本文選題：cTnT + 5-氮雜胞苷�。� 參考：《新鄉(xiāng)醫(yī)學(xué)院》2017年碩士論文

【摘要】：背景當(dāng)心肌組織受損,成纖維細(xì)胞可以很快形成瘢痕組織,而心肌細(xì)胞再生能力有限,嚴(yán)重影響心肌組織的結(jié)構(gòu)穩(wěn)定性及心臟的正常功能。心肌組織工程的理想種子細(xì)胞脂肪間充質(zhì)干細(xì)胞(Adipose-derived Mesenchymal Stem Cells,ADMSCs)有多向分化潛能,可向成脂、成骨、成心肌等分化,并且具有取材方便,倫理限制較少,增殖速率高等優(yōu)點(diǎn)。目前將ADMSCs誘導(dǎo)分化為心肌細(xì)胞的方法多為化學(xué)試劑誘導(dǎo)。由于化學(xué)試劑具有一定的細(xì)胞毒性,在一定程度上制約了其在基礎(chǔ)研究和臨床中的應(yīng)用,尋找安全有效的誘導(dǎo)方法是心肌細(xì)胞治療和心肌組織工程的重要條件。目的探討體內(nèi)、外心肌微環(huán)境與體外5-氮雜胞苷(5-aza)誘導(dǎo)ADMSCs分化為心肌樣細(xì)胞的作用差異,為進(jìn)一步開(kāi)展細(xì)胞治療心肌疾患提供實(shí)驗(yàn)參考。方法1.取小鼠腹股溝的皮下脂肪組織,分離培養(yǎng)ADMSCs,流式細(xì)胞術(shù)對(duì)其表面標(biāo)記檢測(cè);分別用成骨、成脂誘導(dǎo)劑誘導(dǎo),并使用茜素紅與油紅O鑒定ADMSCs多向分化能力。2.取小鼠乳鼠的心臟組織,分離培養(yǎng)心肌細(xì)胞。3.選取生長(zhǎng)狀態(tài)良好的第3代ADMSCs分為:5-aza體外誘導(dǎo)組、體外與心肌細(xì)胞共培養(yǎng)組和ADMSCs心肌內(nèi)移植組,體外誘導(dǎo)以及體內(nèi)移植1~3周后,檢測(cè)ADMSCs的心肌特異性肌鈣蛋白(cTnT)的免疫熒光表達(dá)。結(jié)果1.小鼠ADMSCs表達(dá)間充質(zhì)干細(xì)胞的表面標(biāo)記CD29,不表達(dá)造血干細(xì)胞標(biāo)記CD45;且具有成脂、成骨的分化能力。2.三種誘導(dǎo)方法ADMSCs均表達(dá)cTnT:5-aza誘導(dǎo)組3周表達(dá)率(32.65±3.79)%,心肌細(xì)胞共培養(yǎng)組3周表達(dá)率(36.31±5.12)%,心肌內(nèi)移植組1周表達(dá)率(42.93±4.04)%,3周表達(dá)率(78.43±1.32)%;心肌內(nèi)移植組1周及3周較5-aza誘導(dǎo)組和心肌細(xì)胞共培養(yǎng)組3周均具有更高的分化效率(P0.05)。結(jié)論ADMSCs在體外經(jīng)5-aza化學(xué)誘導(dǎo)可分化為心肌樣細(xì)胞,但分化效率明顯低于ADMSCs體外與心肌細(xì)胞共培養(yǎng)及體內(nèi)移植兩種心肌微環(huán)境誘導(dǎo)的分化效率。
[Abstract]:Background when myocardial tissue is damaged, fibroblasts can quickly form scar tissue, but the ability of myocardial regeneration is limited, which seriously affects the structural stability of myocardial tissue and the normal function of heart. Adipose-derived mesenchymal stem cells (Adipose-derived mesenchymal stem cells), which are ideal seed cells for myocardial tissue engineering, have the potential to differentiate into adipogenic, osteogenic and myocardial cells, and have the advantages of convenient selection, less ethical limitation and high proliferation rate. At present, the methods of inducing ADMSCs to differentiate into cardiomyocytes are chemical reagents. Because of the cytotoxicity of chemical reagent, its application in basic research and clinic is restricted to a certain extent. It is an important condition for cardiomyocyte therapy and myocardial tissue engineering to find a safe and effective induction method. Objective to investigate the differentiation of 5-azacytidine (5-aza) into cardiomyocytes in vitro and in vivo, and to provide an experimental reference for the further development of cell therapy for myocardial diseases. Method 1. The subcutaneous adipose tissue of mouse groin was isolated and cultured, and the surface markers were detected by flow cytometry. ADMSCs were induced by osteogenesis and lipogenic inducer, and ADMSCs were identified by alizarin red and oil red O. Cardiomyocytes were isolated and cultured from the heart tissue of neonatal mice. The third generation of ADMSCs in good growth state was divided into three groups: 1: 5-aza in vitro, co-cultured with cardiomyocytes in vitro, and in vivo transplanted with ADMSCs, then induced in vitro and transplanted in vivo for 1 to 3 weeks. The expression of cardiac troponin (cTnT) in ADMSCs was detected. Result 1. Mouse ADMSCs expressed CD29 on the surface of mesenchymal stem cells, but not CD45 on the surface of hematopoietic stem cells, and had the ability of adipogenic and osteogenic differentiation. The expression rate of cTnT: 5-aza was (32.65 鹵3.79) in three weeks, 36.31 鹵5.12 in co-cultured cardiomyocytes, (42.93 鹵4.04) in intramyocardial transplantation in 1 week and (78.43 鹵1.32) in intramyocardial transplantation. The differentiation efficiency was higher in the co-culture group for 3 weeks (P0.05). Conclusion AdMSCs can differentiate into cardiomyocyte-like cells induced by 5-aza chemically in vitro, but the differentiation efficiency is significantly lower than that induced by co-culture with cardiomyocytes and transplantation in vivo.
【學(xué)位授予單位】：新鄉(xiāng)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)】：R54

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