体外诱导骨髓间充质干细胞分化为肾小管上皮样细胞的研究

发布时间：2018-03-22 00:07

本文选题：骨髓间充质干细胞　切入点：肾小管上皮样细胞　出处：《福建医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:探讨体外诱导骨髓间充质干细胞(mesenchyml stem cells, MSCs)向肾小管上皮样细胞分化的可能性。并观察不同条件下骨髓间充质干细胞体外诱导分化后的差异。方法:抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的骨髓间充质干细胞,以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的骨髓间充质干细胞,分别用含有胎牛血清(空白对照组);胎牛血清+缺血再灌注肾脏匀浆上清+全反式维甲酸ATRA(ATRA对照组);胎牛血清+缺血再灌注肾脏匀浆上清+表皮生长因子(EGF)(EGF组);胎牛血清+缺血再灌注肾脏匀浆上清+肝细胞生长因子(HGF)(HGF组);胎牛血清+缺血再灌注肾脏匀浆上清+骨形成蛋白(BMP-7)(BMP-7组);胎牛血清+缺血再灌注肾脏匀浆上清+促红细胞生成素(EPO)(EPO组);胎牛血清+缺血再灌注肾脏匀浆上清+上述四种因子(联合诱导组)的培养基进行培养。诱导7天后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达,免疫细胞化学法检测cytokeratin-18蛋白的表达,免疫荧光细胞化学法检测E-cadherin蛋白的表达,RT-PCR检测E-cadherinmRNA的表达。结果: (1)流式细胞仪显示体外分离培养的第3代大鼠骨髓干细胞表面抗原表达: CD44阳性细胞表达率98.7%,CD90阳性细胞表达率98.1%, CD29阳性细胞表达率96.1%,而CD11b/c阳性细胞表达率14.7%, CD34阳性细胞表达率1.98%;(2)诱导7天后,与空白对照组相比,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)部分细胞变圆形、短梭形单层排列,联合诱导组的大部分细胞变圆形、短梭形,细胞密集处呈鹅卵石样排列;(3)碱性磷酸酶染色示:空白对照组细胞为阴性,ATRA对照组和单因子诱导组(EGF组、HGF组、BMP-7组、EPO组)有出现部分的阳性细胞,而在联合诱导组中阳性细胞数明显的增多;(4)免疫细胞化学示:空白对照组无表达,cytokeratin-18各组表达的阳性细胞率分别为:ATRA对照组27.40±2.70%;单因子EGF组29.60±4.51%;单因子HGF组26.20±3.70%;单因子BMP-7组26.80±5.00%;单因子EPO组27.80±3.03%;联合诱导组44.00±3.16%,与空白对照组相比均明显增加(P0.01 vs空白对照组),其中联合诱导组增加更为明显;(5)免疫荧光细胞化学示:在高倍视野下,空白对照组无表达, E-cadherin各组表达的阳性细胞均数分别为:ATRA对照组16.40±2.69;单因子EGF组18.25±3.50;单因子HGF组19.24±3.41;单因子BMP-7组16.06±2.00;单因子EPO组19.33±3.57;联合诱导组30.26±5.16,与空白对照组相比均明显增加(P0.01 vs空白对照组),其中联合诱导组增加更为明显;(6)RT-PCR方法检测到联合诱导组MSC的E-cadherin的mRNA表达明显高于ATRA对照组和单因子诱导组(P㩳0.01),而空白对照组不表达。结论:以上四中因子在体外模拟的急性肾衰微环境中可诱导骨髓间充质干细胞分化为肾小管上皮样细胞。
[Abstract]:Aim: to investigate the possibility of inducing mesenchymal stem cells (MSCs) to differentiate into renal tubular epithelioid cells (TECs) in vitro. Methods: bone marrow of SD rats was isolated by density gradient centrifugation and purified bone marrow mesenchymal stem cells were obtained by adherent screening. The surface markers of mesenchymal stem cells (MSCs) were identified by flow cytometry. Fetal bovine serum (blank control group); fetal bovine serum ischemia reperfusion renal homogenate supernatant total trans retinoic acid (ATRA(ATRA) control group; fetal bovine serum ischemia reperfusion kidney homogenate supernatant epidermal growth factor (EGF) group; Fetal bovine serum homogenate supernatant hepatocyte growth factor HGFGF-HGF group; fetal bovine serum homogenate supernatant bone morphogenetic protein (BMP-7); fetal bovine serum homogenate homogenate homogenate supernatant promotion; fetal bovine serum ischemia reperfusion renal homogenate homogenate supernatant supernatant of HGF group; fetal bovine serum ischemia reperfusion renal homogenate supernatant supernatant. Erythropoietin EPO-EPO group and fetal bovine serum ischemia reperfusion kidney homogenate supernatant of the above four factors (combined induction group) were cultured. After 7 days of induction, The expression of alkaline phosphatase was detected by chemical staining, the expression of cytokeratin-18 protein was detected by immunocytochemistry, the expression of E-cadherin protein was detected by immunofluorescence cytochemistry and the expression of E-cadherinmRNA was detected by RT-PCR. Results:. Flow cytometry showed that the expression rate of CD44 positive cells was 98.70.The expression rate of CD90 positive cells was 98.1. The expression rate of CD29 positive cells was 96.1.The expression rate of CD11b/c positive cells was 14.7g and CD34 positive cells was 14.7%. The expression rate of sex cells was 1.98%, 7 days after induction, Compared with the blank control group, the cells of ATRA control group and EGF group HGF group (HGF group) became round, short fusiform monolayer arrangement, and most of the cells in the combined induction group became round and fusiform. The results of alkaline phosphatase staining showed that the cells in the blank control group were negative ATRA control group and single factor induction group (EGF group, HGF group, BMP-7 group, EPO group). Immunocytochemistry showed that the positive rate of cytokeratin-18 in the blank control group was 27.40 卤2.70 in the control group, 29.60 卤4.51 in the single-factor EGF group, 26.20 卤3.70 in the single-factor HGF group and 27.40 卤2.70 in the single-factor BMP-7 group. Group 26.80 卤5.00; single factor EPO group 27.80 卤3.03; combined induction group 44.00 卤3.16; compared with blank control group, P 0.01 vs blank control group; The average number of positive cells in E-cadherin group was 16.40 卤2.69 in control group, 18.25 卤3.50 in single-factor EGF group, 19.24 卤3.41 in single-factor HGF group, 16.06 卤2.00 in single-factor BMP-7 group, 19.33 卤3.57 in single-factor EPO group and 30.26 卤5.16 in combined induction group, respectively. The mRNA expression of MSC in the co-induction group was significantly higher than that in the ATRA control group and the univariate induction group by RT-PCR, and the expression of E-cadherin in the co-induction group was significantly higher than that in the ATRA control group and single-factor induction group, and the expression of E-cadherin in the co-induction group was significantly higher than that in the co-induction group. It was not expressed in the blank control group. Conclusion: the above four factors can induce the differentiation of bone marrow mesenchymal stem cells into renal tubular epithelioid cells in acute renal decay environment in vitro.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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