CD73在ADMSCs向心肌分化和细胞因子VEGF、TNF-α分泌中的作用

发布时间：2018-06-23 09:44

本文选题：CD73 + 血管内皮生长因子　；参考：《新乡医学院》2016年硕士论文

【摘要】：背景脂肪间充质干细胞(ADMSCs)目前是心肌细胞移植和心肌组织工程最具应用前景的干细胞之一。ADMSCs由具有不同的生物学特性和分化潜能的亚群组成,目前研究多为混合细胞群,缺少研究的针对性和实效性,这也许也是影响疗效的重要原因之一。筛选心肌分化的优势亚群并研究其生物学特性,有助于提高研究的针对性和细胞治疗的效果。目的探讨CD73在脂肪间充质干细胞向心肌分化和细胞因子分泌中的作用,证明CD73分子在心肌修复中的功能。方法1.体外培养鉴定小鼠脂肪源间充质干细胞(ADMSCs),检测表面标记CD29、CD44、CD73和CD45的表达及成骨、成脂、成心肌分化潜能。2.流式细胞术分选ADMSCs CD73+亚群和CD73-亚群。使用CD73特异性抑制剂5′-α,β-亚甲基二磷酸腺苷(adenosine 5′-α,β-methylene diphosphate,APCP)抑制ADMSCs CD73的表达。实验分组:CD73+组、CD73-组和CD73+-APCP组。3.构建CD73过表达(CD73-OE)、CD73干扰(CD73-RNAi)和GFP(C-GFP)慢病毒载体,感染ADMSCs。实验分组:CD73-OE组、CD73-RNAi组和C-GFP组。免疫荧光法检测病毒转染效率。4.分别对CD73+组、CD73-组和CD73+-APCP组进行心肌化诱导,免疫荧光法和流式细胞术检测心肌特异性肌钙蛋白T(cTnT)表达情况。5.提取各组细胞培养上清液,ELISA法检测细胞因子VEGF、TNF-α的浓度。结果1.ADMSCs表面标记检测和诱导分化结果显示:CD29、CD44的表达率分别为53.1±1.1%、34.8±2.1%,CD73表达不稳定,在1.8%~19.7%之间波动,不表达造血干细胞标记CD45;在体外特定培养条件下,ADMSCs可诱导分化为成骨细胞、脂肪细胞和心肌样细胞。2.5-aza诱导21天,免疫荧光显示各组均表达心肌特异性蛋白cTnT,CD73+组cTnT阳性率为85.0±6.3%,CD73-组cTnT阳性率为2.1±0.6%,CD73+-APCP组cTnT阳性率为4.8±1.7%,CD73+组心肌化诱导效率高于CD73+-APCP组和CD73-组(P0.05)。流式细胞术检测结果显示cTnT表达率:CD73+组为44.9%,CD73-组为1.5%,CD73+-APCP组为3.3%。3.ELISA法检测VEGF结果显示,5-aza诱导前:CD73+-APCP组明显低于CD73+组和CD73-组,P0.01;5-aza诱导10天:CD73+-APCP组明显低于CD73+组和CD73-组,P0.01;CD73+组高于CD73-组,P0.05;5-aza诱导20天:CD73+-APCP组明显低于CD73+组和CD73-组,P0.01;CD73+组高于CD73-组,P0.05。ELISA法检测TNF-α结果显示:分别在5-aza诱导前,诱导10天,诱导20天,CD73+-APCP组明显低于CD73+组和CD73-组,P0.01;CD73+组与CD73-组比较,差异无统计学意义。4.慢病毒转染后,ELISA法检测VEGF结果显示:CD73-OE组高于CD73-RNAi组和C-GFP组,P0.05;TNF-α浓度:CD73-OE低于CD73-RNAi组和C-GFP组,P0.05。结论CD73能促进ADMSCs向心肌分化,促进VEGF分泌,抑制TNF-α分泌。
[Abstract]:Background Adipose mesenchymal stem cells (ADMSCs) are currently one of the most promising stem cells in cardiomyocyte transplantation and myocardial tissue engineering. The lack of pertinence and effectiveness may also be one of the important factors affecting the efficacy. Screening the dominant subsets of myocardial differentiation and studying its biological characteristics are helpful to improve the pertinence of the research and the effect of cell therapy. Objective to investigate the role of CD73 in myocardial differentiation and cytokine secretion of adipose mesenchymal stem cells. Method 1. Mouse adipose derived mesenchymal stem cells (ADMSCs) were identified in vitro. The expression of CD29, CD4, CD4, CD73, CD45, osteogenesis, adipogenesis and myocardial differentiation potential were detected. CD73 subsets and CD73-subsets of ADMSCs were separated by flow cytometry. CD73 specific inhibitor (adenosine 5- 伪, 尾 -methylene diphosphate- 伪, 尾 -methylene diphosphate- APCP) was used to inhibit the expression of CD73. Experimental group: CD73 group: CD73- group and CD73 -APCP group. 3. CD73 overexpression (CD73-OE) interference (CD73-RNAi) and GFP (C-GFP) lentivirus vectors were constructed and infected with ADMSCs. The experiment was divided into two groups: CD73-RNAi group and C-GFP group. Immunofluorescence assay was used to detect the efficiency of virus transfection. The expression of cardiac troponin T (cTnT) was detected by immunofluorescence and flow cytometry respectively in CD73 group and CD73 -APCP group. The concentration of VEGF TNF- 伪 was detected by Elisa. Results 1. The expression rates of CD29 + CD44 in ADMSCs were 53.1 卤1.1 and 34.8 卤2.1respectively, which fluctuated between 1.8% and 19.7%, and did not express hematopoietic stem cell marker CD45.The ADMSCs could be induced to differentiate into osteoblasts under specific culture conditions in vitro. After 21 days of induction of adipocytes and cardiomyocytes, immunofluorescence showed that the positive rate of cTnT in CD73 group was 85.0 卤6.3and that in CD73- group was 2.1 卤0.60.The positive rate of cTnT in CD73 -APCP group was 4.8 卤1.75.The positive rate of cTnT in CD73 group was higher than that in CD73 -APCP group and CD73- group (P0.05). Flow cytometry showed that the expression rate of cTnT was 44.9 and CD73- group was 1.5 and CD73-APCP was 3.30.3.The results of Elisa showed that the percentage of VEGF expression in CD73-APCP group was significantly lower than that in CD73 group and CD73- group P0.015-aza induction for 10 days, which was significantly lower than that in CD73 group and CD73- group P0.01a CD73 group before induction of CD73 and CD73-APCP group was significantly lower than that in CD73 group and CD73 group P0.01A PCP group was significantly higher than that in CD73 group and CD73 group P0.01a CD73 group before induction. The TNF- 伪 levels in CD73- group were significantly lower than those in CD73 group and CD73-group P0.01 + CD73 group after 20 days of induction by P0.055-aza. The results showed that: before 5-aza induction, TNF- 伪 was detected by Elisa. After 10 days of induction and 20 days of induction, CD73-APCP group was significantly lower than CD73 group and CD73-group P0. 01 + CD73 group and CD73-group. There was no significant difference between CD73-group and CD73-group. After lentivirus transfection, the expression of VEGF was detected by Elisa. The results showed that the concentration of TNF- 伪 was higher in 10% CD73-OE group than that in CD73-RNAi group and C-GFP group, and the concentration of TNF- 伪 was lower than that in CD73-RNAi group and C-GFP group. Conclusion CD73 can promote the differentiation of ADMSCs into myocardium, promote the secretion of VEGF and inhibit the secretion of TNF- 伪.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R542.2

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