摘要目的 通过观察低氧致大鼠肺动脉平滑肌细胞(PASMC)与细胞张力蛋白同源在10号染色体有缺失的磷酸酶(PTEN)和丝氨酸/苏氨酸蛋白激酶1(Akt1)mRNA及其蛋白表达水平的变化与低氧PASMC增殖的关系,探讨PTEN/Akt1信号途径在低氧肺血管重建中的可能调控作用.方法 用组织块法培养PASMC.采用半定量逆转录-PCR技术检测常氧组、低氧2、8、12、24 h组PTEN、Akt1基因mRNA的表达水平,采用Western blot技术检测相应的蛋白表达水平.采用噻唑蓝比色法和氚-胸腺嘧啶脱氧核苷(3H-TdR)掺入法检测PASMC的增殖改变.数据用x±s表示,采用Excel 2003软件进行t检验,P<0.05为差异有统计学意义.结果低氧刺激PASMC不断增殖,3H-TdR法检测的吸光度值低氧12 h组(0.70±0.10)比常氧组(0.37±0.06)明显增高(t=14.29,P<0.01);噻唑蓝法检测的吸光度值低氧24 h组(11 208±679)比常氧组(8374±545)明显增高(t=19.56,P<0.01).各组均检测出PTEN、Akt1基因mRNA及蛋白表达的变化.常氧组Akt1的mRNA、总蛋白、磷酸化蛋白的吸光度值分别为0.76±0.09、25±6和48±8,随着低氧培养时间延长,吸光度值逐渐升高,低氧8 h组达到高峰,分别为1.05±0.09、41±7和79±14,与常氧组比较,差异均有统计学意义(t值为8.31～168.00,P<0.05和P<0.01),随后开始下降,低氧24 h组恢复至常氧组水平.常氧组PTEN的mRNA、磷酸化蛋白的吸光度值分别为0.25±0.06和98±8,并随着低氧培养时间延长而逐渐升高,低氧24 h组达到高峰,分别为0.38±0.05和232±12,与常氧组比较,差异均有统计学意义(t值分别为22.04和50.46,均P<0.01).结论 PTEN/Akt1的转录和激活与低氧肺血管重建的PASMC增殖密切相关.

abstractsObjective To observe the changes of PTEN/Akt1 mRNA and protein expression level in pulmonary arterial smooth muscle cells (PASMC) induced by hypoxia in rats, and to investigate the role of PTEN/Akt1 signaling pathway in hypoxic pulmonary hypertension (HPH). Methods Pure PASMCs were grown and cultured from rat pulmonary arterial tissues. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) were used to examine PTEN and Akt1 mRNA expression after PASMCs were exposed to hypoxia for 2 h, 8 h, 12 h and 24 h respectively. PTEN/Akt1 protein expressions were determined by Western blotting. The changes of PASMC proliferation were determined by MTT and 3H-TdR incorporation. Results were expressed as x±s. Statistical comparisons of results were performed by t test of Excel 2003. Differences were considered significant if P < 0. 05. Results PASMCs were induced to keep proliferating by hypoxia. The value of3H-TdR was 0. 37±0. 06 under normoxia, but was increased with prolonged exposure to hypoxia, reaching 0.70±0. 10 at 12 h (t = 14.29, P < 0.01), being significantly different compared to the normoxia group. The value of MTT was 8374±545 under normoxia,but was increased by exposure to hypoxia, reaching 11 208±679 at 24 h (t = 19. 56, P < 0. 01), being significantly different compared to the normoxia group. The mRNA and protein of PTEN/Akt1 were detectable in all the groups. The values of mRNA, total protein and phosphorylated protein of Akt1 were 0. 76±0. 09, 25±6, 48±8 respectively in the normoxia group. After exposure to hypoxia, the values were 1.05±0.09, 41±7, 79±14 respectively at 8 h, being significantly different compared to the normoxia group (t = 168.00,58. 54,8.31, P <0. 01 and P <0. 05), but the values decreased thereafter and returned to the level comparable to the normoxia group at 24 h. The values of mRNA, phosphorylated protein of PTEN were 0. 25±0. 06, and 98±8 respectively in the normoxia group, but were increased after exposure to hypoxia, reaching 0. 38±0. 05, and 232±12 respectively at 24 h, being significantly different compared to the normoxia group (t =22. 04, 50. 46, all P <0. 01). Conclusion The results suggested that PTEN/Akt1 signaling pathway might play an important role in hypoxic pulmonary hypertension.