间充质干细胞在角膜移植排斥反应中作用的研究

发布时间：2018-03-25 23:51

本文选题：间充质干细胞　切入点：角膜移植　出处：《天津医科大学》2010年硕士论文

【摘要】：目的角膜移植术是目前器官和组织移植成功率最高的手术,但术后的免疫排斥反应仍是手术失败的最主要的原因。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和／或环孢霉素A(cyclosporin A,CsA)治疗大鼠角膜移植免疫排斥反应,检测MSCs在角膜移植大鼠体内能否发挥免疫调节作用,以及其可能的作用机制,试图建立MSCs移植治疗角膜移植排斥反应的技术平台。方法 1.原代培养Lewis或Wistar大鼠MSCs,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.制作大鼠角膜移植动物模型,随机分为6组。A组给予等量不含药物的PBS,B组术后肌注CsA,C组术前尾静脉注射MSCs,D组术后尾静脉注射MSCs,E组术前注射MSCs+术后肌注CsA, F组术后注射MSCs+术后肌注CsA。裂隙灯下对角膜植片进行临床观察,以混浊、水肿和新生血管3项指标作为临床评估标准,比较6组角膜植片的平均存活时间。术后10d,A组与D组病理组织学检查角膜的结构变化。 3.角膜移植模型建立后10天,收集A组与D组大鼠脾脏以及腹股沟淋巴结,分离单个核细胞,在刀豆蛋白A (concanavalin A, ConA)刺激下孵育72h后,采用BrdU试剂盒检测各组大鼠免疫应答强度。 4.取ConA刺激下A组与D组大鼠脾脏和淋巴结单个核细胞孵育72h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.角膜移植模型建立后10天,取A组和D组大鼠外周血,流式细胞仪测定CD4+CD25+T细胞占CD4+T细胞的百分比。 6.角膜移植模型建立后10天,收集A组和D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FOXP3mRNA表达水平。结果 1.成功分离并鉴定了MSCs, MSCs呈间质细胞特性：梭形,漩涡状排列。VonKossa染色、油红染色贴壁细胞呈骨样细胞及脂肪样细胞分化。 2.成功建立大鼠角膜移植动物模型。 3.采用MSCs治疗角膜移植术后大鼠,除C组外,B组、D组、E组和F组角膜植片的存活时间与A组相比明显延长,D组与B组、E组与F组相比有显著差异,但B组、E组与F组3者之间统计学无显著性差异(P0.05)。组织学检查发现D组角膜植片中炎性细胞和新生血管明显轻于A组。 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,D组Th1细胞因子分泌显著降低(P0.05),而Th2细胞因子(IL-4)分泌升高(P0.05),但IL-10分泌两者无显著性差异。 6.与对照组相比,D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D组表达量显著高于对照组(P0.05)。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,有效预防并治疗角膜移植免疫排斥反应。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
Corneal transplantation is currently the highest success rate of organ and tissue transplantation surgery, but postoperative immune rejection is the main reason for the failure of surgery is the reaction. Mesenchymal stem cells (mesenchymal stem cells, MSCs) can inhibit a variety of immune cells (including T lymphocytes, B lymphocytes, NK cells and dendritic cells) the proliferation and function. This research adopts MSCs and / or cyclosporine A (cyclosporin A CsA) on rat corneal allograft rejection, the detection of MSCs in corneal transplantation rats can play a role in immune regulation, and its possible mechanism, trying to establish a technology platform MSCs transplantation for the treatment of corneal allograft rejection.
Method
1. the primary culture of Lewis or Wistar rat MSCs reached 90% fusion and was passaged. The phenotype of cultured cells was detected by flow cytometry, and in vitro induced differentiation experiments were used for subsequent experiments.
2. making rat corneal transplantation animal model, randomly divided into 6 groups:.A group was given the same amount of free drugs, PBS, B group after intramuscular injection of CsA, C group of preoperative intravenous injection of MSCs, group D after intravenous injection of MSCs, E group after injection of MSCs+ after intramuscular injection of CsA, F group after operation injection of MSCs+ after intramuscular injection of CsA. on the slit lamp corneal graft were observed, with opacity, edema and neovascularization of 3 indicators as a standard clinical evaluation, comparison of 6 groups of corneal graft. The mean survival time of postoperative 10d, structural changes of A group and D group histopathological examination of the cornea.
3. days after establishment of corneal transplantation model, mononuclear cells were isolated from spleen and groin lymph nodes of rats in group A and group D, and 72h was incubated under concanavalin A (concanavalin A, ConA) stimulation. Then BrdU test kit was used to detect immune response in each group of rats. A was used to detect the immune response of rats in each group.
4. after ConA stimulation, the supernatant of spleen and lymph node mononuclear cells of A group and D group were incubated with 72h, and the secretion of Thl and Th2 cytokines were detected by ELISA kit.
10 days after the establishment of 5. corneal transplantation models, the peripheral blood of rats in group A and group D was taken, and the percentage of CD4+CD25+T cells in CD4+T cells was measured by flow cytometry.
6. days after the establishment of corneal transplantation model, the spleen and lymph nodes of rats in group A and D were collected, and the mononuclear cells were isolated. The total RNA was extracted, and the expression level of FOXP3mRNA was detected by RT-PCR. The expression level of FOXP3mRNA was detected by RT-PCR.
Result
1., MSCs and MSCs were successfully isolated and identified, showing interstitial cell characteristics: spindle shape, whirlpool arrangement,.VonKossa staining, and oil red staining adherent cells were differentiated into osteoid cells and fat like cells.
2. the rat model of corneal transplantation was successfully established.
By 3. MSCs after corneal transplantation in rats, except C group, B group, D group, E group and F group of corneal graft survival time was significantly prolonged compared with A group, D group and B group, E group and F groups have significant differences, but in B group, no significant difference statistics between E group and F group 3 (P0.05). Histological findings of D group of corneal inflammatory cells and neovascularization significantly lighter than A group.
4. the proliferation of T cells in vitro and in vivo showed that MSCs could significantly inhibit the proliferation of pathogenetic T cells (P0.05).
5.ELISA results showed that the secretion of Th1 cytokines in the D group decreased significantly (P0.05), while the secretion of Th2 cytokine (IL-4) increased (P0.05), but there was no significant difference between the secretion of IL-10 and the secretion of IL-10.
6. compared with the control group, the percentage of regulatory T cells in the D group increased significantly (P0.05), and the real-time quantitative PCR test also showed that the expression of FOXP3-mRNA in D group was significantly higher than that in the control group (P0.05).
conclusion
MSCs can effectively prevent and treat corneal allograft rejection by inhibiting the T cell immune response, changing the balance of Th1/Th2 response and upregulating regulatory T cells. This study confirms that MSCs plays a therapeutic role in 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】：R779.65

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