miRNA-138-5p过表达对香烟烟雾暴露所致大鼠睾丸支持细胞损伤的调控作用  

Regulatory effect of over-expression of miRNA-138-5p on testicular sertoli cell injury of rats induced by cigarette smoking exposure

作　　者：仲春雪 徐华[2] 张晨 何丽娟 ZHONG Chunxue;XU Hua;ZHANG Chen;HE Lijuan(Teaching and Scientific Research Office and Central Laboratory,Fifth Affiliated Hospital,Xinjiang Medical University,Urumqi 830054,China;Center of Reproduction,First Affiliated Hospital,Xinjiang Medical University,Urumqi 830054,China;Department of Social Medicine,College of Public Health,Xinjiang Medical University,Urumqi 830054,China;Post Doctoral Mobile Station,School of Pharmacy,Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第五附属医院教学科研办公室和中心实验室,新疆乌鲁木齐830054 [2]新疆医科大学第一附属医院生殖助孕中心,新疆乌鲁木齐830054 [3]新疆医科大学公共卫生学院社会医学教研室,新疆乌鲁木齐830054 [4]新疆医科大学药学院博士后流动站,新疆乌鲁木齐830054

出　　处：《吉林大学学报（医学版）》2022年第3期692-701,共10页Journal of Jilin University：Medicine Edition

基　　金：新疆维吾尔自治区科技厅特殊环境与健康研究重点实验室开放课题资助项目(SKL-SEHR-2021-05)。

摘　　要：目的:探讨香烟烟雾暴露对大鼠睾丸支持细胞的损伤作用,阐明微小RNA-138-5p(miR-138-5p)在此过程中发挥的保护作用。方法:200只SPF级雄性SD大鼠随机分为对照组和低、中及高剂量香烟暴露组(每天给予每只大鼠10、20和30支香烟),分别于2、4、6、8和12周麻醉处死动物,检测各组大鼠血浆中抑制素B水平,实时荧光定量PCR(RT-qPCR)法检测各组大鼠睾丸组织中miR-138-5p表达水平,免疫组织化学法检测各组大鼠睾丸支持细胞中波形蛋白表达水平。体外培养睾丸TM4细胞(对照组),将制备好的香烟烟雾提取物(CSE)原液稀释至5%和10%,并处理TM4细胞,作为5%CSE组和10%CSE组。采用MTT法检测各组细胞存活率和各组细胞中乳酸脱氢酶(LDH)活性,流式细胞术和TUNEL法检测各组细胞凋亡率。睾丸TM4细胞转染过表达miR-138-5p质粒后,分为对照组、5%CSE组、5%CSE+Pri-miRNA-138-5p Ctrl组、5%CSE+Pri-miRNA-138-5p组、10%CSE组、10%CSE+Pri-miRNA-138-5p Ctrl组和10%CSE+Pri-miRNA-138-5p组,采用上述方法检测各组细胞生存率、细胞中LDH活性和细胞凋亡率。结果:与对照组比较,各剂量香烟暴露组大鼠血浆中抑制素B水平降低;与对照组比较,第8周高剂量香烟暴露组和第12周各剂量香烟暴露组大鼠血浆中抑制素B水平降低(P<0.05)。光镜下观察,对照组大鼠睾丸细胞胞质出现阳性黄染,香烟暴露组大鼠睾丸TM4细胞中睾丸组织波形蛋白表达水平逐渐降低;与对照组比较,不同时间高剂量香烟暴露组大鼠睾丸TM4细胞中睾丸波形蛋白表达水平降低(P<0.05)。与对照组比较,第8周时中和高剂量香烟暴露组及12周时各剂量香烟暴露组大鼠睾丸组织中miR-138-5p表达水平均降低(P<0.05)。与对照组比较,5%CSE组和10%CSE组睾丸TM4细胞存活率明显降低(P<0.01)。与对照组比较,5%CSE组和10%CSE组大鼠睾丸TM4细胞中LDH活性降低(P<0.05或P<0.01)。过表达miR-138-5p后,与对照组比较,5%CSE组、5%Objective:To investigate the damage effect of cigarette smoking exposure on the testicular sertoli cells of the rats,and to clarify the protective effect of miR-138-5p in this process.Methods:A total of 200 SPF male SD rats were randomly divided into control group and low,middle,and high doses of cigarette exposure groups(given 10,20,and 30 cigarettes per rat per day).The rats were anesthetized at 2rd,4th,6th,8th,and 12th weeks,respectively.The plasma inhibin B levels of the rats in various groups were detected,the expression levels of miR-138-5p in testicular tissue of the rats in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,and the expression levels of vimentin in testicular TM4 cells of the rats in various groups were detected by immunohistochemistry.The testicular TM4 cells(control group)were cultured in vitro.The cigarette smoke extract(CSE)stock solution was diluted to 5%and 10%and to treat the TM4 cells,and used as 5%CSE group and 10%CSE group.The survival rates of the testicular TM4 cells and lactate dehydrogenase(LDH)activities in the testicular TM4 cells in various groups were detected by MTT method,and the apoptotic rates of the cells in various groups were detected by flow cytometry and TUNEL method.After transfected with miR-138-5p plasmid,the testicular TM4 cells were divided into control group,5%CSE group,5%CSE+Pri-mirna-138-5p Ctrl group,5%CSE+Pri-mirna-138-5p group,10%CSE group,10%CSE+Pri-mirna-138-5p Ctrl group,and 10%CSE+Pri-mirna-138-5p group.The survival rates of the cells,LDH activities in the cells and apoptotic rates of the cells in various groups were detected by the above methods.Results:Compared with control group,the plasma inhibin B levels of the rats in different doses of cigarette exposure groups were decreased;compared with control group,the plasma inhibin levels of the rats in high dose of cigarette exposure group at the 8 weeks and in middle and high doses of cigarette expasure groups at the 12 th week were decreased(P<0.05).The light mic

关 键 词：香烟烟雾暴露 睾丸 支持细胞 细胞损伤 微小RNA138-5p 

分 类 号：R34[医药卫生—基础医学]