本文選題：骨髓 + 間充質(zhì)干細(xì)胞��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2008年博士論文

【摘要】： 目的:探討豬骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)自發(fā)分化和5-氮胞苷誘導(dǎo)分化為心肌細(xì)胞的潛能,以及體外向心肌細(xì)胞分化的規(guī)律性。 方法:抽取豬髂骨骨髓,密度梯度離心法分離單個(gè)核細(xì)胞,貼壁培養(yǎng)法篩選和培養(yǎng)MSCs,選取第9,10,11,12,15和20代MSCs,研究其向心肌樣細(xì)胞自發(fā)分化潛能和5-氮胞苷誘導(dǎo)后4周能否分化為心肌細(xì)胞,并探索豬MSCs體外向心肌細(xì)胞分化的規(guī)律性。各代MSCs采用連續(xù)顯微鏡下觀察、流式細(xì)胞儀、免疫細(xì)胞化學(xué)染色法、Real Time-PCR、透射電鏡檢測(cè)相關(guān)指標(biāo)。 結(jié)果:體外自然培養(yǎng)條件下,P9,P11,P12,P15,P20和大部分P10 MSCs形態(tài)一致,呈梭形成纖維細(xì)胞樣形態(tài),但部分P10細(xì)胞多核、胞漿延長(zhǎng),轉(zhuǎn)變?yōu)榧」軜有螒B(tài)。5-氮胞苷誘導(dǎo)1周后,部分P9,P11,P12,P15和P20MSCs表現(xiàn)為較長(zhǎng)的胞漿形態(tài)和多核化,4周后更多細(xì)胞相互連接形成不規(guī)則的管樣結(jié)構(gòu),而P10MSCs則形成相對(duì)規(guī)則的肌管樣結(jié)構(gòu)。 流式法顯示,P10MSCs的G0/G1期比例顯著增多(88.00±3.90)%,P10與P9相比有顯著性差異(P＜0.0001),出現(xiàn)細(xì)胞生長(zhǎng)停滯。貼壁細(xì)胞CD90、CD29陽(yáng)性,而CD34、CD45陰性,說明貼壁細(xì)胞為MSCs。 免疫細(xì)胞化學(xué)染色顯示,自然培養(yǎng)和誘導(dǎo)4周后各代MSCs均表達(dá)心肌特異性結(jié)構(gòu)蛋白Cx43和α-sarcomeric actin,而誘導(dǎo)后P10MSCs分化率與其他各代相比有顯著性差異(P=0.011)。 Real time-PCR結(jié)果表明,自然培養(yǎng)和誘導(dǎo)4周后的各代MSCs均有心肌特異性轉(zhuǎn)錄因子GATA4和結(jié)構(gòu)基因MLC、α-SKA、Cx43、cTNI基因表達(dá),而P10MSCs自然培養(yǎng)和誘導(dǎo)后4周,各基因的表達(dá)豐度均高于其他代在相同條件下的分化細(xì)胞,而α-SKA誘導(dǎo)后增高顯著(P＜0.0001),而P20MSCs表達(dá)的基因減少。 透射電鏡顯示在自然培養(yǎng)狀態(tài)下和誘導(dǎo)后4周,P9,P10,P11,P12,P15 MSCs均有較多不規(guī)則細(xì)肌絲,而P10MSCs經(jīng)誘導(dǎo)后出現(xiàn)較多不規(guī)則排列粗肌絲,P20MSCs肌絲減少。 結(jié)論:1.豬骨髓MSCs在體外自然培養(yǎng)條件下,各代形態(tài)、增殖能力和分化潛能是不均一的,傳代次數(shù)的增加MSCs增殖逐漸變慢、部分細(xì)胞自發(fā)分化為心肌樣細(xì)胞。2.MSCs于第10代出現(xiàn)細(xì)胞生長(zhǎng)停滯,但并沒有喪失多向分化潛能。3,在體外長(zhǎng)期培養(yǎng)過程中,MSCs出現(xiàn)轉(zhuǎn)化能力。4.5-氮胞苷可誘導(dǎo)MSCs分化為心肌樣細(xì)胞,P10MSCs分化更具有成熟心肌細(xì)胞的特異性表型。4.結(jié)合本實(shí)驗(yàn)研究組前期的結(jié)果,推測(cè)P10可能是豬MSCs體外分化為心肌細(xì)胞過程中的拐點(diǎn)。
[Abstract]:Aim: to investigate the spontaneous differentiation of porcine bone marrow mesenchymal stem cells (MSCs) and the potential of 5-azacytidine (5-azacytidine) induced differentiation into cardiomyocytes, and the regularity of differentiation into cardiomyocytes in vitro. Methods: mononuclear cells were isolated from porcine iliac bone marrow by density gradient centrifugation. MSCs were selected and cultured by adherent culture method. The spontaneous differentiation potential of MSCs into cardiomyocyte-like cells and whether 5-azacytidine could differentiate into cardiomyocytes 4 weeks after induction were studied, and the regularity of porcine MSCs differentiation to cardiomyocytes in vitro was explored. All generations of MSCs were observed under a continuous microscope, flow cytometry, immunocytochemical staining, Real Time-PCR, and transmission electron microscopy (TEM) were used to detect the relative indexes. Results: in natural culture in vitro, the morphology of P9, P11, P12, P15, P20 and P10 MSCs was the same as that of most P10 cells, but some P10 cells were polynucleated and the cytoplasm was lengthened, which was transformed into myotube like form. 5-azacytidine was induced for 1 week. Some of P9 P11 P12 P15 and P20MSCs showed long cytoplasmic morphology and more cells connected to each other to form irregular tubular structure after 4 weeks of polynucleation, while P10MSCs formed a relatively regular myotube structure. Flow cytometry showed that the proportion of P10 MSCs in G0/G1 phase was significantly increased (88.00 鹵3.90), and there was significant difference between P10 and P9 (P < 0.0001). The adherent cells were positive for CD90 and CD29, but negative for CD34 and CD45, indicating that the adherent cells were MSCs. Immunocytochemical staining showed that MSCs expressed myocardial specific structural protein Cx43 and 偽 -sarcomeric actinin in all generations after 4 weeks of natural culture and induction, but the differentiation rate of P10MSCs was significantly different from that of other generations. Real time-PCR results showed that myocardial specific transcription factor (GATA4) and structural genes (MLC, 偽 -SKAN Cx43 cTNI) were expressed in all generations of MSCs after 4 weeks of natural culture and induction, while P10MSCs was expressed at 4 weeks after natural culture and induction. The expression abundance of each gene was higher than that of other generation differentiation cells under the same condition, but 偽 -SKA increased significantly (P < 0. 0001), while the gene expression of P20MSCs decreased. Transmission electron microscope showed that there were more irregular fine filaments in P9 P10P10P11P12P15 MSCs in natural culture and 4 weeks after induction, but more irregular arrangement of coarse myofilament P20MSCs decreased after P10MSCs induction. Conclusion 1. Under the condition of natural culture of porcine bone marrow MSCs in vitro, the morphology, proliferative ability and differentiation potential of each generation were different, and the increase of passage number of MSCs gradually slowed down the proliferation of MSCs. Some of the cells spontaneously differentiated into cardiomyocyte-like cells. 2. MSCs had cell growth arrest in the 10th generation. However, it did not lose the potential of multidirectional differentiation. During the long-term culture in vitro, the ability of transforming MSCs into cardiomyocyte-like cells could be induced by 4.5-azacytidine, and the differentiation of P10MSCs was more specific phenotypic than that of mature cardiomyocytes. Based on the previous results of this study group, we speculated that P10 might be the inflection point in the process of porcine MSCs differentiation into cardiomyocytes in vitro.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【共引文獻(xiàn)】

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6 畢文浩;超順磁性氧化鐵標(biāo)記西藏小型豬骨髓間充質(zhì)干細(xì)胞體內(nèi)示蹤的實(shí)驗(yàn)研究[D];廣州醫(yī)學(xué)院;2011年

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