醋酸铅对PC12细胞的增殖与凋亡研究  

Research on proliferation and apoptosis of PC12 cells induced by lead acetate

作　　者：张谊 欧玉龙[1] 王惠霞 陈月芳 李永金[2] ZHANG Yi;OU Yu-long;WANG Hui-xia;CHEN Yue-fang;LI Yong-jin(Affiliated Danyang Hospital of Nantong University,Danyang 212300,Jiangsu Province,China;Department of Pharmacology,Jiangsu University School of Medicine,Zhenjiang 212013,Zhenjiang Province,China)

机构地区：[1]南通大学附属丹阳医院,江苏丹阳212300 [2]江苏大学医学院药理学教研室,江苏镇江212013

出　　处：《中国临床药理学杂志》2021年第10期1214-1217,共4页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(31700444);丹阳市社会发展科技支撑计划基金资助项目(SF201804)。

摘　　要：目的探讨醋酸铅(Pb(Ac)2)对大鼠肾上腺嗜铬细胞瘤PC12细胞的增殖与凋亡中低氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)与Rho关联卷曲螺旋蛋白激酶(rho associated coiled coil forming protein kinase, ROCK)信号通路的关系以及潜在的作用机制。方法 (1)将体外培养的PC12细胞分为对照组和实验组。对照组加入含血清的培养基,低、中、高剂量实验组加入100,200,400μmol·L-1的Pb(Ac)2染毒,用噻唑蓝还原法测定药物对细胞增殖率的影响,以乳酸脱氢酶(lactate dehydrogenase, LDH)漏出率试剂盒检测细胞损伤程度,用DCFH-DA探针染色法检测细胞内活性氧(reactive oxygen species, ROS)水平,用Annexin V-FITC/PI细胞凋亡检测试剂盒检测细胞凋亡率,以蛋白质印迹法检测细胞内HIF-1α、 ROCK-1、ROCK-2、淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)以及Bcl-2相关X蛋白(Bcl-2 Associated X Protein, Bax)的蛋白表达。(2)采用siRNA技术沉默PC12细胞HIF-1α基因技术,将细胞分为对照组、实验组、阴性对照组、干扰组,观察细胞中HIF-1α、ROCK-1,ROCK-2蛋白表达水平的变化。结果随着Pb(Ac)2剂量的增大,药物对PC12细胞的损伤作用加深。对照组和高剂量实验组的存活率分别为(100.00±3.22)%,(47.21±4.98)%,差异有统计学意义(P<0.01)。与对照组相比,中、高2个剂量实验组细胞凋亡率差异有统计学意义(P<0.01),表明Pb(Ac)2诱导细胞凋亡。Pb(Ac)2可上调细胞内HIF-1α、ROCK-1、ROCK-2、Bax/Bcl-2的蛋白表达比例(均P<0.01)。当采用小RNA干扰HIF-1α后,发现醋酸铅对PC12细胞损伤的程度降低,HIF-1α、ROCK-1、ROCK-2的蛋白表达水平下降(均P<0.01)。结论 Pb(Ac)2诱导PC12细胞发生凋亡可能与HIF-1α和ROCK信号通路的表达以及自由基损伤有关。Objective To investigate the relationship between hypoxia inducible factor-1α(HIF-1α) and rhoassociated coiled coil protein kinase(ROCK) signaling pathway on proliferation and apoptosis of PC12 cells induced by lead acetate. Methods The PC12 cells cultured in vitro were divided into control group and experimental group. Control group was added with serum-containing medium, low, medium and high dose experimental groups were given 100, 200, and 400 μmol·L-1Pb(Ac)2. The cell viability was determined by MTT reduction assay and lactate dehydrogenase(LDH) assay. The intracellular levels of oxygen species was measured by assessing. Annexin-V/PI double staining to detect cell apoptosis rate, the expressions of HIF-1α, ROCK-1 ROCK-2,Bcl-2 and Bax were determined by Western blot analysis. Silence siR NA technology was used to detected the HIF-1α gene of PC12 cells,the cells are divided into control group,experimental group,negative control group,and interference group,and observing the changes of HIF-1α,ROCK-1,ROCK-2 protein expression in the cells.Results Lead acetate induced cell injury in PC12 cells in a dose-dependent manner. The survival rates of the cells in control group and high dose experimental group were( 100. 00 ± 3. 22) %,( 47. 21 ± 4. 98) %,with significant difference( P < 0. 01). Compared with control group,the apoptosis rate of middle and high dose experimental groups were with significant difference( P < 0. 01),indicating that Pb( Ac)2 induced apoptosis. Pb( Ac) 2 could up-regulate the protein expression ratio of HIF-1α,ROCK-1,ROCK-2 and Bax/Bcl-2 in cells( all P < 0. 01). When HIF-1α was interfered with by small RNA,the damage of PC12 cells was reduced by lead acetate,and the protein expression levels of HIF-1α,ROCK-1 and ROCK-2 were decreased( all P < 0. 01). Conclusion Lead acetate induced PC12 cell apoptosis,which may be related with the expressions of of HIF-1α and ROCK signal path. And cellular oxidative stress may contribute to the injury as well.

关 键 词：醋酸铅 PC12细胞 细胞凋亡 缺氧诱导因子-1Α ROCK信号通路 

分 类 号：R97[医药卫生—药品]