MSC移植诱导移植胰、肾免疫耐受的实验研究

发布时间：2018-06-15 06:48

本文选题：骨髓间充质干细胞 + 胰腺　；参考：《昆明医科大学》2014年硕士论文

【摘要】：目的分离、体外培养、鉴定兔的骨髓间充质干细胞(MSCs),建立兔胰、肾联合移植(SPKT)小动物模型,探讨MSC能否诱导胰肾联合移植免疫耐受。材料和方法 1.采用健康幼龄日本大耳白兔,麻醉致死后冲洗骨髓收集骨髓液,采用直接贴壁法分离培养MSCs,对MSCs进行形态学观察,同时通过流式细胞分析术对CD34、CD44.CD45.CD904种表面抗原进行鉴定。取第5代MSCs,加入50μmol/L抗坏血酸盐2磷酸盐、50μmol/L消炎痛0.5μmol/L地塞米松诱导其分化。 2.选用日本大耳兔作SPKT的供、受体,切除受体双侧肾脏,供体肾脏原位吻合,供体胰腺吻合于另一侧；用袖套法结扎完成供体门静脉、肾静脉与受体肾静脉断端的吻合；用缝合法完成动脉吻合,输尿管和胰管置管行膀胱内引流。 3.术后存活的移植兔分组(A.B.C.D每组3只),分别给予不同处理,A组：受体胰肾移植后予MSC静脉输注,B组：受体胰肾移植后予MSC静脉输注+免疫抑制剂(他克莫司胶囊),C组：受体胰肾移植后只给免疫抑制剂(他克莫司胶囊),D组：受体胰肾移植后不行干预；取各组移植兔术前和术后第1、3天共3个时间段静脉血,应用流式细胞仪检测CD4+T细胞和CD8+T细胞百分率,并计算其比值及CD4+CD25+Treg细胞的比例。结果 1.第2代MSCs经流式细胞技术鉴定出细胞的CD34(-).CD45(-).CD44(+). CD90(+)符合MSCs的表面抗原,第5代骨髓间充质干细胞,成脂诱导10d后油红O染色显示有大量脂质沉积并相互融合,细胞由长梭形变为多边形,证明了干细胞的分化特性,培养的细胞就是骨髓间充质干细胞。 2.15只供体顺利完成胰腺和肾脏切取术：15只受体顺利完成胰肾联合移植术；供体肾脏、胰腺移植后迅速恢复血液循环,无出血及漏血,术后3只死于持续性低血压,实验组的3只受体术后平均生存期(4.0±0.7)d；对照组的9只受体术后平均生存期(4.0±0.5)d,成功建立兔胰肾联合移植模型。 3.术后第1、3天输注MSC组(n=3)比未干预组(n=3)CD4+T细胞数和CD4+CD25+Treg细胞数比例高,差异有统计学意义(p0.05)。 4.术后第1、3天输注MSC组(n=3)比输注免疫抑制剂组(n=3)CD4+T细胞数和CD4+CD25+Treg细胞数比例低,差异有统计学意义(p0.05)。 5.术后第1、3天输注MSC+免疫抑制剂组(n=3)比输注免疫抑制剂(n=3)CD4+T细胞数和CD4+CD25+Treg细胞数比例高,差异有统计学意义(p0.05)。结论 MSC能调节移植胰、肾免疫反应,减轻排斥反应,但效果没有免疫抑制剂明显,不能替代免疫抑制剂。
[Abstract]:Objective to isolate and culture rabbit bone marrow mesenchymal stem cells (MSCs) and to establish a small animal model of pancreas and kidney transplantation (SPKT) in order to investigate whether MSC can induce the immune tolerance of simultaneous pancreaticorenal transplantation. Materials and methods 1. Bone marrow fluid was collected from the bone marrow of young Japanese white rabbits after anaesthesia. MSCs were isolated and cultured by direct adherent method. The morphology of MSCs was observed, and CD34, CD44.CD45.CD904 surface antigens were identified by flow cytometry. The fifth generation of MSCs was induced by 50 渭 mol / L ascorbate 2 phosphate 50 渭 mol / L indomethacin 0.5 渭 mol / L dexamethasone. Japanese big ear rabbits were used as donor and recipient of SPKT, bilateral kidney was resected, donor kidney was anastomosed in situ, donor pancreas was anastomosed on the other side, portal vein of donor was ligated with cuff method, anastomosis of renal vein and renal vein of recipient was completed. Arterial anastomosis was performed by suture and intravesical drainage of ureter and pancreatic duct was performed. The surviving rabbits were divided into three groups of A.B.C.D in each group and treated with different treatments: group A: group B: after transplantation of recipient pancreas and kidney, immunosuppressive agent MSC (tacrolimus capsule) was given. Group D: only immunosuppressive agents were given to recipients after pancreaticorenal transplantation (tacrolimus capsule group D: no intervention after recipient pancreaticorenal transplantation; The percentage of CD4 T cells and CD8 T cells were measured by flow cytometry, and the ratio of CD4 CD25 Treg cells were calculated. Result 1. The second passage of MSCs was identified by flow cytometry. CD90 () conformed to the surface antigen of MSCs. After 10 days of adipogenic induction, oil red O staining showed a large amount of lipid deposition and fusion, and the cells changed from fusiform to polygon, which proved the differentiation of stem cells. The cultured cells were bone marrow-derived mesenchymal stem cells. 2.15 donors successfully completed pancreas and kidney excision, 15 recipients successfully completed pancreaticorenal transplantation, donor kidneys, pancreas transplantation, and rapid recovery of blood circulation. Without bleeding or bleeding, 3 died of persistent hypotension after operation. The mean survival time of 3 recipients in the experimental group was 4.0 卤0.7 days, and the average survival time of 9 recipients in the control group was 4.0 卤0.5 days. The model of simultaneous pancreaticorenal transplantation was successfully established in rabbits. The numbers of CD4 T cells and CD4 CD25 Treg cells in MSC group were higher than those in control group on the 1st day after operation, and the difference was statistically significant (P 0.05). The number of CD4 T cells and CD4 CD25 Treg cells in MSC group was lower than that in immunosuppressive group on the 1st day after operation (P < 0.05). The ratio of CD4 T cells and CD4 CD25 Treg cells in MSC immunosuppressant group was significantly higher than that in MSC immunosuppressant group on the 1st day after operation (P 0.05). Conclusion MSC can regulate the immune response of pancreas and kidney and alleviate the rejection, but the effect is less than that of immunosuppressant and can not replace the immunosuppressant.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R657.5;R699.2

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