猪骨髓间充质干细胞体外自然培养及不同诱导方式下分化和旁分泌功能的实验研究

发布时间：2018-03-14 05:48

本文选题：骨髓间充质干细胞　切入点：分化　出处：《北京协和医学院》2009年博士论文　论文类型：学位论文

【摘要】：目的：探讨猪骨髓间充质干细胞体外自发分化及不同诱导方式分化为心肌细胞的潜能及分分泌VEGF的规律。方法：抽取猪骨髓,密度梯度离心法分离单个核细胞,贴壁培养法筛选和培养MSCs,研究自然培养条件下第1、2、3、4、5代细胞向心肌细胞分化潜能和分泌VEGF的规律；选取P1MSCs,分别用5-氮胞苷、心肌组织裂解液、5-氮胞苷+心肌组织裂解液诱导1周、2周、3周,研究其分化、分泌的规律。各代细胞采用连续显微镜下观察、免疫细胞化学染色法、Real Time-PCR、ELISA.透射电镜检测相关指标。结果：体外自然培养条件下,P1-P5MSCs形态基本一致,呈梭形成纤维细胞样外观。经心肌组织裂解液处理1周后,MSCs增殖活跃,细胞形态均一,呈旋涡状排列。各诱导组诱导3周后,细胞胞质内颗粒增粗,培养基中碎片增多,D组MSCs有部分不再贴壁。提取后传代的P1细胞,100%表达CD29、CD90,而0%的细胞表达CD45,说明贴壁培养的细胞为MSCs。免疫细胞化学染色显示,自然培养状态下,P4MSCs表达Connexin43、cTnT、 α-sarcomeric actin,与其它代MSCs相比有显著性差别(P0.001)。诱导2周后,5-氮胞苷与心肌组织裂解液共同诱导组表达Connexin43、cTnT、a-sarcomeric actin,与其它组MSCs相比有显著性差别(P0.001)。 Real Time-PCR结果显示,P1-P5MSCs在体外自然培养状态下都可表达connexin43、VEGF及心肌特异性蛋白基因cTnI, P4细胞表达量最高(P0.001)而P0细胞不表达cTnI,仅表达少量connexin43。A、B、C、D各组细胞诱导1周、2周、3周都可表达Connexin43, cTnI和VEGF。诱导1周、2周后,C组表达量高于其它各组(P0.001)。各组细胞Connexin43, cTnl和VEGF的表达量在诱导2周时最高,3周时最低。 ELISA检测结果显示,各处理组细胞条件培养基浓缩20倍,VEGF含量均低于37.5pg/ml。透射电镜显示自然培养状态下,部分P1-P5MSCs胞质内可见细肌丝束。P4MSCs胞质内可见多束细肌丝,密体结构清晰。A、B、C、D四组MSCs处理2周后,胞质内均可见细肌丝束,密体结构清晰可见。B、C两组可见质膜相贴,局部电子密度增高,出现非特异的细胞连接。C组细胞内偶见肌节样结构形成。结论：1.猪骨髓MSCs体外自然培养条件下,各代之间形态、分化潜能存在着差异,向心肌细胞分化能力随细胞年龄先增强,后减低。本实验中的拐点为P4。2.5-氮胞苷与心肌组织裂解液共同诱导相比单独使用5-氮胞苷或心肌组织裂解液诱导效率更高,诱导2周后分化效率最高,诱导3周基本丧失向心肌细胞分化能力。3.体外短期培养过程中(3周)猪骨髓MSCs旁分泌VEGF可能与其向心肌样细胞分化程度一致。
[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, 伪 -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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329.2

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