CD4^+CD25^(high)FoxP3^+Treg与帕金森病干细胞移植后排斥反应间的相关性研究  

Relationship between CD4^+CD25^(high)FoxP3^+Treg and the rejection after stem cells transplant for Parkinson's disease

作　　者：赵永波[1] 沈楠[1] 

机构地区：[1]上海交通大学附属第一人民医院神经科,上海200080

出　　处：《中国医学前沿杂志（电子版）》2013年第12期23-43,共21页Chinese Journal of the Frontiers of Medical Science(Electronic Version)

基　　金：上海市科委基础研究重点项目(11JC1411402)

摘　　要：目的通过应用骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)移植治疗帕金森病(Parkinson's disease,PD)大鼠,检测大鼠外周血中CD4+CD25highFoxP3+调节性T细胞(regulatory T cell,Treg)比例的变化及相关细胞因子的漂移,观察脑内免疫排斥反应的发生状况。以期探讨干细胞移植治疗PD后,外周血中Treg的改变与脑内免疫排斥反应间的相关性及其在干细胞移植后免疫耐受中的作用。方法 SD大鼠随机分为正常对照组、PD组、(PD+PBS)组、(PD+BMSCs)组。正常对照组大鼠不进行任何处理,为完全空白对照;余下大鼠均通过脑内立体定向注射6-羟基多巴胺(6-hydmxydopamine,6-OHDA)建立PD大鼠模型。成功建模后第3周,(PD+PBS)组大鼠脑内定向注射PBS缓冲液;(PD+BMSCs)组大鼠脑内定向注射应用全骨髓贴壁法培养的大鼠BMSCs悬液;PD组大鼠不做任何处理。PBS缓冲液和BMSCs悬液注射2周后,各组大鼠均应用流式细胞术(flow cytometry,FCM)检测其外周血中CD4+CD25highFoxP3+Treg占T淋巴细胞比例的变化,并运用酶联免疫吸附测定(ELISA)检测外周血中白介素(IL)-4、IL-10和干扰素-γ(INF-γ)等相关细胞因子的改变。另外,通过免疫组织化学染色法观察大鼠脑内酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性神经元、离子钙接头蛋白分子-1(ionized calcium bindingadaptor molecule-1,iba-1)阳性细胞、主要组织相容性复合体-Ⅱ(major histocompatibility complex-Ⅱ,MHC-Ⅱ)分子的变化。同时,(PD+BMSCs)组大鼠采用EdU染色法监测移植后BMSCs的存活情况。结果脑内立体定向注射6-OHDA 3周后,部分大鼠腹腔注射阿扑吗啡可诱导出旋转行为,表现为向健侧旋转,呈首尾相接状,以210圈/30分钟为标准,建模成功率达65.45%,并且免疫组织化学染色显示,建模成功的PD大鼠偏侧TH阳性神经元毁损率可达90%以上。体外培养的大鼠BMSCs至P4时,细胞形态良好且均一,经流式细胞术鉴定,其纯度达95%以上�Objective To detect the changes of CD4+CD25highFoxp3+regulatory T cells (Treg) and related cytokines in peripheral blood, and observe the immune rejection in the brain, the bone marrow mesenchymal stem cells (BMSCs) transplangted Parkinson's disease (PD) rats were use. To investigate the correlation between the changes of Treg in the peripheral blood and the immune rejection in the brain, and the role of Treg in the immune tolerance after stem cells transplantation. Methods All Sprague-Dawley rats were randomly divided into four groups:control group, PD group, (PD+PBS) group and (PD+BMSCs) group. Control group rats were blank control, the other rats were established rat models of PD by injection of 6-hydroxydopamine (6-OHDA). Three weeks after modeling, (PD + PBS) group rats were injected PBS to brain directly, (PD + BMSCs) group rats were injected BMSCs to brain which obtained by whole bone marrow adherent culture method, and PD group rats were blank control. Two weeks after PBS injection and BMSCs transplantation, the proportion of CD4+CD25highFoxp3+Treg in the peripheral blood of all rats were detected through lfow cytometry method (FCM). The changes of related cytokines such as IL-4, IL-10 and INF-γwere examined by ELISA at the same time. Moreover, tyrosine hydroxylase (TH)-positive cells, ionized calcium bindingadaptor molecule-1 (iba-1)-positive cells and the expression of Major histocompatibility complex-Ⅱ(MHC-Ⅱ) were showed employing immunolfuorescence staining. Meanwhile, the survival of BMSCs in PD+BMSCs group rats were tested by EdU staining. Results Three weeks after stereotactic injection of 6-OHDA, parts of PD rats could be induced to rotation behavior after intraperitoneal injection of apomorphine, which performed as the contralateral rotation and end-to-end. Taking 210 circle/min as a standard, the success rate of modeling was 65.45%. The damage rate of TH-positive neurons of those PD rats model was more than 90%. Cell morphology of cultured BMSCs of SD rats was good when cultured to P4, and

关 键 词：帕金森病 骨髓间充质干细胞 CD4+CD25highFoxP3+调节性T细胞 免疫排斥反应 

分 类 号：R742.5[医药卫生—神经病学与精神病学]