脂肪基质细胞分化为心肌细胞的研究
发布时间：2019-05-18 14:41
【摘要】：干细胞治疗在心血管疾病的临床应用研究中具有巨大的发展潜力。虽然一些体内和体外的实验结果令人振奋,然而,最佳干细胞来源以及干细胞成心肌细胞分化的分子机理仍有待进一步研究和证实。脂肪基质细胞(ADSCs)以其干细胞含量丰富、易采集、易培养、对供体损伤小、具备多谱系分化潜能和可自体移植等优势受到干细胞研究领域的广泛关注。虽然ADSCs可定向分化成心肌细胞的能力已被证实,但这一能力的验证目前仅限于兔、小鼠和人三个物种,而且对ADSCs成心肌细胞分化的机理也不甚清楚。 本研究以6-8周龄小鼠为实验动物,从腹股沟皮下脂肪组织中分离获得ADSCs,培养于仅添加青霉素、链霉素和20% NBS的DMEM培养液中观察其自发分化,采用流式细胞术分析ADSCs表面标记CD29、CD31、CD34和CD105的表达;细胞免疫荧光染色、免疫组织化学染色和流式细胞术分析α-actin、MF20、Connexin45、cMHC、cTnI、Nkx2.5和GATA4的表达;细胞免疫荧光染色和Ca2+成像分析验证ADSCs源心肌细胞E-C coupling的畅通;接着使用Touchdown-PCR方法克隆cardiacα-actin基因CDS序列,并构建其重组腺病毒表达载体(pAd- Nkx2.5),使用pAd-Nkx2.5和pAd-actin侵染原代培养的小鼠ADSCs,Real time-PCR和Western Blot检测超表达效果;并使用如上所述方法量化分析过表达前后心肌标志蛋白的变化,最后从整体上探讨过表达Nkx2.5和cardiacα-actin对小鼠ADSCs成心肌细胞分化的影响,主要研究结果如下: 1、原代小鼠ADSCs在体外培养条件下表达CD29和CD105,但不表达CD31和CD34,它们可在体外较低的培养条件下(DMEM+20% NBS,未添加任何细胞因子、5-azacytidine或抗氧化剂等)自发分化成心肌细胞,这些细胞可自发收缩,表达心肌标志蛋白α-actin、MF20、Connexin45、cMHC、cTnI、Nkx2.5和GATA4,收缩细胞中E-C coupling畅通并且表现出Ca2+瞬变的特性; 2、在ADSCs成心肌分化过程中过表达Nkx2.5和α-cardiac actin虽然增加了表达心肌标志蛋白(包括GATA4、cTnI、Connexin45和DHPR/RYR2)的表达,但减少了收缩心肌细胞数目,这可能与β-MHC蛋白表达的上调有关。 此外,本研究还以18日龄SD大鼠为实验动物,从腹股沟皮下脂肪组织中分离获得SVF,分别采用5-azacytidine和培养于半固体甲基纤维素培养基两种方法诱导ADSCs/SVF成心肌细胞分化,采用PCR分析心肌标志基因GATA4、MEF-2C、α- MHC、β-MHC、α-cardiac actin、cTnT和ANP的表达;细胞免疫化学染色、细胞免疫荧光染色、免疫组织化学染色和流式细胞术分析α-actin、MF20、Connexin45、cMHC、cTnI和Nkx2.5的表达;细胞免疫荧光染色和Ca2+成像分析验证SVF源心肌细胞E-C coupling的畅通;接着利用ROCK的特异性抑制剂Y-27632阻断Rho/ROCK通路,通过Actin-Tracker Green染色观察大鼠SVF细胞成心肌分化前后actin细胞骨架的变化;Western Blot分析调控细胞骨架改变的重要通路Rho/ROCK通路中关键激酶ROCK的表达,以及ROCK下游三条与细胞骨架相关通路(JNK、p38和Akt)的变化,最后从整体上探讨细胞骨架相关信号通路调控大鼠ADSCs成心肌细胞分化的机理,主要研究结果如下: 1、5-azacytidine可启动大鼠ADSCs成心肌分化的程序,诱导后的ADSCs表达部分心肌标志蛋白(Desmin、α-actinin、α-actin、MF20和Connexin45)并形成肌管样结构,但未见自发收缩; 2、大鼠SVF细胞在半固体甲基纤维素培养基中培养时可在体外分化为收缩的心肌细胞,这些收缩细胞表达心肌特异性mRNA(GATA4、MEF-2C、α- MHC、β-MHC、α-cardiac actin、cTnT和ANP)和蛋白(MF20、Connexin45、Nkx2.5、cTnI, cMHC和α-actin),并且表现出Ca2+瞬变的特性; 3、细胞骨架对大鼠SVF分化为心肌细胞的过程起着重要调控作用,抑制细胞骨架相关蛋白ROCK信号通路,减少了心肌标志蛋白的表达和跳动心肌细胞的形成,而后者可能是通过抑制JNKMAPK的磷酸化,以及促进p38MAPK和Akt磷酸化起作用的。
[Abstract]:Stem cell therapy has a great potential for development in the clinical application of cardiovascular disease. Although some in vivo and in vitro experimental results are encouraging, the molecular mechanism of the optimal stem cell origin and the differentiation of stem cells into cardiomyocytes remains to be further studied and confirmed. Adipose stromal cells (ADSCs) have been widely concerned in the field of stem cell research with the advantages of rich stem cell content, easy acquisition, easy culture, small donor damage, multi-lineage differentiation potential and autotransplantation. Although the ability of ADSCs to differentiate into cardiomyocytes has been demonstrated, this ability is currently limited to three species of rabbits, mice and humans, and the mechanism of the differentiation of ADSCs into cardiomyocytes is not clear. In this study,6-8-week-old mice were used as experimental animals, and ADSCs were isolated from the inguinal subcutaneous fat tissue. The spontaneous differentiation was observed in DMEM medium supplemented with penicillin, streptomycin and 20% NBS, and the surface markers of ADSCs, CD29, CD31, CD34 and CD105 were analyzed by flow cytometry. The expression of C-actin, MF20, Connexin45, cMHC, cTnI, NkX2.5 and GATA4 was analyzed by immunofluorescent staining, immunohistochemical staining and flow cytometry. And then using the Touchdown-PCR method to clone the CDS sequence of the cardic-actin gene and construct the recombinant adenovirus expression vector (pAd-Nkx2.5), and using pAd-Nkx2.5 and pAd-actin to infect the primary cultured mouse ADSCs, the Real time-PCR and the Western Blot to detect the super-expression effect. The effect of the expression of Nkx2.5 and cardic-actin on the differentiation of the mouse ADSCs into the cardiac myocyte was discussed, and the main results of the study were as follows: Next: 1. The primary mouse ADSCs express CD29 and CD105 under in vitro culture conditions, but do not express CD31 and CD34, which can spontaneously differentiate into cardiomyocytes under low in vitro culture conditions (DMEM + 20% NBS, no cytokine,5-azacytidine, or antioxidant, etc.), which may The expression of E-C clinking in the contractile cells was smooth and the Ca 2 + transient was expressed. The expression of Nkox2.5 and C-cardic actin in the course of the differentiation of ADSCs has increased the expression of the myocardial marker protein (including GATA4). , cTnI, Connexin45, and DHPR/ RYR2), but reduced the number of contracted cardiomyocytes, which may be related to the expression of the antigen-MHC protein In addition,18-day-old SD rats were used as experimental animals, and SVF was obtained from the inguinal subcutaneous fat tissue. The differentiation of ADSCs/ SVF into cardiomyocytes was induced by using 5-azacytidine and two methods of culture in semi-solid methyl cellulose medium, and the myocardial marker gene GATA4 was analyzed by PCR. , MEF-2C, I-MHC, I-MHC, I-cardic actin, cTnT and ANP; immunocytochemical staining, cellular immunofluorescent staining, immunohistochemical staining and flow cytometry to analyze the expression of HCO3-actin, MF20, Connexin45, cMHC, cTnI and Nk The expression of X2.5 was confirmed by means of immunofluorescence staining and Ca 2 + imaging, and the expression of E-C clinking in the SVF source was verified by means of the specific inhibitor Y-27632 of ROCK, and the actin-Tracker Green staining was used to observe the actin in the rat SVF cells. Cytoskeleton changes; Western Blot analysis regulates the expression of the key kinase ROCK in the important pathway Rho/ ROCK pathway of the cell skeleton change, as well as three of the ROCK downstream and cytoskeleton-related pathways (JNK, p38, and The changes of Akt (Akt) and the mechanism of the control of ADSCs of the rat ADSCs into cardiomyocytes were discussed in the last part. The results of this study are as follows:1,5-azacytidine can initiate the procedure for the differentiation of ADSCs in rats, and the induced ADSCs express part of the myocardial marker protein (Desmin, P-actini n,1-actin, MF20, and Connexin45) and form a myotube-like knot but did not show spontaneous contraction;2. The rat SVF cells were cultured in a semi-solid methylcellulose medium to be differentiated into a contracted cardiomyocytes in vitro, which express the myocardial specific mRNA (GATA4, MEF-2C, HCO3-MHC, HCO3-MHC, HCO3-c, ardiactin, cTnT and ANP) and proteins (MF20, Connexin45, Nkx2.5, cTnI, cMHC, and HCO3-actin), and Table The results show that the cytoskeleton plays an important role in regulating the differentiation of SVF in the rat and inhibiting the cell bone. The shelf-related protein ROCK signaling pathway reduces the expression of the myocardial marker protein and the formation of the beating cardiac muscle cells, which may be by inhibiting the phosphorylation of JNKMAPK and promoting p38MA
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
[Abstract]:Stem cell therapy has a great potential for development in the clinical application of cardiovascular disease. Although some in vivo and in vitro experimental results are encouraging, the molecular mechanism of the optimal stem cell origin and the differentiation of stem cells into cardiomyocytes remains to be further studied and confirmed. Adipose stromal cells (ADSCs) have been widely concerned in the field of stem cell research with the advantages of rich stem cell content, easy acquisition, easy culture, small donor damage, multi-lineage differentiation potential and autotransplantation. Although the ability of ADSCs to differentiate into cardiomyocytes has been demonstrated, this ability is currently limited to three species of rabbits, mice and humans, and the mechanism of the differentiation of ADSCs into cardiomyocytes is not clear. In this study,6-8-week-old mice were used as experimental animals, and ADSCs were isolated from the inguinal subcutaneous fat tissue. The spontaneous differentiation was observed in DMEM medium supplemented with penicillin, streptomycin and 20% NBS, and the surface markers of ADSCs, CD29, CD31, CD34 and CD105 were analyzed by flow cytometry. The expression of C-actin, MF20, Connexin45, cMHC, cTnI, NkX2.5 and GATA4 was analyzed by immunofluorescent staining, immunohistochemical staining and flow cytometry. And then using the Touchdown-PCR method to clone the CDS sequence of the cardic-actin gene and construct the recombinant adenovirus expression vector (pAd-Nkx2.5), and using pAd-Nkx2.5 and pAd-actin to infect the primary cultured mouse ADSCs, the Real time-PCR and the Western Blot to detect the super-expression effect. The effect of the expression of Nkx2.5 and cardic-actin on the differentiation of the mouse ADSCs into the cardiac myocyte was discussed, and the main results of the study were as follows: Next: 1. The primary mouse ADSCs express CD29 and CD105 under in vitro culture conditions, but do not express CD31 and CD34, which can spontaneously differentiate into cardiomyocytes under low in vitro culture conditions (DMEM + 20% NBS, no cytokine,5-azacytidine, or antioxidant, etc.), which may The expression of E-C clinking in the contractile cells was smooth and the Ca 2 + transient was expressed. The expression of Nkox2.5 and C-cardic actin in the course of the differentiation of ADSCs has increased the expression of the myocardial marker protein (including GATA4). , cTnI, Connexin45, and DHPR/ RYR2), but reduced the number of contracted cardiomyocytes, which may be related to the expression of the antigen-MHC protein In addition,18-day-old SD rats were used as experimental animals, and SVF was obtained from the inguinal subcutaneous fat tissue. The differentiation of ADSCs/ SVF into cardiomyocytes was induced by using 5-azacytidine and two methods of culture in semi-solid methyl cellulose medium, and the myocardial marker gene GATA4 was analyzed by PCR. , MEF-2C, I-MHC, I-MHC, I-cardic actin, cTnT and ANP; immunocytochemical staining, cellular immunofluorescent staining, immunohistochemical staining and flow cytometry to analyze the expression of HCO3-actin, MF20, Connexin45, cMHC, cTnI and Nk The expression of X2.5 was confirmed by means of immunofluorescence staining and Ca 2 + imaging, and the expression of E-C clinking in the SVF source was verified by means of the specific inhibitor Y-27632 of ROCK, and the actin-Tracker Green staining was used to observe the actin in the rat SVF cells. Cytoskeleton changes; Western Blot analysis regulates the expression of the key kinase ROCK in the important pathway Rho/ ROCK pathway of the cell skeleton change, as well as three of the ROCK downstream and cytoskeleton-related pathways (JNK, p38, and The changes of Akt (Akt) and the mechanism of the control of ADSCs of the rat ADSCs into cardiomyocytes were discussed in the last part. The results of this study are as follows:1,5-azacytidine can initiate the procedure for the differentiation of ADSCs in rats, and the induced ADSCs express part of the myocardial marker protein (Desmin, P-actini n,1-actin, MF20, and Connexin45) and form a myotube-like knot but did not show spontaneous contraction;2. The rat SVF cells were cultured in a semi-solid methylcellulose medium to be differentiated into a contracted cardiomyocytes in vitro, which express the myocardial specific mRNA (GATA4, MEF-2C, HCO3-MHC, HCO3-MHC, HCO3-c, ardiactin, cTnT and ANP) and proteins (MF20, Connexin45, Nkx2.5, cTnI, cMHC, and HCO3-actin), and Table The results show that the cytoskeleton plays an important role in regulating the differentiation of SVF in the rat and inhibiting the cell bone. The shelf-related protein ROCK signaling pathway reduces the expression of the myocardial marker protein and the formation of the beating cardiac muscle cells, which may be by inhibiting the phosphorylation of JNKMAPK and promoting p38MA
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
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