广州管圆线虫感染后脑组织及小胶质细胞组织蛋白酶D的表达  

Cathepsin D expression in brain tissue and microglia of mice after Angiostrongylus cantonensis infection

作　　者：魏洁[1] 邓健 许莲 曾幸达 吴忠道 WEI Jie;DENG Jian;XU Lian;ZENG Xing-da;WU Zhong-dao(Department of Clinical Laboratory,the Second Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong 510260,China;Department of Parasitology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou,Guangdong 510080,China)

机构地区：[1]广州医科大学附属第二医院检验科,广东广州510260 [2]中山大学中山医学院寄生虫学教研室,广东广州510080

出　　处：《热带医学杂志》2022年第11期1473-1477,1492,1612,共7页Journal of Tropical Medicine

基　　金：广东省医学科学技术研究基金(B2019093);广州市医药卫生一般引导项目(20191A011084)

摘　　要：目的研究广州管圆线虫感染后小鼠脑内组织蛋白酶(CTS)表达情况。方法建立广州管圆线虫感染BALB/c小鼠模型,分离脑组织,提取RNA和总蛋白。分光光度计检测RNA吸光度和浓度,合格后送RNA测序。设计组织蛋白酶A、B、C、D、E、F、G、H、K、L、O、S、W、Z引物,Real-time PCR扩增特异性片段,检测正常及感染广州管圆线虫后7、14、21 d小鼠脑组织内组织蛋白酶转录水平。Western blotting检测正常组及感染后7、14、21 d组小鼠脑组织内组织蛋白酶蛋白表达水平。免疫荧光显示组织蛋白酶D(CTSD)在正常组及21 d组小鼠脑组织内细胞定位。Iba-1是小胶质细胞特异性标记分子,与CTSD共标记。收集广州管圆线虫四期幼虫,提取可溶性虫体抗原。体外培养小胶质细胞系BV2。可溶性虫体抗原(25μg/mL)、脂多糖(LPS)(1μg/mL)、IL-13(ng/mL)刺激BV2细胞,24 h后收集细胞,分离总蛋白。BCA蛋白浓度测定试剂盒检测浓度后,WB检测CTSD蛋白表达。结果正常组和感染广州管圆线虫7、14、21 d组小鼠脑组织转录组测序,14种组织蛋白酶即CTSL、S、F、A、H、B、Z、D、O、K、C、E、W、G存在差异表达。CTSJ、M、P、Q、R未在转录组测序中查到结果。经Real-time PCR验证,各CTS在感染不同时间表达水平不尽相同。随感染时间延长,CTSD表达水平逐渐升高,21 d组明显高于正常组,差异有统计学意义(P<0.05)。免疫荧光显示正常组和21 d组小鼠脑内小胶质细胞与CTSD可共定位,且21 d组荧光强于正常组。可溶性虫体抗原、LPS、IL-13刺激BV2细胞,WB结果显示,虫体抗原刺激CTSD表达水平最高,其次是LPS组,IL-13组最低。结论广州管圆线虫感染可诱导BALB/c小鼠脑组织及小胶质细胞表达CTSD,后者可能参与脑炎的发生发展。Objective To study the expression of cathepsin(CTS)in the brain of BALB/c mice infected withAngiostrongylus cantonensis.MethodsBALB/c mouse model of A.cantonensis infection was established.The brain tissuewas isolated and RNA and total protein were extracted.The cathepsin transcription levels were determined by Real-time PCRusing designed primers of cathepsin(A,B,C,D,E,F,G,H,K,L,O,S,W and Z)in the brain tissues of normaland A.cantonensis infected mice on post-infection day 7,14,and 21.Western blotting(WB)was used to detect cathepsinprotein expression in normal group and 7,14,21 d group of mice brain tissues.Immunofluorescence showed the localizationof cathepsin D(CTSD)in normal group and 21 d group mice brain.Iba-1 and microglia specific marker were co-labeledwith CTSD.The fourth stage larvae of A.cantonensis were collected and soluble worm antigens were extracted.Microglia cellline BV2 was cultured in vitro.BV2 cells were stimulated with soluble worm antigen(25μg/m L),lipopolysaccha-ride(LPS)(1μg/m L),and IL-13(ng/m L).After 24 hours,BV2 cells were collected and total proteins were isolated.After BCAdetection,the expression of CTSD protein was detected by WB.ResultsThe transcriptome results of normal group and 7,14 and 21 d group mice brain showed that the transcriptional levels of 14 cathepsin including CTSL,S,F,A,H,B,Z,D,O,K,C,E,W and G were different.CTSJ,M,P,Q and R were not found in transcriptome sequencing.As verifiedby Real-time PCR,the expression levels of CTS were different at different times of infection.With the prolongation ofinfection,the expression level of CTSD increased gradually,and the 21 d group level was significantly higher than that ofthe normal group,the difference was statistically significant(P<0.05).Immunofluorescence showed that microglia couldcolocalize with CTSD in the brain of normal group and 21 d group mice.The fluorescence in 21 d group was stronger thanthat in the normal group.BV2 cells were stimulated by soluble insect antigen,LPS,and IL-13.WB results showed that theexpressio

关 键 词：广州管圆线虫 组织蛋白酶D 小胶质细胞 脑组织 

分 类 号：R532.1[医药卫生—内科学]