CAJ | 학술논문

背景:膜联蛋白Ⅴ是近年来被作为肝细胞膜上存在的与乙型肝炎病毒感染相关受体研究的热点之一,并在胎盘组织中也有表达.目的:探讨膜联蛋白Ⅴ在HBV宫内感染中的作用;揭示HBV进入胎盘组织细胞的方式,为HBV的宫内感染的机理提供理论依据.设计:随机对照实验.单位:泰山医学院.对象:选择2003-01/2004-12济南市妇幼保健院、泰安市中心医院、泰安市妇幼保健院收集的30例HBsAg阳性孕妇足月分娩的胎盘组织,同时收集母血分离血清.实验经过孕妇知情同意及医院伦理委员会批准.兔抗人膜联蛋白Ⅴ亲和纯化抗体(第一抗体)、鼠抗人HBs mAb(第一抗体)、生物素化羊抗小鼠IgG(第二抗体)均购自武汉博士德生物工程有限公司.方法:用即用型SABC免疫组织化学染色试剂,检测30例HBsAg阳性的足月胎盘组织中,有18例检测到HBsAg,随机选取10例进行荧光双标染色,其染色主要过程为将待测石蜡切片标本常规脱蜡至水;抗原微波热修复;加入第一种一抗(兔抗人AnV亲和纯化抗体1:60,为单克隆抗体)置湿盒中,4 ℃冰箱过夜;第二种一抗(鼠抗人HBs mAb1:50),置湿盒中,4 ℃冰箱过夜;加入与第二种一抗相应的二抗(如生物素化的羊抗鼠IgG1:100);加入以PBS作适当稀释的第一种荧光抗体(如FITC-羊抗兔IgG1:50);加入第二种荧光抗体(Avidin-Cy3);缓冲甘油封固;激光共聚焦显微镜下观察并进行图像分析.用IPP4.5图像分析法,先进入IPP4.5系统的Image Analysis程序,然后调入分析文件进行分析.主要观察指标:乙型肝炎病毒感染的胎盘组织中HBsAg/膜联蛋白:Ⅴ的存在与分布情况.结果:选取10例进行荧光双标染色结合激光共聚焦显微镜检测胎盘组织上滋养层细胞乙型肝炎病毒/膜联蛋白Ⅴ的存在与分布模式.胎盘组织中存在着丰富的膜联蛋白Ⅴ,位于绒毛合体滋养层细胞,在基质细胞和血管内皮细胞都有膜联蛋白Ⅴ的表达.并发现在同一细胞中存在着乙型肝炎病毒和膜联蛋白Ⅴ的共存现象.结论:乙型肝炎病毒感染胎盘细胞可能是通过与细胞上的受体膜联蛋白Ⅴ结合,促使病毒进入胎盘细胞,导致宫内感染. 揹景:膜聯蛋白Ⅴ是近年來被作為肝細胞膜上存在的與乙型肝炎病毒感染相關受體研究的熱點之一,併在胎盤組織中也有錶達.目的:探討膜聯蛋白Ⅴ在HBV宮內感染中的作用;揭示HBV進入胎盤組織細胞的方式,為HBV的宮內感染的機理提供理論依據.設計:隨機對照實驗.單位:泰山醫學院.對象:選擇2003-01/2004-12濟南市婦幼保健院、泰安市中心醫院、泰安市婦幼保健院收集的30例HBsAg暘性孕婦足月分娩的胎盤組織,同時收集母血分離血清.實驗經過孕婦知情同意及醫院倫理委員會批準.兔抗人膜聯蛋白Ⅴ親和純化抗體(第一抗體)、鼠抗人HBs mAb(第一抗體)、生物素化羊抗小鼠IgG(第二抗體)均購自武漢博士德生物工程有限公司.方法:用即用型SABC免疫組織化學染色試劑,檢測30例HBsAg暘性的足月胎盤組織中,有18例檢測到HBsAg,隨機選取10例進行熒光雙標染色,其染色主要過程為將待測石蠟切片標本常規脫蠟至水;抗原微波熱脩複;加入第一種一抗(兔抗人AnV親和純化抗體1:60,為單剋隆抗體)置濕盒中,4 ℃冰箱過夜;第二種一抗(鼠抗人HBs mAb1:50),置濕盒中,4 ℃冰箱過夜;加入與第二種一抗相應的二抗(如生物素化的羊抗鼠IgG1:100);加入以PBS作適噹稀釋的第一種熒光抗體(如FITC-羊抗兔IgG1:50);加入第二種熒光抗體(Avidin-Cy3);緩遲甘油封固;激光共聚焦顯微鏡下觀察併進行圖像分析.用IPP4.5圖像分析法,先進入IPP4.5繫統的Image Analysis程序,然後調入分析文件進行分析.主要觀察指標:乙型肝炎病毒感染的胎盤組織中HBsAg/膜聯蛋白:Ⅴ的存在與分佈情況.結果:選取10例進行熒光雙標染色結閤激光共聚焦顯微鏡檢測胎盤組織上滋養層細胞乙型肝炎病毒/膜聯蛋白Ⅴ的存在與分佈模式.胎盤組織中存在著豐富的膜聯蛋白Ⅴ,位于絨毛閤體滋養層細胞,在基質細胞和血管內皮細胞都有膜聯蛋白Ⅴ的錶達.併髮現在同一細胞中存在著乙型肝炎病毒和膜聯蛋白Ⅴ的共存現象.結論:乙型肝炎病毒感染胎盤細胞可能是通過與細胞上的受體膜聯蛋白Ⅴ結閤,促使病毒進入胎盤細胞,導緻宮內感染.
배경:막련단백Ⅴ시근년래피작위간세포막상존재적여을형간염병독감염상관수체연구적열점지일,병재태반조직중야유표체.목적:탐토막련단백Ⅴ재HBV궁내감염중적작용;게시HBV진입태반조직세포적방식,위HBV적궁내감염적궤리제공이론의거.설계:수궤대조실험.단위:태산의학원.대상:선택2003-01/2004-12제남시부유보건원、태안시중심의원、태안시부유보건원수집적30례HBsAg양성잉부족월분면적태반조직,동시수집모혈분리혈청.실험경과잉부지정동의급의원윤리위원회비준.토항인막련단백Ⅴ친화순화항체(제일항체)、서항인HBs mAb(제일항체)、생물소화양항소서IgG(제이항체)균구자무한박사덕생물공정유한공사.방법:용즉용형SABC면역조직화학염색시제,검측30례HBsAg양성적족월태반조직중,유18례검측도HBsAg,수궤선취10례진행형광쌍표염색,기염색주요과정위장대측석사절편표본상규탈사지수;항원미파열수복;가입제일충일항(토항인AnV친화순화항체1:60,위단극륭항체)치습합중,4 ℃빙상과야;제이충일항(서항인HBs mAb1:50),치습합중,4 ℃빙상과야;가입여제이충일항상응적이항(여생물소화적양항서IgG1:100);가입이PBS작괄당희석적제일충형광항체(여FITC-양항토IgG1:50);가입제이충형광항체(Avidin-Cy3);완충감유봉고;격광공취초현미경하관찰병진행도상분석.용IPP4.5도상분석법,선진입IPP4.5계통적Image Analysis정서,연후조입분석문건진행분석.주요관찰지표:을형간염병독감염적태반조직중HBsAg/막련단백:Ⅴ적존재여분포정황.결과:선취10례진행형광쌍표염색결합격광공취초현미경검측태반조직상자양층세포을형간염병독/막련단백Ⅴ적존재여분포모식.태반조직중존재착봉부적막련단백Ⅴ,위우융모합체자양층세포,재기질세포화혈관내피세포도유막련단백Ⅴ적표체.병발현재동일세포중존재착을형간염병독화막련단백Ⅴ적공존현상.결론:을형간염병독감염태반세포가능시통과여세포상적수체막련단백Ⅴ결합,촉사병독진입태반세포,도치궁내감염.
BACKGROUND: Recently, one focus of research has been Annexin Ⅴ (AnV) existing on hepatic cells membranes as a fundamental receptor related to hepatitis B virus (HBV) infection. Also its expression in placental tissues has been a matter of debate. The study of the relationships between placental cells infected with HBV and their AnV expression will be of great value in future prevention strategies and treatments.OBJECTIVE: To investigate the presence of AnV in HBV infected human's placental cells and its potential role in HBV intrauterine transmission.DESIGN: Randomized controlled experiment.SETTING: Taishan Medical College.MATERIALS: Placental tissue was collected from HBsAg positive full term pregnant women (30 cases) admitted to Jinan Institute for Maternal and Child Health, Taian Central Hospital and Taian Institute for Maternal and Child Health from January 2003 to December 2004. Maternal serum was also obtained. Informed consents for participating in this study were obtained from all the involved pregnant women and this experiment was approved by the Hospital Ethics Committee. Rabbit-anti-human AnV purified affinity antibody (first antibody), rat-anti-human HBs mAb (first antibody),and biotinylated goat-anti-mouse IgG (secondary antibody) were supplied by Wuhan Boster Bioengineering Company. METHODS: Using SABC immunohistochemical staining reagent, 18 HBsAg positive placentas were obtained from 30HBsAg infected patients in full term pregnancy. These were considered as the positive group and the other 12 were used as negative controls. The staining process included dewaxing, dehydration of embedded slides and microwave antigen restoration. In the wet box, rabbit-anti-human AnV purified antibody (first antibody, 1:60, monoclonal antibody)was added on the slides and kept at 4 ℃ overnight. Rat-anti-human antibody HBs mAb(secondary antibody, 1:50) was added and kept at 4 ℃ ovemight, after this procedure, biotinylated goat-anti-mouse IgG(1:100), the first fluorescent antibody such as FITC-goat anti-rabbit IgG (1:50) and the second fluorescent antibody (Avidin-Cy3) were used,respectively. The slides were sealed with buffered glycerol and examined under a confocal laser scanning microscope.The images on the slides were analyzed with IPP 4.5 image programs.MAIN OUTCOME MEASURES: Detecting the simultaneous existence and distribution of HBsAg/AnV in placental cells with HBV infection.RESULTS: Ten cases from the positive group were simultaneously detected for HBsAg/AnV by double-labeled immunofluorenscence assay and confocal laser scanning microscope. AnV expression was detected in the trophoblastic, interstitial cells and vascular endothelial cells of villi interstitial blood vessels, and the coexistence of HBsAg/AnV was found even in one cell.CONCLUSION: HBsAg combined with the receptor AnV in the same placental cells is a common finding in HBV infected full term pregnant women. This finding is very suggestive of a mechanism where AnV could promote hepatitis B virus to enter the placental cells and cause intrauterine infection.