树鼩睾丸间质细胞的分离培养及寨卡病毒感染特性的研究  

Isolation and culture of Leydig cells from tree shrews and studies on the characteristics of Zika virus infection

作　　者：邓伟 金亮子 奎秀莹 王文广[1] 李娜[1] 仝品芬[1] 代解杰[1] DENG Wei;JIN Liangzi;KUI Xiuying;WANG Wenguang;LI Na;TONG Pinfen;DAI Jiejie(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650031,China;School of Life Sciences,Yunnan University,Kunming 650091;Kunming Medical University,Kunming 650500)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650031 [2]云南大学生命科学学院,昆明650091 [3]昆明医科大学,昆明650500

出　　处：《中国比较医学杂志》2022年第6期48-54,共7页Chinese Journal of Comparative Medicine

基　　金：云南省科技人才和平台计划项目(2017HC019);昆明市科技创新团队(2019-1-R-24483)。

摘　　要：目的建立树鼩原代睾丸间质细胞分离培养方法,探究寨卡病毒(Zika virus,Zikv)对该细胞的感染特性,为Zikv感染导致的睾丸损伤及不育提供研究基础。方法3月龄的雄性树鼩0.4 mL 3%戊巴比妥钠麻醉处死后采集睾丸,采用IV胶原酶和胰蛋白酶联合消化法获得单细胞悬液,通过percoll密度梯度离心结合差异贴壁法对细胞进行纯化,3β-羟类固醇脱氢酶(3β-HSD)对细胞荧光染色鉴定,ELISA检测细胞分泌睾酮的能力;用Zikv感染树鼩睾丸间质细胞,并用对寨卡病毒易感的非洲绿猴肾细胞(Vero)作为对比参照,测定感染不同时间点的细胞及其培养上清液中病毒载量,镜下观察细胞病变,ELISA检测睾酮含量。结果分离培养的睾丸间质细胞经3β-HSD鉴定阳性率高达98%以上,且增殖迅速,能够连续分泌睾酮,第4代睾丸间质细胞在0~24 h和24~48 h睾酮分泌量分别为1.253 ng/mL和1.163 ng/mL;用Zikv感染树鼩睾丸间质细胞,6 h内病毒拷贝数可达2.15×10^(5) copies/mL,且感染48 h后基本丧失睾酮分泌能力,感染第3天可见明显的细胞病变。结论成功建立树鼩睾丸间质细胞的分离培养方法,Zikv可感染树鼩睾丸间质细胞并能导致该细胞睾酮分泌功能缺失,为Zikv入侵生殖系统机制研究提供基础。Objective To establish a method for the isolation and culture of primary Leydig cells in tree shrew,to explore the infection characteristics of Zika virus(Zikv)on these cells,and to provide a basis for research on the damage and sterility of testis caused by Zikv infection.Methods A 3-month-old male tree shrew was sacrificed with 0.4 mL of 3%sodium pentobarbital anesthetized and the testis was collected.A single-cell suspension was obtained by combined digestion with collagenase IV and trypsin.After purification,cells were identified by fluorescent staining with 3β-hydroxysteroid dehydrogenase,and the ability of cells to secrete testosterone was detected by ELISA.Tree shrew Leydig cells were infected with Zikv,as were kidney cells from African green monkeys susceptible to Zika virus for comparison.The viral loads in the cells and their culture supernatants at different time points of infection were determined,cytopathic changes were observed under a microscope,and testosterone content was determined by ELISA.Results The positive rate of isolated and cultured Leydig cells identified by 3β-hydroxysteroid dehydrogenase was as high as 98%.They proliferated rapidly and continuously secreted testosterone.The forth-generation Leydig cells secreted 1.253 and 1.163 ng/mL testosterone at 0~24 h and 24~48 h,respectively.Upon infection of Leydig cells of tree shrew with Zikv,the virus copy number reached 2.15×10^(5) copies/mL within 6 h,the cells’ability to secrete testosterone was basically lost after 48 h of infection,and cytopathic effects were seen on the third day of infection.Conclusions A method for isolating and culturing Leydig cells from tree shrew was successfully established.The findings showed that Zikv could infect Leydig cells of tree shrew and lead to the loss of their testosterone secretion function,providing a basis for research on the mechanism by which Zikv affects the reproductive system.

关 键 词：树鼩 睾丸间质细胞 3Β-HSD 睾酮水平 Zikv 

分 类 号：R-33[医药卫生]