本文選題：CD73 + 血管內(nèi)皮生長因子　； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2016年碩士論文

【摘要】：背景脂肪間充質(zhì)干細胞(ADMSCs)目前是心肌細胞移植和心肌組織工程最具應(yīng)用前景的干細胞之一。ADMSCs由具有不同的生物學(xué)特性和分化潛能的亞群組成,目前研究多為混合細胞群,缺少研究的針對性和實效性,這也許也是影響療效的重要原因之一。篩選心肌分化的優(yōu)勢亞群并研究其生物學(xué)特性,有助于提高研究的針對性和細胞治療的效果。目的探討CD73在脂肪間充質(zhì)干細胞向心肌分化和細胞因子分泌中的作用,證明CD73分子在心肌修復(fù)中的功能。方法1.體外培養(yǎng)鑒定小鼠脂肪源間充質(zhì)干細胞(ADMSCs),檢測表面標記CD29、CD44、CD73和CD45的表達及成骨、成脂、成心肌分化潛能。2.流式細胞術(shù)分選ADMSCs CD73+亞群和CD73-亞群。使用CD73特異性抑制劑5′-α,β-亞甲基二磷酸腺苷(adenosine 5′-α,β-methylene diphosphate,APCP)抑制ADMSCs CD73的表達。實驗分組:CD73+組、CD73-組和CD73+-APCP組。3.構(gòu)建CD73過表達(CD73-OE)、CD73干擾(CD73-RNAi)和GFP(C-GFP)慢病毒載體,感染ADMSCs。實驗分組:CD73-OE組、CD73-RNAi組和C-GFP組。免疫熒光法檢測病毒轉(zhuǎn)染效率。4.分別對CD73+組、CD73-組和CD73+-APCP組進行心肌化誘導(dǎo),免疫熒光法和流式細胞術(shù)檢測心肌特異性肌鈣蛋白T(cTnT)表達情況。5.提取各組細胞培養(yǎng)上清液,ELISA法檢測細胞因子VEGF、TNF-α的濃度。結(jié)果1.ADMSCs表面標記檢測和誘導(dǎo)分化結(jié)果顯示:CD29、CD44的表達率分別為53.1±1.1%、34.8±2.1%,CD73表達不穩(wěn)定,在1.8%~19.7%之間波動,不表達造血干細胞標記CD45;在體外特定培養(yǎng)條件下,ADMSCs可誘導(dǎo)分化為成骨細胞、脂肪細胞和心肌樣細胞。2.5-aza誘導(dǎo)21天,免疫熒光顯示各組均表達心肌特異性蛋白cTnT,CD73+組cTnT陽性率為85.0±6.3%,CD73-組cTnT陽性率為2.1±0.6%,CD73+-APCP組cTnT陽性率為4.8±1.7%,CD73+組心肌化誘導(dǎo)效率高于CD73+-APCP組和CD73-組(P0.05)。流式細胞術(shù)檢測結(jié)果顯示cTnT表達率:CD73+組為44.9%,CD73-組為1.5%,CD73+-APCP組為3.3%。3.ELISA法檢測VEGF結(jié)果顯示,5-aza誘導(dǎo)前:CD73+-APCP組明顯低于CD73+組和CD73-組,P0.01;5-aza誘導(dǎo)10天:CD73+-APCP組明顯低于CD73+組和CD73-組,P0.01;CD73+組高于CD73-組,P0.05;5-aza誘導(dǎo)20天:CD73+-APCP組明顯低于CD73+組和CD73-組,P0.01;CD73+組高于CD73-組,P0.05。ELISA法檢測TNF-α結(jié)果顯示:分別在5-aza誘導(dǎo)前,誘導(dǎo)10天,誘導(dǎo)20天,CD73+-APCP組明顯低于CD73+組和CD73-組,P0.01;CD73+組與CD73-組比較,差異無統(tǒng)計學(xué)意義。4.慢病毒轉(zhuǎn)染后,ELISA法檢測VEGF結(jié)果顯示:CD73-OE組高于CD73-RNAi組和C-GFP組,P0.05;TNF-α濃度:CD73-OE低于CD73-RNAi組和C-GFP組,P0.05。結(jié)論CD73能促進ADMSCs向心肌分化,促進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- 偽, 尾 -methylene diphosphate- 偽, 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- 偽 was detected by Elisa. Results 1. The expression rates of CD29 + CD44 in ADMSCs were 53.1 鹵1.1 and 34.8 鹵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 鹵6.3and that in CD73- group was 2.1 鹵0.60.The positive rate of cTnT in CD73 -APCP group was 4.8 鹵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- 偽 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- 偽 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- 偽 was higher in 10% CD73-OE group than that in CD73-RNAi group and C-GFP group, and the concentration of TNF- 偽 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- 偽.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R542.2

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