东方田鼠HSP90α和KPNA2基因表达及体内抗日本血吸虫效果比较观察  

Comparative expression of HSP90α and KPNA2 genes in different tissues of Microtus fortis and their effects on the resistance against Schistosoma japonicum infection in recipient mice

作　　者：成钢 王芊薄 龚强[2] 熊德慧[2] CHENG Gang;WANG Qian-bo;GONG qiang;XIONG De-hui(Zoology Key Laboratory of Hunan Higher Education,Research Center on Conservation and Utilization of Biological Resources in Dongting Lake Area,Animal Health Research Institute of College of Life and Environmental Sciences,Research Center of Agricultural Biology Macromolecules,Hunan University of Arts and Science,Changde 415000,China;Molecular Biology Research Center,School of Biological Science and Technology,Central South University,Changsha 410078,China)

机构地区：[1]湖南文理学院/生命与环境科学学院,动物学湖南省高校重点实验室,湖南文理学院环洞庭湖生物资源保育与利用研究中心,湖南文理学院生命与环境科学学院动物健康养殖研究所,湖南文理学院农业生物大分子研究中心,常德415000 [2]中南大学生物科学与技术学院分子生物学研究中心,长沙410078

出　　处：《中国寄生虫学与寄生虫病杂志》2019年第5期552-555,562,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：湖南文理学院大学生创新创业研究项目（No.ZC1626,No.YB1719,No.YB1926)~~

摘　　要：目的比较东方田鼠抗日本血吸虫基因HSP90α和KPNA2的表达差异及体内抗虫效果差异。方法RT-PCR扩增并比较健康东方田鼠新分离的心、肝、脾、肺、肾、脑、肌肉、皮肤、骨髓组织中HSP90α和KPNA2的表达情况。40只雄性昆明小鼠随机分为空白对照组(DMEM培养基)、阴性对照组(pLXSN质粒转染病毒)、pLXSN-HSP90α组和pLXSN-KPNA2组等4组,每组10只,各组分别于第1、3、7 d每鼠尾静脉注射重组质粒转染病毒(2×10^6 cfu/ml)或培养基0.2 ml。注射后第2天,各组小鼠腹部皮肤感染日本血吸虫尾蚴(30±2)尾/鼠。感染后第42天,剖杀小鼠,门脉灌注检获成虫,测量死亡后合抱及单性虫体长度,计算虫荷、减虫率、每克肝虫卵数(LEPG)、肝脏减卵率,HE染色观察肝脏病理变化,观察比较HSP90α和KPNA2对日本血吸虫感染小鼠的保护效果。结果RT-PCR结果显示,HSP90α在东方田鼠脑、骨髓中高表达,而KPNA2在心、肾和肌肉中高表达。体内实验结果显示,pLXSN-HSP90α组和pLXSN-KPNA2组小鼠的肝脏颜色分别呈灰黄色和灰黑色,虫荷分别为(11.3±1.1)、(11.6±1.3)条,血吸虫成虫体长分别为(1.19±0.04)、(1.21±0.05)cm,LEPG分别为1852.0±392.4、1035±485.4,与阴性对照组[(16.7±1.3)条、(1.39±0.06)cm、3644.0±523.6]比较差异均有统计学意义(P<0.05)。两个实验组的虫荷、成虫体长差异无统计学意义(P>0.05),而LEPG指标差异有统计学意义(P<0.05)。两组减虫率分别为40.8%和39.4%(P>0.05),肝减卵率分别为57.9%和76.5%(P<0.05),与阴性对照组(12.6%、17.3%)比较差异有统计学意义(P<0.01)。肝组织切片HE染色镜下观察显示,pLXSN-HSP90α组和pLXSN-KPNA2组小鼠的肝脏虫卵结节数量均远少于阴性对照。结论东方田鼠抗日本血吸虫基因HSP90α和KPNA2在脑、骨髓、心脏、肾脏和肌肉组织中表达存在差异;两者对日本血吸虫感染小鼠保护效果在减卵率和LEPG存在差异。Objective To determine the expression levels of Schistosoma-resistant genes HSP90α(heat shock protein-90α)and KPNA2(Karyopherinα2)in different tissues of rodent Microtus fortis and their effects on the resistance against S.japonicum infection in recipient mice.Methods RT-PCR was used to determine the levels of mRNA expression of HSP90αand KPNA2 in different tissues(heart,liver,spleen,lung,kidney,brain,muscle,skin and bone marrow)of normal M.fortis.To determine the effect of HSP90αand KPNA2 on their resistance against S.japonicum infection,the recombinant retroviral vectors containing target genes,pLXSN-HSP90αand pLXSN-KPNA2,were used to make recombinant viruses.The viruses containing the target HSP90αand KPNA2 were used to transfect naive Kunming mice on day 1,3,7 by tail vein injection of 4×10^5 cfu viruses to make them expressed in the recipient mice.The mice were then infected with(30±2)cercaria of S.japonicum 2 days after the final injection of viruses.The mice received empty vector or PBS were used as negative controls.All mice were euthanized 42 days after cercaria challenge and adult worms were collected by portal vein drainage and livers collected to measure the egg count and the egg granuloma by HE staining.The HSP90αand KPNA2-induced resistance against S.japonicum infection was measured by the adult worm reduction,egg count reduction and the reduced liver egg-granuloma compared to mice received empty vector or PBS only.Results RT-PCR results showed that HSP90αgene was highly expressed in the brain and bone marrow of M.fortis,while KPNA2 gene highly expressed in heart,kidney and muscle.The cercaria challenge study showed that mice transfected with pLXSN-HSP90αand pLXSN-KPNA2 viruses produced 40.80%and 39.42%adult worm reduction,and 57.90%and 76.50%egg reduction,respectively,with statistical significance compared with mice received pLXSN vector only(P<0.01).However,there was no significant difference in adult worm reduction between mice received pLXSN-HSP90αand pLXSN-KPNA2,but with significan

关 键 词：东方田鼠 HSP90Α KPNA2 日本血吸虫 基因表达 

分 类 号：R532.21[医药卫生—内科学]