脂肪間充質(zhì)干細(xì)胞移植改善糖尿病心肌病大鼠心肌損傷的研究
發(fā)布時(shí)間：2018-07-29 20:29
【摘要】：目的:觀察間充質(zhì)干細(xì)胞對(duì)大鼠糖尿病心肌病的心肌損傷及心功能的影響及可能的作用機(jī)制。方法:第一部分:構(gòu)建糖尿病心肌病(Diabetic cardiomyopathy, DCM)大鼠模型,分離培養(yǎng)SD大鼠的脂肪間充質(zhì)干細(xì)胞,SD大鼠隨機(jī)分為3組,正常組(Normal)、DCM組、DCM+ADMSCs組,18周時(shí)連續(xù)4次經(jīng)尾靜脈進(jìn)行ADMSCs移植后觀察3組大鼠血糖水平、口服葡萄糖耐量試驗(yàn)(OGTTs)、腹腔注射胰島素耐量試驗(yàn)(IPITTs)變化,心肌組織行蘇木精-伊紅(HE)、馬松(Masson)、油紅O染色,Cobas8000生化儀檢測(cè)心肌損傷因子,心臟超聲儀檢測(cè)大鼠心功能,免疫熒光觀察心肌MG53蛋白表達(dá)水平,蛋白印記(Western blot)法檢測(cè)心肌組織MG53、IRS1、IR、p-Akt表達(dá)水平。第二部分:分離培養(yǎng)SD乳鼠原代心肌細(xì)胞,隨機(jī)分為3組,正常組(Normal)、高糖組(HG)、高糖+脂肪間充質(zhì)干細(xì)胞培養(yǎng)上清組(HG+ADMSCs-CM),干預(yù)后于倒置相差顯微鏡下觀察心肌細(xì)胞形態(tài)及搏動(dòng)情況,并計(jì)算表面積,采用定量實(shí)時(shí)聚合酶鏈反應(yīng)(Q-PCR)檢測(cè)各組心肌細(xì)胞中ANP、BNP、β -MHC mRNA表達(dá)水平。HG+ADMSCs-CM組再次分為不同濃度組(0.25mL、0.5mL、1mL、2mL)。Western blot法檢測(cè)各組心肌細(xì)胞中MG53、IRS1、IR、p-Akt表達(dá)水平。結(jié)果:1、連續(xù)4次ADMSCs移植后,DCM+ADMSCs組FBG及PBG水平與DCM組比較,均明顯降低(P0.01),心臟收縮及舒張功能明顯提高(P0.05),心肌肥大與基質(zhì)纖維化明顯改善。MG53蛋白表達(dá)水平:DCM組與Normal組相比明顯增高,DCM+ADMSCs組與DCM組相比明顯降低,差異具有統(tǒng)計(jì)學(xué)意義(P均0.05)。IRS1、IR、p-Akt蛋白表達(dá)水平:DCM組與Normal組相比明顯降低,DCM+ADMSCs組與DCM組相比明顯增高,差異具有統(tǒng)計(jì)學(xué)意義(P均0.05)。2、心肌細(xì)胞體外培養(yǎng)干預(yù)72h后,HG組心肌細(xì)胞與Normal組相比表面積增大,搏動(dòng)頻率減弱,ANP、BNP、β-MHCmRNA表達(dá)升高;HG+ADMSCs-CM組與HG組相比心肌細(xì)胞表面積減小,搏動(dòng)頻率增強(qiáng),ANP、BNP、β-MHCmRNA表達(dá)降低,差異均具有統(tǒng)計(jì)學(xué)意義(P均0.01)。給予ADMSCs-CM梯度干預(yù)72h后,MG53蛋白表達(dá)水平:2mL組與0.25ml組相比減低,差異具有統(tǒng)計(jì)學(xué)意義(P均0.01); IRS1、IR、p-Akt蛋白表達(dá)水平:2mL組與0.25mL組相比明顯增高,差異具有統(tǒng)計(jì)學(xué)意義(P均0.01)。MG53蛋白相對(duì)表達(dá)水平與IRS1、IR、p-Akt蛋白表達(dá)水平呈負(fù)相關(guān)(r=-0.75, -0.94, -0.84)。結(jié)論:1高糖血癥是DCM大鼠心肌損傷的危險(xiǎn)因素,心肌肥大及基質(zhì)纖維化是DCM的主要病理改變。2、MSCs可以降低DCM大鼠血糖水平,改善心肌損傷及心功能障礙。3、MG53蛋白的高表達(dá)參與DCM的發(fā)生發(fā)展,MSCs可能通過(guò)下調(diào)DCM造成的MG53的高表達(dá),進(jìn)而上調(diào)IRS1、IR、p-Akt的表達(dá)緩解胰島素抵抗,改善心肌損傷。用MSCs-CM進(jìn)行干預(yù)可以觀察到相同效果,說(shuō)明MSCs既可以通過(guò)細(xì)胞直接發(fā)揮心肌損傷保護(hù)作用,又可以通過(guò)分泌途徑作用于MG53蛋白及胰島素信號(hào)通路發(fā)揮以上作用。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1;R542.2
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1;R542.2
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