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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。
The purpose of this study was to investigate the relationship between the expression changes of TRPC1 and TRPC4 and the proliferation and apoptosis of pulmonary artery smooth muscle cells in hypoxia and hypercapnia. After immunofluorescence, the cells were randomly divided into 5 groups: normoxic control group (N), hypoxic hypercapnia group (H), dimethylsulfoxide (DMSO) control group (D), TRPC1 / 4 inhibition The group SKF-96365 (S) and the TRPC1 / 4 agonist cyclopiazonic acid (CPA) group (C). The rats in group N were cultured in normoxic incubator (21% O2, 5% CO2, 37 ° C) for 24 h, and the other 4 groups were placed in hypoxic high CO2 incubator (5% O2, 6% CO2, 37 ° C) In training 24 h. The mRNA and protein levels of TRPC1 and TRPC4 were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. Cell proliferation was detected by CCK-8 assay. The apoptotic index (AI) of each group was detected by TdT-mediated dUTP nick-end labeling (TUNEL). The intracellular calcium concentration [Ca2 +] i was detected by Fura 2-AM assay. The results showed that m RNA and protein levels and cell proliferation and [Ca2 +] i of TRPC1 and TRPC4 in group H were significantly higher than those in group N (P <0.05), while apoptotic index was lower in group H than that in group N (P <0.05). The mRNA and protein levels of TRPC1 / 4 and the cell proliferation and [Ca2 +] i in group S were significantly lower than those in group H (P <0.05) (P <0.05). However, there was no significant difference in the mRNA and protein expression of TRPC4 in group C (P> 0.05). The above results show that in the condition of hypoxia and hypercapnia, the mRNA and protein levels of TRPC1 / 4 and [Ca2 +] i are increased, and the proliferation and apoptosis of TRPC1 / 4 are related to the changes of intracellular TRPC1 expression.