【摘要】：干細(xì)胞治療在心血管疾病的臨床應(yīng)用研究中具有巨大的發(fā)展?jié)摿�。雖然一些體內(nèi)和體外的實(shí)驗(yàn)結(jié)果令人振奮,然而,最佳干細(xì)胞來(lái)源以及干細(xì)胞成心肌細(xì)胞分化的分子機(jī)理仍有待進(jìn)一步研究和證實(shí)。脂肪基質(zhì)細(xì)胞(ADSCs)以其干細(xì)胞含量豐富、易采集、易培養(yǎng)、對(duì)供體損傷小、具備多譜系分化潛能和可自體移植等優(yōu)勢(shì)受到干細(xì)胞研究領(lǐng)域的廣泛關(guān)注。雖然ADSCs可定向分化成心肌細(xì)胞的能力已被證實(shí),但這一能力的驗(yàn)證目前僅限于兔、小鼠和人三個(gè)物種,而且對(duì)ADSCs成心肌細(xì)胞分化的機(jī)理也不甚清楚。 本研究以6-8周齡小鼠為實(shí)驗(yàn)動(dòng)物,從腹股溝皮下脂肪組織中分離獲得ADSCs,培養(yǎng)于僅添加青霉素、鏈霉素和20% NBS的DMEM培養(yǎng)液中觀察其自發(fā)分化,采用流式細(xì)胞術(shù)分析ADSCs表面標(biāo)記CD29、CD31、CD34和CD105的表達(dá);細(xì)胞免疫熒光染色、免疫組織化學(xué)染色和流式細(xì)胞術(shù)分析α-actin、MF20、Connexin45、cMHC、cTnI、Nkx2.5和GATA4的表達(dá);細(xì)胞免疫熒光染色和Ca2+成像分析驗(yàn)證ADSCs源心肌細(xì)胞E-C coupling的暢通;接著使用Touchdown-PCR方法克隆cardiacα-actin基因CDS序列,并構(gòu)建其重組腺病毒表達(dá)載體(pAd- Nkx2.5),使用pAd-Nkx2.5和pAd-actin侵染原代培養(yǎng)的小鼠ADSCs,Real time-PCR和Western Blot檢測(cè)超表達(dá)效果;并使用如上所述方法量化分析過(guò)表達(dá)前后心肌標(biāo)志蛋白的變化,最后從整體上探討過(guò)表達(dá)Nkx2.5和cardiacα-actin對(duì)小鼠ADSCs成心肌細(xì)胞分化的影響,主要研究結(jié)果如下: 1、原代小鼠ADSCs在體外培養(yǎng)條件下表達(dá)CD29和CD105,但不表達(dá)CD31和CD34,它們可在體外較低的培養(yǎng)條件下(DMEM+20% NBS,未添加任何細(xì)胞因子、5-azacytidine或抗氧化劑等)自發(fā)分化成心肌細(xì)胞,這些細(xì)胞可自發(fā)收縮,表達(dá)心肌標(biāo)志蛋白α-actin、MF20、Connexin45、cMHC、cTnI、Nkx2.5和GATA4,收縮細(xì)胞中E-C coupling暢通并且表現(xiàn)出Ca2+瞬變的特性; 2、在ADSCs成心肌分化過(guò)程中過(guò)表達(dá)Nkx2.5和α-cardiac actin雖然增加了表達(dá)心肌標(biāo)志蛋白(包括GATA4、cTnI、Connexin45和DHPR/RYR2)的表達(dá),但減少了收縮心肌細(xì)胞數(shù)目,這可能與β-MHC蛋白表達(dá)的上調(diào)有關(guān)。 此外,本研究還以18日齡SD大鼠為實(shí)驗(yàn)動(dòng)物,從腹股溝皮下脂肪組織中分離獲得SVF,分別采用5-azacytidine和培養(yǎng)于半固體甲基纖維素培養(yǎng)基兩種方法誘導(dǎo)ADSCs/SVF成心肌細(xì)胞分化,采用PCR分析心肌標(biāo)志基因GATA4、MEF-2C、α- MHC、β-MHC、α-cardiac actin、cTnT和ANP的表達(dá);細(xì)胞免疫化學(xué)染色、細(xì)胞免疫熒光染色、免疫組織化學(xué)染色和流式細(xì)胞術(shù)分析α-actin、MF20、Connexin45、cMHC、cTnI和Nkx2.5的表達(dá);細(xì)胞免疫熒光染色和Ca2+成像分析驗(yàn)證SVF源心肌細(xì)胞E-C coupling的暢通;接著利用ROCK的特異性抑制劑Y-27632阻斷Rho/ROCK通路,通過(guò)Actin-Tracker Green染色觀察大鼠SVF細(xì)胞成心肌分化前后actin細(xì)胞骨架的變化;Western Blot分析調(diào)控細(xì)胞骨架改變的重要通路Rho/ROCK通路中關(guān)鍵激酶ROCK的表達(dá),以及ROCK下游三條與細(xì)胞骨架相關(guān)通路(JNK、p38和Akt)的變化,最后從整體上探討細(xì)胞骨架相關(guān)信號(hào)通路調(diào)控大鼠ADSCs成心肌細(xì)胞分化的機(jī)理,主要研究結(jié)果如下: 1、5-azacytidine可啟動(dòng)大鼠ADSCs成心肌分化的程序,誘導(dǎo)后的ADSCs表達(dá)部分心肌標(biāo)志蛋白(Desmin、α-actinin、α-actin、MF20和Connexin45)并形成肌管樣結(jié)構(gòu),但未見(jiàn)自發(fā)收縮; 2、大鼠SVF細(xì)胞在半固體甲基纖維素培養(yǎng)基中培養(yǎng)時(shí)可在體外分化為收縮的心肌細(xì)胞,這些收縮細(xì)胞表達(dá)心肌特異性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),并且表現(xiàn)出Ca2+瞬變的特性; 3、細(xì)胞骨架對(duì)大鼠SVF分化為心肌細(xì)胞的過(guò)程起著重要調(diào)控作用,抑制細(xì)胞骨架相關(guān)蛋白R(shí)OCK信號(hào)通路,減少了心肌標(biāo)志蛋白的表達(dá)和跳動(dòng)心肌細(xì)胞的形成,而后者可能是通過(guò)抑制JNKMAPK的磷酸化,以及促進(jìn)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
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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