摘要
目的探讨跨膜蛋白16A（TMEMl6A）对原代培养大鼠门静脉平滑肌细胞增殖的作用及其机制。方法原代培养大鼠门静脉平滑肌细胞，慢病毒转染上调其TMEMl6A表达，分别加T16Ainh-A01和U0126后用Westernblot法检测蛋白表达变化，流式细胞术观察细胞周期变化，细胞计数试剂盒（CCK-8）观察细胞增殖。结果成功培养出大鼠门静脉平滑肌细胞并转染了TMEMl6A，转染效率约为80％，流式细胞术结果显示过表达TMEMl6A组s期细胞比例为（34．44±3．72）％，高于对照组的（15．56±2．36）％，差异有统计学意义（t=7．423，P〈0．01）。Westernblot显示各组细胞外信号调节激酶1／2（ERKl／2）表达水平差异无统计学意义（t=2．338，P〉0．05）。各组磷酸化ERKl／2（p-ERKl／2）表达差异均有统计学意义（P〈0．05）。结论TMEMl6A可能通过影响丝裂原活化蛋白激酶（MAPK）-ERKl／2通路活性来调控门静脉平滑肌细胞增殖，其表达在原代培养的大鼠门静脉平滑肌细胞增殖中起重要作用。
Objective To investigate the effects of transmembrane protein 16A （TMEM16A） on proliferation of primary cultured smooth muscle cells of rat portal vein （ rPVSMCs ） and the mechanism. Methods The primary rPVSMCs were cultured, and transfected with lentivirus to investigate the effect of TMEM16A on proliferation of rPVSMCs. T16Ainh - A01 and U0126 were used in both control group and lentivirns transfection group, respectively. Western blotting analysis was used to detect the expression of TMEM16A, phosphorylated extracellular signal - regulated kinase 1/2 （ p - ERK1/2 ） and extracellular signal- regulated kinase 1/2 （ERK1/2） in rPVSMCs. Flow cytometry and cell counting kit- 8 （CCK -8） were used to measure the cell cycle and proliferation of rPVSMCs. Results We successfully cultured the rPVSMCs and up - regulated the expression of TMEM16A. Flow cytometry showed that the cell ratio of S phase [ （ 34. 44 ± 3.72 ） % ] in the over - expressed TMEM16A group was significantly higher than in the control group [ （ 15.56 ± 2. 36） %, P 〈 0. 05 ]. Western blotting showed that ERK1/2 was ex- pressioned stablely in 7 groups （ P 〉 0. 05 ）. There were significant differences in p - ERK1/2 among groups. Conclusion TMEM16A may regulate the proliferation of rPVSMCs by influencing the activity of mitogen- activated protein kinase （MAPK） -ERK1/2 signal pathway, and its expression may play an im- portant role in the proliferation of orimary cultured rPVSMCs.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第10期2423-2426,共4页
Chinese Journal of Experimental Surgery
关键词
跨膜蛋白16A
门脉高压症
血管重构
丝裂原活化蛋白激酶-细胞外信号调
节激酶1/2通路
Transmembrane 16A
Portal hypertension
Vascular remodeling
Mitogen - ac-tivated protein kinase - extracellular signal - regulated kinase 1/2 signal pathway
