本文選題：骨髓間充質(zhì)干細胞　切入點：分化　出處：《北京協(xié)和醫(yī)學院》2009年博士論文　論文類型：學位論文

【摘要】：目的：探討豬骨髓間充質(zhì)干細胞體外自發(fā)分化及不同誘導方式分化為心肌細胞的潛能及分分泌VEGF的規(guī)律。 方法：抽取豬骨髓,密度梯度離心法分離單個核細胞,貼壁培養(yǎng)法篩選和培養(yǎng)MSCs,研究自然培養(yǎng)條件下第1、2、3、4、5代細胞向心肌細胞分化潛能和分泌VEGF的規(guī)律；選取P1MSCs,分別用5-氮胞苷、心肌組織裂解液、5-氮胞苷+心肌組織裂解液誘導1周、2周、3周,研究其分化、分泌的規(guī)律。各代細胞采用連續(xù)顯微鏡下觀察、免疫細胞化學染色法、Real Time-PCR、ELISA.透射電鏡檢測相關指標。 結(jié)果：體外自然培養(yǎng)條件下,P1-P5MSCs形態(tài)基本一致,呈梭形成纖維細胞樣外觀。經(jīng)心肌組織裂解液處理1周后,MSCs增殖活躍,細胞形態(tài)均一,呈旋渦狀排列。各誘導組誘導3周后,細胞胞質(zhì)內(nèi)顆粒增粗,培養(yǎng)基中碎片增多,D組MSCs有部分不再貼壁。 提取后傳代的P1細胞,100%表達CD29、CD90,而0%的細胞表達CD45,說明貼壁培養(yǎng)的細胞為MSCs。 免疫細胞化學染色顯示,自然培養(yǎng)狀態(tài)下,P4MSCs表達Connexin43、cTnT、 α-sarcomeric actin,與其它代MSCs相比有顯著性差別(P0.001)。誘導2周后,5-氮胞苷與心肌組織裂解液共同誘導組表達Connexin43、cTnT、a-sarcomeric actin,與其它組MSCs相比有顯著性差別(P0.001)。 Real Time-PCR結(jié)果顯示,P1-P5MSCs在體外自然培養(yǎng)狀態(tài)下都可表達connexin43、VEGF及心肌特異性蛋白基因cTnI, P4細胞表達量最高(P0.001)而P0細胞不表達cTnI,僅表達少量connexin43。A、B、C、D各組細胞誘導1周、2周、3周都可表達Connexin43, cTnI和VEGF。誘導1周、2周后,C組表達量高于其它各組(P0.001)。各組細胞Connexin43, cTnl和VEGF的表達量在誘導2周時最高,3周時最低。 ELISA檢測結(jié)果顯示,各處理組細胞條件培養(yǎng)基濃縮20倍,VEGF含量均低于37.5pg/ml。 透射電鏡顯示自然培養(yǎng)狀態(tài)下,部分P1-P5MSCs胞質(zhì)內(nèi)可見細肌絲束。P4MSCs胞質(zhì)內(nèi)可見多束細肌絲,密體結(jié)構清晰。A、B、C、D四組MSCs處理2周后,胞質(zhì)內(nèi)均可見細肌絲束,密體結(jié)構清晰可見。B、C兩組可見質(zhì)膜相貼,局部電子密度增高,出現(xiàn)非特異的細胞連接。C組細胞內(nèi)偶見肌節(jié)樣結(jié)構形成。 結(jié)論：1.豬骨髓MSCs體外自然培養(yǎng)條件下,各代之間形態(tài)、分化潛能存在著差異,向心肌細胞分化能力隨細胞年齡先增強,后減低。本實驗中的拐點為P4。2.5-氮胞苷與心肌組織裂解液共同誘導相比單獨使用5-氮胞苷或心肌組織裂解液誘導效率更高,誘導2周后分化效率最高,誘導3周基本喪失向心肌細胞分化能力。3.體外短期培養(yǎng)過程中(3周)豬骨髓MSCs旁分泌VEGF可能與其向心肌樣細胞分化程度一致。
[Abstract]:Aim: to investigate the potential of spontaneous differentiation of porcine bone marrow mesenchymal stem cells into cardiomyocytes in vitro and the regularity of VEGF secretion. Methods: porcine bone marrow was extracted and mononuclear cells were isolated by density gradient centrifugation. MSCs were screened and cultured by adherent culture method. P1MSCs were induced by 5-azacytidine and 5-azacytidine for 1 week, 2 weeks and 3 weeks, respectively, to study the regularity of differentiation and secretion. The immunocytochemical staining method was used to detect the relative indexes by transmission electron microscope (TEM). Results: the morphology of P1-P5MSCs in vitro was basically the same as that of fusiform fibroblasts. After treated with myocardial tissue lysate for 1 week, MSCs proliferated actively, the cells were uniform in shape and arranged in swirl shape. The cytoplasmic granules were thickened and the fragments increased in the culture medium. Some of the MSCs in group D were no longer adhered to the wall. CD29 CD90 was expressed in 100% of P1 cells, while CD45 was expressed in 0% cells, indicating that the cells in adherent culture were MSCs. Immunocytochemical staining showed that. The expression of Connexin 43 cTnTnT, 偽 -sarcomeric actinin was significantly different from that of other generations of MSCs in natural culture. After 2 weeks of induction, the expression of Connexin43 cTnTnTa-sarcomeric actinin was significantly different from that of other MSCs groups. The results of Real Time-PCR showed that connexin 43 Real and cardiomyocyte specific protein gene cTnI, the highest expression of cTnI in P4 cells, could be expressed in all cultured MSCs in natural culture in vitro. However, P0 cells did not express cTnI, but only a small amount of connexin 43. Agnin C D cells could be induced for 1 week, 2 weeks and 3 weeks after induction. The expression of Connexin 43, cTnI and VEGF. After 1 week and 2 weeks of induction, the expression of Connexin 43 in group C was higher than that in other groups (P 0.001). The expression of cTnl and VEGF in the cells of each group was the highest at 2 weeks and the lowest at 3 weeks after induction. The results of ELISA detection showed that the concentration of ELISA in conditioned medium was lower than that in 37.5 PG / ml. Transmission electron microscopy (TEM) showed that in some P1-P5MSCs cytoplasm, several fine myofilaments could be seen in the cytoplasm of P1-P5MSCs. After 2 weeks of MSCs treatment, the fine muscle filaments could be seen in the cytoplasm of P1-P5MSCs. The dense body structure was clearly seen in the two groups. The plasma membrane was attached to each other, the local electron density was increased, and the formation of sarcoid structure was occasionally seen in the cells of the group of non-specific cell junctions. Conclusion 1. In vitro natural culture of porcine bone marrow MSCs, there are differences in morphology and differentiation potential between generations, and the ability of differentiation into cardiomyocytes increases with cell age. The inflexion point in this experiment was that P4.2.5- azacytidine was more efficient than 5-azacytidine or myocardial tissue lysate alone, and the differentiation efficiency was the highest after 2 weeks of induction. After 3 weeks of induction, the ability of differentiation into cardiomyocytes was basically lost. 3. During the short period of culture in vitro, the paracrine VEGF in porcine bone marrow might be consistent with the differentiation of porcine bone marrow into cardiomyocytes.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329.2

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本文編號：1609875