诱导骨髓间充质干细胞向心肌样组织分化的初步研究

发布时间：2018-05-02 20:46

  本文选题：间充质干细胞 + 心肌样组织　； 参考：《广西医科大学》2011年硕士论文

【摘要】：目的:采用心肌细胞培养上清液、心肌组织裂解液诱导MSCs向心肌样细胞分化、形成心肌组织样结构。 方法:优化间充质干细胞体外培养体系,采用全骨髓贴壁培养法培养MSCs,建立间充质干细胞系;采用差速贴壁法纯化培养心肌细胞,为实验提供参照。选取生长良好的第3代MSCs分为三组:I组空白对照组,用含10%FBS的H-DMEM培养MSCs。II组实验对照组(5-aza诱导组),用不同浓度5-aza作用于MSCs24小时后换用含有10%FBS的H-DMEM培养; III组心肌细胞培养上清液组,收集心肌细胞培养上清液制备条件培养液诱导培养MSCs。IV组心肌组织裂解液组,用心肌组织裂解液配置条件培养液诱导培养MSCs。各组同时培养4W,在倒置相差显微镜下观察各组细胞形态学变化,HE染色、电子显微镜下观察心肌样细胞的结构。行α-Actin、connexin43免疫细胞化学染色、Vimentin、Ⅷ因子相关抗原免疫组织化学染色,鉴定心肌样组织内各种细胞及其相关结构。 结果:在连续诱导培养4周内,心肌细胞培养的上清液不足以诱导骨髓MSCs分化为心肌样细胞; 5-aza、心肌组织裂解液能够诱导骨髓MSCs分化为心肌样细胞,且具有自律性搏动,搏动频率约90-110次/分;HE染色可见由波形蛋白免疫组织化学染色阳性的成纤维样细胞包裹的肌束样结构;心肌组织裂解液诱导组可见大量中空性结构,管壁波形蛋白免疫组织化学染色阳性,Ⅷ因子相关抗原免疫组织化学染色阴性。 结论:心肌组织裂解液能模拟体内微环境,诱导骨髓间充质干细胞分化为普通型心肌样细胞和具有自律性搏动的心肌样细胞;分化为成纤维样细胞并包裹心肌样细胞形成肌束样结构,形成含内皮样细胞的中空性结构,最终分化形成具有“心肌样组织”特点的结构。
[Abstract]:Aim: myocardial cell culture supernatant was used to induce MSCs to differentiate into cardiomyocyte-like cells and form myocardial tissue-like structure. Methods: to optimize the culture system of mesenchymal stem cells in vitro, to culture MSCs by whole bone marrow adherent culture, to establish mesenchymal stem cell lines, and to purify and culture cardiac myocytes by differential adhesion method. The third generation of MSCs with good growth was divided into three groups: group I: blank control group, The MSCs.II group was cultured with H-DMEM containing 10s, the control group was induced by 5-aza, and treated with different concentrations of 5-aza for an hour, then replaced with H-DMEM containing 10s, and the supernatant of myocardial cell culture in III group. Cardiomyocyte culture supernatant was collected to prepare conditioned culture medium and MSCs.IV group was induced to culture myocardial tissue lysate. The morphological changes of the cells in each group were observed under inverted phase contrast microscope and the structure of cardiomyocytes was observed under electron microscope. The immunocytochemical staining of 伪 -Actinine connexin 43 and the immunohistochemical staining of factor 鈪，

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