内源性二氧化硫对氯化钴诱导人肺动脉内皮细胞凋亡的影响  

Effect of endogenous sulfur dioxide on the apoptosis induced by cobalt chloride in the human pulmonary arterial endothelial cells

作　　者：刘欣[1] 张达 李坤[2] 余孝其[2] 唐朝枢 杜军保[1] 金红芳[1] 黄娅茜[1] Liu Xin;Zhang Da;Li Kun;Yu Xiaoqi;Tang Chaoshu;Du Junbao;Jin Hongfang;Huang Yaqian(Department of Pediatrics,Peking University First Hospital,Belting 100034,China（Liu X,Zhang D,Du JB,Jin HF,Huang YQ;College of Chemistry,Sichuan University,Chengdu 610064,China（Li K,Yu XQ;Department of Physiology and Pathophysiology,Peking University Health Science Center,Belling 100191,China（Tang CS)

机构地区：[1]北京大学第一医院儿科,100034 [2]四川大学化学院,成都610064 [3]北京大学医学部生理与病理生理教研室,100191

出　　处：《中华实用儿科临床杂志》2018年第13期999-1003,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（91439110）

摘　　要：目的研究内源性二氧化硫（SO2）对氯化钴（CoCl2）诱导人肺动脉内皮细胞（HPAECs）凋亡的调节作用。方法采用CoCl2诱导原代HPAECs制备低氧刺激细胞凋亡模型，采用慢病毒转染过表达内源性SO2生成关键酶天冬氨酸氨基转移酶1（AAT1），HPAECs分为4组：空载体（vehicle）组、vehicle＋CoCl2组、AAT1组及AAT1＋CoCl2组。采用Western blot法检测AAT1、B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2相关X蛋白（bax）、Caspase-3蛋白和活化的Caspase-3（cleaved Caspase-3）的表达，采用比色法检测AAT活性，采用荧光探针原位检测HPAECs中SO2水平，采用依赖于末端脱氧核苷酸转移酶的dUTP末端标记法（TUNEL）测定细胞凋亡。结果4组HPAECs中SO2含量、AAT1和bcl-2蛋白表达及cleaved Caspase-3/Caspase-3比值差异均有统计学意义（F＝147.364、23.738、6.521、64.884，均P〈0.05），但bax蛋白表达差异无统计学意义（F＝1.620，P〉0.05）。与vehicle组相比，vehicle＋CoCl2组HPAECs中AAT活性[（0.96±0.24） Carmen′s unit/μg比（2.21±0.60） Carmen′s unit/μg]、SO2水平（40.71±7.72比105.60±16.20）和bcl-2蛋白表达（0.59±0.19比1.02±0.20）均显著下降，细胞凋亡和cleaved Caspase-3/Caspase-3比值（1.56±0.25比0.95±0.13）明显增加，差异均有统计学意义（均P〈0.05）；但AAT1蛋白表达差异无统计学意义（0.50±0.12比0.53±0.11，P〉0.05）。与vehicle组相比，AAT1组HPAECs中SO2水平（351.50±42.43比105.60±16.20）和AAT1蛋白表达（1.22±0.33比0.53±0.11）均显著升高，差异均有统计学意义（均P〈0.05）。与AAT1组相比，AAT1＋CoCl2组HPAECs中SO2水平（333.50±46.22比351.50±42.43）、AAT1蛋白表达（1.26±0.36比1.22±0.33）和bcl-2蛋白表达（1.14±0.38比1.03±0.27）差异无统计学意义，细胞凋亡和cleaved Caspase-3/Caspase-3比值（0.51±0.17比0.50±0.11）差异无统计学意义（均P〉0.05）。结论内源性SO2可抑制CoCl2诱导HPAObjectiveTo explore the effect of endogenous sulfur dioxide （SO2） on the apoptosis induced by cobalt chloride （CoCl2） in the human pulmonary arterial endothelial cells （HPAECs）.MethodsCoCl2 was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1（AAT1）, and the key enzyme generating endogenous SO2 were over-expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups： vehicle group, vehicle＋ CoCl2 group, AAT1 group and AAT1＋ CoCl2 group.The expressions of AAT1, B-cell lymphoma-2 （bcl-2）, bcl-associated X protein （bax）, Caspase-3 and activated Caspase-3 （cleaved Caspase-3） in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） assay.ResultsThere were significant differences in the endogenous SO2 content, the expre-ssions of AAT1 and bcl-2, and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were （F=147.364, 23.738, 6.521, 64.884, all P〈0.05）. However, there was no difference in the expression of bax among 4 groups of HPAECs （F=1.620, P〉0.05）. Compared with vehicle group, AAT activity [（0.96±0.24） Carmen′s unit/μg vs.（2.21±0.60） Carmen′s unit/μg], endogenous SO2 content （40.71±7.72 vs.105.60±16.20） and bcl-2 expression （0.59±0.19 vs.1.02±0.20） in the HPAECs of vehicle＋ CoCl2 group were significantly decreased, while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 （1.56±0.25 vs.0.95±0.13） were significantly increased （all P〈0.05）. However, there were no differences in the expression of AAT1 （0.50±0.12 vs.0.53±0.11） in the HPAECs between vehicle group and vehicle＋ CoCl2 group （P〉0.05）. The SO2 content （351.50±42.43 vs.10

关 键 词：二氧化硫 天冬氨酸氨基转移酶1 氯化钴 人肺动脉内皮细胞 凋亡 

分 类 号：R725.4[医药卫生—儿科]