间充质干细胞对脑死亡大鼠肾脏免疫调节及移植后功能改善的研究

发布时间：2018-04-28 22:39

本文选题：间充质干细胞 + 移植　；参考：《暨南大学》2014年硕士论文

【摘要】：目的：利用间充质干细胞（Mesenchymal stem cells，MSCs）免疫调节作用和低免疫原性的特性，探讨其对脑死亡大鼠肾脏免疫调节及移植后肾脏长期功能的影响。方法：（1）近交系、雄性F344大鼠骨髓来源的间充质干细胞贴壁分离、培养、纯化及流式细胞仪鉴定、传代、冻存。（2）将近交系、雄性F344大鼠（体质量200g~250g）作为供体，近交系、雄性Lewis大鼠（体质量200g~250g）作为受体。分3组：①正常对照组（G1，n=5）：取F344大鼠左肾，原位植入已切除左肾的Lewis大鼠；②脑死亡组（G2，n=5）：将F344大鼠诱导脑死亡，6小时候后取其左肾，原位植入已切除左肾的Lewis大鼠；③脑死亡+MSCs组（G3，n=5）：将F344大鼠诱导脑死亡后，输注MSCs，6小时候后取左肾，再植入已切除左肾的Lewis大鼠；（3）所有Lewis大鼠肾移植手术当天起连续10天每天给予肌注环孢素A（0.15mg/100g大鼠体重），术后第10天切除右肾，术后第14天、21天、28天、35天分别抽血，检测血清肌酐（Scr）;（4）将术后35天获取的移植肾标本和术后第10天切除的右肾标本行①免疫组化检测；②移植肾病理组织学检测。结果：本研究将贴壁分离法获得的细胞传至第三代，经流式细胞仪检测具有低表达CD34、CD45，高表达CD29、CD105和CD44的细胞特征，符合MSCs表型特点。这些细胞光镜下呈成纤维样、梭形，形态较均一，另外，它们可诱导分化成为脂肪细胞，证实从近交系、雄性F344大鼠骨髓分离、培养、纯化的细胞是MSCs。所有建立的脑死亡模型大鼠均符合脑死亡供体要求：（深昏迷，自主呼吸停止，无条件反射，瞳孔对光反射消失，角膜反射消失，观察6小时大鼠平均动脉压仍大于80mmHg）。检测肾移植术后不同时间三组大鼠Scr来评估移植肾的功能，G2组大鼠的Scr均比G1、G3组的高，，且差异具有统计学意义（P0.01），G3组大鼠的Scr略高于G1组，差异无统计学意义（P0.05）。病理学检测取各组肾组织行HE染色，在光镜下观察，发现G2组出现了较明显的单核细胞浸润及肾小管上皮炎症（P0.05），G3组和G1组无明显差异（P0.05）；三组移植肾的动脉血管都有明显炎症改变，且G2组的比G1和G3组的严重，但三组差异无统计学意义（P0.05）。免疫组化检测显示G2组移植肾肾小球、肾小管及间质细胞IL-1β、TNF-α表达呈阳性，G1、G3组的表达均低于G2组（P0.05）。结论：MSCs可调节脑死亡供体大鼠肾脏免疫状态，减轻供肾的炎症损害，移植后对受体肾脏具有保护作用，减少移植肾组织IL-1β、TNF-α炎症因子产生，移植肾能获得良好的长期功能，。
[Abstract]:Aim: to investigate the effects of mesenchymal stem cells (MSCs) on renal immunomodulation and long-term renal function after transplantation in brain-dead rats. Methods the bone marrow-derived mesenchymal stem cells from male F344 rats were isolated, cultured, purified and identified by flow cytometry. The inbred lines and male F344 rats (200g / 250g) were used as inbred lines. Male Lewis rats (200g / kg) were used as receptors. The rats were divided into 3 groups: 1 normal control group: the left kidney of F344 rats was taken from the left kidney and implanted into the brain dead group of Lewis rats with resected left kidney in situ. The left kidney of F344 rats was harvested after brain death induced 6 hours later and implanted into the Lewis rats with resected left kidney in situ. 3 brain death group (MSCs group): after inducing brain death in F344 rats, the left kidney was taken after 6 hours of infusion. All Lewis rats were given intramuscular injection of cyclosporine (A(0.15mg/100g) daily for 10 days from the day of renal transplantation. The right kidney was resected on the 10th day after operation. The blood was taken from the 14th day to the 21st day and from the 28th day to the 35th day after operation. Serum creatinine (creatinine) the grafted kidney specimens obtained 35 days after operation and the right kidney specimens removed on the 10th day after operation were examined by immunohistochemistry. Results: the cells obtained by adherent method were transferred to the third generation in this study. Flow cytometry was used to detect the cells with low expression of CD34, CD45, high expression of CD29, CD105 and CD44, which were in line with the phenotypic characteristics of MSCs. These cells were fibroblast-like, fusiform and homogenous under light microscope. In addition, they could be induced to differentiate into adipocytes. It was confirmed that the cells isolated, cultured and purified from the bone marrow of inbred and male F344 rats were MSCs. All of the established brain death model rats were in accordance with the brain death donor requirement: (deep coma, spontaneous respiratory arrest, no conditioned reflex, pupil light reflex disappeared, corneal reflex disappeared. The mean arterial pressure of 6 hours was still more than 80 mm HgN. The Scr of group G _ 2 was higher than that of group G _ 3, and the Scr of group G _ 3 was higher than that of group G _ 1 after renal transplantation, and the difference was not statistically significant (P 0.05). The renal tissues in each group were stained with HE and observed under light microscope. It was found that there were obvious monocyte infiltration and tubular epithelial inflammation in G2 group and there was no significant difference between G3 group and G1 group, and there were obvious inflammatory changes in artery vessels of grafts in all three groups, and the severity of inflammation in G2 group was more serious than that in G1 and G3 groups. However, there was no significant difference among the three groups (P 0.05). Immunohistochemical staining showed that the expression of IL-1 尾 -TNF- 伪 in glomeruli, tubules and interstitial cells in G2 group was lower than that in G2 group (P 0.05). Conclusion\
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2

【参考文献】