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、24h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Excd 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12h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24h组（11208±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8h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24h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24h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。
Objective 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 Aktl 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 of 3 H-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 Aktl 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/Aktl signaling pathway might play an important role in hypoxic pulmonary hypertension.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2008年第8期586-590,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家自然科学基金资助项目（30400200,30700347）
重庆市自然科学基金资助项目（2007BB5045）
关键词
肌细胞
平滑肌
蛋白质丝氨酸苏氨酸激酶
高血压
肺性
Myocytes, smooth muscle
Protein-serine-threonine kinases
Hypertension, pulmonary
