCR）. TRPC1/4 protein levels were detected by Western blot. The proliferation assay of PASMCs was performed by CCK-8 kit. The apoptosis of PASMCs was detected using the terminal deoxyribonucleotide transferase-mediated d UTP nick end-labeling（TUNEL） assay. [Ca2＋]i was measured in PASMCs using fura 2-AM fluorescence. The results showed that the expression of TRPC1/4 m RNA and proteins and [Ca2＋]i were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted PASMCs proliferation and inhibited apoptosis. TRPC1/4 inhibitor SKF96365 reversed the effect of hypoxia and hypercapnia. CPA increased TRPC1 m RNA and protein levels, but neither TRPC4. The levels of TRPC1/4 m RNA and proteins and [Ca2＋]i were upregulated under hypoxic and hypercapnic conditions. TRPC1 has a relationship with PASMCs proliferation and apoptosis.