间充质干细胞在自体免疫性葡萄膜炎中作用的研究

发布时间：2018-04-26 06:11

本文选题：间充质干细胞 + 实验性自体免疫性葡萄膜炎　；参考：《天津医科大学》2010年硕士论文

【摘要】： 目的葡萄膜炎是一类最常见的眼部自体免疫性疾病,其中多种类型具有反复发作、并发症多、治疗困难以及预后较差和致盲率较高等特点。间充质干细胞(mesenchymal stem cells, MSCs)能够抑制多种免疫细胞(包括T淋巴细胞、B淋巴细胞、NK细胞以及树突状细胞)的增殖和功能。本研究采用MSCs治疗大鼠实验性自体免疫性葡萄膜炎(experimental autoimmune uveoretinitis, EAU),检测MSCs在患有葡萄膜炎的大鼠体内能否发挥免疫调节作用,以及其可能的作用机制,试图为临床治疗葡萄膜炎提供新的思路以及可供选择的治疗方法。方法1.原代培养Lewis或Wistar大鼠骨髓间充质干细胞,达90%融合时传代培养。用流式细胞仪检测培养细胞的表型,并做体外诱导分化实验,以备后续实验使用。2.使用光感受器间维生素A类结合蛋白(interphotoreceptor retinoid-binding protein, IRBP)与含有结核杆菌H37RA的完全弗氏佐剂等体积混合,充分乳化后,在双后足部皮下免疫Lewis大鼠,24小时内经腹腔注射百日咳毒素,制作大鼠EAU动物模型。将动物随机分组,为了观察在免疫同时输注MSCs对EAU的预防作用以及MSCs对已经发生的EAU的治疗作用,从不同时间开始连续三天(免疫后0,1,2天,EAU发生前；免疫后第9,10,11天,EAU初发期；免疫后第12,13,14,EAU高峰期；及免疫后第16,17,18天,EAU慢性期)静脉输注自体或异体来源的间充质干细胞,空白对照组输注PBS。裂隙灯下定期进行临床观察,20天后进行病理学观察,参照Caspi分级对临床体征和病理学改变进行评分,观察葡萄膜炎的发生和转归情况。3.收集各组大鼠脾脏以及腹股沟淋巴结,分离单个核细胞,在IRBP刺激下孵育72小时后,采用BrdU试剂盒检测各组大鼠免疫应答强度。4.取IRBP刺激下各组大鼠脾脏和淋巴结单个核细胞孵育72小时后的上清液,采用ELISA试剂盒检测Thl类和Th2类细胞因子分泌情况。5.免疫20天后取大鼠外周血,流式细胞仪测定CD4+CD25+T细胞占CD4+T细胞的百分比。 6.收集各组大鼠脾脏以及淋巴结,分离单个核细胞,提取总RNA,采用RT-PCR测定FOXP3 mRNA表达水平。结果 1.成功分离并鉴定MSCs。 2.建立了EAU动物模型。 3.采用MSCs治疗EAU,与对照组相比,免疫的同时、免疫后第9天及免疫后第12天给EAU大鼠输注MSCs后,临床症状均显著减轻(P0.05),在免疫同时注射MSCs的预防效果尤为明显,而免疫后第16天,在疾病慢性期注射MSCs则无作用(P0.05)。与临床表现一致,除外免疫后第16天注射MSCs组,病理学显示其他三组实验组大鼠视网膜结构损害明显较对照组轻(P0.05)。 4.体外和体内T细胞增殖实验表明,MSCs可以显著抑制致病性T细胞增殖(P0.05)。 5. ELISA结果显示,实验组Th1细胞因子分泌显著降低(P0.05),而Th2细胞因子分泌升高(P0.05),表明MSCs可以调节免疫应答平衡,导致Thl向Th2应答漂移。 6.与对照组相比,MSCs治疗组大鼠体内调节性T细胞比例显著增高(P0.05),实时定量PCR检测也发现FOXP3-mRNA在MSCs治疗组表达量显著高于对照组(P0.05),这些结果表明MSCs可能通过上调调节性T细胞来发挥作用。结论 MSCs可以通过抑制T细胞免疫应答、改变Th1/Th2应答平衡以及上调调节性T细胞,有效预防并治疗EAU。本研究证实了通过调节机体免疫系统,MSCs对免疫性疾病具有治疗作用。
[Abstract]:objective
Uveitis is one of the most common autoimmune diseases of the eye. Many of them have recurrent attacks, many complications, difficult treatment, poor prognosis and high blindness. Mesenchymal stem cells (MSCs) can inhibit a variety of immune cells (including T lymphocytes, B lymphocytes, NK cells, and trees. This study used MSCs to treat experimental autoimmune uveitis (experimental autoimmune uveoretinitis, EAU) in rats, and to detect the immune regulation of MSCs in rats with uveitis, as well as its possible mechanism to provide new clinical treatment for uveitis. Methods and alternative treatment methods. Methods 1. Lewis or Wistar rat bone marrow mesenchymal stem cells were cultured in the primary generation, and the cells were cultured at the time of 90% fusion. The phenotype of the cultured cells was detected by flow cytometry, and the induction of differentiation in vitro was done in vitro, in order to prepare the use of.2. to use the vitamin A binding protein (interphotorecep). Tor retinoid-binding protein, IRBP) mixed with complete Freund's adjuvant containing H37RA of Mycobacterium tuberculosis, after full emulsification, immunized Lewis rats under the subcutaneous of two hind feet, and intraperitoneally injected with pertussis toxin within 24 hours to make the rat EAU animal model. The animals were randomly divided in order to observe the prevention of MSCs against EAU at the same time. The effect and the therapeutic effect of MSCs on the already occurring EAU were three days from time to time (0,1,2 days after immunization, before EAU, 9,10,11 days after immunization, EAU initial onset, 12,13,14, EAU peak after immunization, and 16,17,18 days after immunization, and EAU chronic phase) intravenous infusion of mesenchymal stem cells derived from autologous or allogeneic sources, blank pairs The clinical observation under the PBS. slit lamp was carried out regularly. After 20 days, the pathological observation was carried out. The clinical signs and pathological changes were graded according to the Caspi classification. The occurrence and prognosis of uveitis were observed and.3. collected the spleen and inguinal lymph nodes in each group, isolated mononuclear cells, after incubating for 72 hours under the stimulation of IRBP. The immune response intensity of rats in each group was detected by BrdU kit. The supernatant was incubated in each group of spleen and lymph node mononuclear cells for 72 hours under the stimulation of IRBP, and the secretion of Thl and Th2 cytokines was detected by ELISA kit and.5. immunized for 20 days, and the peripheral blood was taken for 20 days. The flow cytometry was used to determine the number of CD4+T cells in CD4+CD25+T cells. The percentage.
6. the spleen and lymph nodes of rats in each group were collected, the mononuclear cells were isolated, the total RNA was extracted, and the expression level of FOXP3 mRNA was measured by RT-PCR.
Result
1. successful separation and identification of MSCs.
2. the animal model of EAU was established.
3. MSCs was used to treat EAU. Compared with the control group, the clinical symptoms were significantly reduced after immunization, Ninth days after immunization and twelfth days after immunization to EAU rats (P0.05). The preventive effect of MSCs injection at the same time was particularly obvious, while the injection of MSCs in the chronic phase of the disease was no effect (P0.05). The MSCs group was injected sixteenth days after immunization. Pathology showed that the retinal structure damage of the other three groups was significantly lighter than that of the control group (P0.05).
4. in vitro and in vivo T cell proliferation assays showed that MSCs could significantly inhibit the proliferation of pathogenic T cells (P0.05).
5. ELISA results showed that the secretion of Th1 cytokines in the experimental group decreased significantly (P0.05), while the secretion of Th2 cytokines increased (P0.05), indicating that MSCs could regulate the balance of the immune response, leading to the drift of Thl to Th2.
6. compared with the control group, the proportion of regulatory T cells in the MSCs treatment group increased significantly (P0.05). Real-time quantitative PCR detection also found that the expression of FOXP3-mRNA in the MSCs treatment group was significantly higher than that of the control group (P0.05). These results suggest that MSCs may play a role by up regulating the regulatory T cells.
conclusion
MSCs can inhibit the immune response of T cells, change the balance of Th1/Th2 response and up-regulate the regulatory T cells, and effectively prevent and treat EAU.. This study confirmed that MSCs has a therapeutic effect on immune diseases by regulating the body's immune system.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R773

【参考文献】