脂肪间充质干细胞移植改善糖尿病心肌病大鼠心肌损伤的研究
发布时间：2018-07-29 20:29
【摘要】：目的:观察间充质干细胞对大鼠糖尿病心肌病的心肌损伤及心功能的影响及可能的作用机制。方法:第一部分:构建糖尿病心肌病(Diabetic cardiomyopathy, DCM)大鼠模型,分离培养SD大鼠的脂肪间充质干细胞,SD大鼠随机分为3组,正常组(Normal)、DCM组、DCM+ADMSCs组,18周时连续4次经尾静脉进行ADMSCs移植后观察3组大鼠血糖水平、口服葡萄糖耐量试验(OGTTs)、腹腔注射胰岛素耐量试验(IPITTs)变化,心肌组织行苏木精-伊红(HE)、马松(Masson)、油红O染色,Cobas8000生化仪检测心肌损伤因子,心脏超声仪检测大鼠心功能,免疫荧光观察心肌MG53蛋白表达水平,蛋白印记(Western blot)法检测心肌组织MG53、IRS1、IR、p-Akt表达水平。第二部分:分离培养SD乳鼠原代心肌细胞,随机分为3组,正常组(Normal)、高糖组(HG)、高糖+脂肪间充质干细胞培养上清组(HG+ADMSCs-CM),干预后于倒置相差显微镜下观察心肌细胞形态及搏动情况,并计算表面积,采用定量实时聚合酶链反应(Q-PCR)检测各组心肌细胞中ANP、BNP、β -MHC mRNA表达水平。HG+ADMSCs-CM组再次分为不同浓度组(0.25mL、0.5mL、1mL、2mL)。Western blot法检测各组心肌细胞中MG53、IRS1、IR、p-Akt表达水平。结果:1、连续4次ADMSCs移植后,DCM+ADMSCs组FBG及PBG水平与DCM组比较,均明显降低(P0.01),心脏收缩及舒张功能明显提高(P0.05),心肌肥大与基质纤维化明显改善。MG53蛋白表达水平:DCM组与Normal组相比明显增高,DCM+ADMSCs组与DCM组相比明显降低,差异具有统计学意义(P均0.05)。IRS1、IR、p-Akt蛋白表达水平:DCM组与Normal组相比明显降低,DCM+ADMSCs组与DCM组相比明显增高,差异具有统计学意义(P均0.05)。2、心肌细胞体外培养干预72h后,HG组心肌细胞与Normal组相比表面积增大,搏动频率减弱,ANP、BNP、β-MHCmRNA表达升高;HG+ADMSCs-CM组与HG组相比心肌细胞表面积减小,搏动频率增强,ANP、BNP、β-MHCmRNA表达降低,差异均具有统计学意义(P均0.01)。给予ADMSCs-CM梯度干预72h后,MG53蛋白表达水平:2mL组与0.25ml组相比减低,差异具有统计学意义(P均0.01); IRS1、IR、p-Akt蛋白表达水平:2mL组与0.25mL组相比明显增高,差异具有统计学意义(P均0.01)。MG53蛋白相对表达水平与IRS1、IR、p-Akt蛋白表达水平呈负相关(r=-0.75, -0.94, -0.84)。结论:1高糖血症是DCM大鼠心肌损伤的危险因素,心肌肥大及基质纤维化是DCM的主要病理改变。2、MSCs可以降低DCM大鼠血糖水平,改善心肌损伤及心功能障碍。3、MG53蛋白的高表达参与DCM的发生发展,MSCs可能通过下调DCM造成的MG53的高表达,进而上调IRS1、IR、p-Akt的表达缓解胰岛素抵抗,改善心肌损伤。用MSCs-CM进行干预可以观察到相同效果,说明MSCs既可以通过细胞直接发挥心肌损伤保护作用,又可以通过分泌途径作用于MG53蛋白及胰岛素信号通路发挥以上作用。
[Abstract]:Aim: to investigate the effects of mesenchymal stem cells (MSCs) on myocardial injury and cardiac function in diabetic cardiomyopathy (DM) rats. Methods: in the first part, the (Diabetic cardiomyopathy, DCM) rat model of diabetic cardiomyopathy was established, and the SD rats of adipose mesenchymal stem cells isolated from SD rats were randomly divided into three groups. The blood glucose levels of the three groups were observed after ADMSCs transplantation through the tail vein for 4 times at 18 weeks in the (Normal) ADMSCs group. The changes of insulin tolerance test (IPITTs) in the three groups were observed by oral glucose tolerance test (OGTTs), intraperitoneal injection of insulin tolerance test (OGTTs), and intraperitoneal injection of insulin tolerance test (IPITTs). Myocardial injury factors were detected by hematoxylin-eosin (HE), Ma Song (Masson), oil red O staining, cardiac damage factor was detected by echocardiography, cardiac function was detected by echocardiography, and expression of myocardial MG53 protein was observed by immunofluorescence. Protein imprinted (Western blot) method was used to detect the expression of MG53, IRS1 and IRP-Akt in myocardium. The second part: isolated and cultured neonatal SD rat primary cardiomyocytes, and were randomly divided into 3 groups. The supernatant group (HG ADMSCs-CM) of (HG), high glucose adipose mesenchymal stem cells (HG ADMSCs-CM) in (Normal), high glucose group was used to observe the morphology and pulsation of cardiomyocytes under inverted phase contrast microscope, and to calculate the surface area. Quantitative real-time polymerase chain reaction (Q-PCR) was used to detect the expression of 尾 -MHC mRNA and 尾 -MHC mRNA. The HG ADMSCs-CM group was subdivided into different concentration groups (0.25mL, 0.5mLL, 1mLL2mL). Western blot assay was used to detect the expression of MG53HHC mRNA and IRP-Akt in cardiomyocytes of each group. Results the levels of FBG and PBG in ADMSCs group after 4 consecutive ADMSCs transplants were compared with those in DCM group. Myocardial hypertrophy and matrix fibrosis were significantly improved. The protein expression level of MG53 in Normal group was significantly higher than that in Normal group. Compared with DCM group, the expression of MG53 protein in DCM ADMSCs group was significantly lower than that in DCM group. The difference was statistically significant (P 0.05). The expression level of IRS1 + -Akt protein was significantly lower in the Normal group than in the Normal group, and increased significantly in the ADMSCs group compared with the DCM group, and the expression level of IRS1 protein in the DCM group was significantly lower than that in the DCM group. The difference was statistically significant (P 0.05) .2After cultured in vitro for 72 hours, the specific surface area of cardiomyocytes in HG group and Normal group increased, the beating frequency decreased, and the expression of 尾 -MHCmRNA increased. The surface area of cardiomyocytes in HG ADMSCs-CM group decreased as compared with that in HG group. The expression of BNPand 尾 -MHC mRNA in ANPs was significantly lower than that in control group (P 0.01). After 72 h of ADMSCs-CM gradient intervention, the expression level of MG53 protein was significantly lower in the 2 mL group than that in the 0.25ml group (all P 0.01), and the expression level of p-Akt protein in the 2 mL group was significantly higher than that in the 0.25mL group (P 0.01), and the expression level of MG53 protein in the 2 mL group was significantly higher than that in the 0.25mL group (P < 0. 01). The difference was statistically significant (P0.01). There was a negative correlation between the relative expression level of MG53 protein and the expression level of IRS1IRP- Akt protein (r-0.75, -0.94, -0.84). Conclusion 1 hyperglycemia is the risk factor of myocardial injury in DCM rats. Myocardial hypertrophy and matrix fibrosis are the main pathological changes of DCM. 2MSCs can reduce the level of blood glucose in DCM rats. The improvement of myocardial injury and the overexpression of MG53 protein in cardiac dysfunction may be involved in the development of DCM, which may alleviate insulin resistance and improve myocardial injury by down-regulating the high expression of MG53 induced by DCM, and then up-regulating the expression of IRS1-IRP-Akt. The same effect can be observed by MSCs-CM intervention, which indicates that MSCs can not only exert the protective effect of myocardial injury directly through cells, but also act on the MG53 protein and insulin signaling pathway through secretory pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R542.2
本文编号：2153923
[Abstract]:Aim: to investigate the effects of mesenchymal stem cells (MSCs) on myocardial injury and cardiac function in diabetic cardiomyopathy (DM) rats. Methods: in the first part, the (Diabetic cardiomyopathy, DCM) rat model of diabetic cardiomyopathy was established, and the SD rats of adipose mesenchymal stem cells isolated from SD rats were randomly divided into three groups. The blood glucose levels of the three groups were observed after ADMSCs transplantation through the tail vein for 4 times at 18 weeks in the (Normal) ADMSCs group. The changes of insulin tolerance test (IPITTs) in the three groups were observed by oral glucose tolerance test (OGTTs), intraperitoneal injection of insulin tolerance test (OGTTs), and intraperitoneal injection of insulin tolerance test (IPITTs). Myocardial injury factors were detected by hematoxylin-eosin (HE), Ma Song (Masson), oil red O staining, cardiac damage factor was detected by echocardiography, cardiac function was detected by echocardiography, and expression of myocardial MG53 protein was observed by immunofluorescence. Protein imprinted (Western blot) method was used to detect the expression of MG53, IRS1 and IRP-Akt in myocardium. The second part: isolated and cultured neonatal SD rat primary cardiomyocytes, and were randomly divided into 3 groups. The supernatant group (HG ADMSCs-CM) of (HG), high glucose adipose mesenchymal stem cells (HG ADMSCs-CM) in (Normal), high glucose group was used to observe the morphology and pulsation of cardiomyocytes under inverted phase contrast microscope, and to calculate the surface area. Quantitative real-time polymerase chain reaction (Q-PCR) was used to detect the expression of 尾 -MHC mRNA and 尾 -MHC mRNA. The HG ADMSCs-CM group was subdivided into different concentration groups (0.25mL, 0.5mLL, 1mLL2mL). Western blot assay was used to detect the expression of MG53HHC mRNA and IRP-Akt in cardiomyocytes of each group. Results the levels of FBG and PBG in ADMSCs group after 4 consecutive ADMSCs transplants were compared with those in DCM group. Myocardial hypertrophy and matrix fibrosis were significantly improved. The protein expression level of MG53 in Normal group was significantly higher than that in Normal group. Compared with DCM group, the expression of MG53 protein in DCM ADMSCs group was significantly lower than that in DCM group. The difference was statistically significant (P 0.05). The expression level of IRS1 + -Akt protein was significantly lower in the Normal group than in the Normal group, and increased significantly in the ADMSCs group compared with the DCM group, and the expression level of IRS1 protein in the DCM group was significantly lower than that in the DCM group. The difference was statistically significant (P 0.05) .2After cultured in vitro for 72 hours, the specific surface area of cardiomyocytes in HG group and Normal group increased, the beating frequency decreased, and the expression of 尾 -MHCmRNA increased. The surface area of cardiomyocytes in HG ADMSCs-CM group decreased as compared with that in HG group. The expression of BNPand 尾 -MHC mRNA in ANPs was significantly lower than that in control group (P 0.01). After 72 h of ADMSCs-CM gradient intervention, the expression level of MG53 protein was significantly lower in the 2 mL group than that in the 0.25ml group (all P 0.01), and the expression level of p-Akt protein in the 2 mL group was significantly higher than that in the 0.25mL group (P 0.01), and the expression level of MG53 protein in the 2 mL group was significantly higher than that in the 0.25mL group (P < 0. 01). The difference was statistically significant (P0.01). There was a negative correlation between the relative expression level of MG53 protein and the expression level of IRS1IRP- Akt protein (r-0.75, -0.94, -0.84). Conclusion 1 hyperglycemia is the risk factor of myocardial injury in DCM rats. Myocardial hypertrophy and matrix fibrosis are the main pathological changes of DCM. 2MSCs can reduce the level of blood glucose in DCM rats. The improvement of myocardial injury and the overexpression of MG53 protein in cardiac dysfunction may be involved in the development of DCM, which may alleviate insulin resistance and improve myocardial injury by down-regulating the high expression of MG53 induced by DCM, and then up-regulating the expression of IRS1-IRP-Akt. The same effect can be observed by MSCs-CM intervention, which indicates that MSCs can not only exert the protective effect of myocardial injury directly through cells, but also act on the MG53 protein and insulin signaling pathway through secretory pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R542.2
【参考文献】
相关期刊论文 前2条
1 杜莉莉;吕润潇;杨晓漪;许静;于艳秋;马廷贤;;胎盘间充质干细胞缺氧培养液对血管内皮细胞生物学特性的影响[J];解剖学研究;2016年02期
2 魏立民;马博清;;糖尿病心脏病的研究进展[J];国外医学(老年医学分册);2002年01期
，本文编号：2153923
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2153923.html
最近更新
教材专著
