心肌细胞共培养诱导骨髓基质干细胞分化为心肌细胞的研究
发布时间：2018-11-27 13:15
【摘要】： 目的 骨髓中存在一定数量的骨髓基质干细胞(BMSCs, bone marrow stromal stem cells),这些细胞具有自我复制能力和巨大的分化潜能,在不同的条件下,可分化为骨、软骨、神经、心肌和脂肪等中胚层来源的组织。有研究结果表明,胞嘧啶类化学物质5-氮胞苷可诱导骨髓基质干细胞向心肌细胞分化,这使BMSCs成为细胞移植治疗缺血性心肌病最有前途的细胞之一。 本实验旨在通过对大鼠骨髓基质干细胞进行体外分离、培养、动态观察BMSCs的生长特点;观察在不经过任何诱导,将扩增的BMSCs与心肌细胞共同培养,观察BMSCs分化为心肌细胞的能力。并且通过GFP质粒转染骨髓基质干细胞,观察GFP在标记BMSCs中的表达能力,以期进一步了解骨髓基质干细胞的生物学特性,为其应用于临床提供更可靠的理论依据。 实验方法 1.全骨髓贴壁培养法体外分离,培养大鼠骨髓基质干细胞,倒置相差显微镜下逐日观察细胞形态及生长状。 2.差速贴壁法体外分离,培养心肌细胞,倒置相差显微镜下逐日观察细胞形态及生长状况。 3.将提取的GFP质粒转染培养好的第3代骨髓基质干细胞,Olympus荧光显微镜下观察细胞的转染效率。 4.将转染后的骨髓基质干细胞离心收集后按1∶3的比例加入到培养好的心肌细胞中共培养,Olympus荧光显微镜逐日观察细胞变化。 实验结果 1.大鼠骨髓基质干细胞的形态学观察:骨髓基质干细胞接种于培养瓶中后24h用倒置显微镜观察大部分细胞已贴壁,贴壁生长的细胞以分散、克隆集落的方式增殖,细胞大多呈梭形,类似成纤维细胞,偶有宽大平坦的多角形细胞。7-10d后克隆集落逐渐增多,随着集落生长的不断扩大,细胞融合。传代培养后细胞不再以集落方式生长,而呈分布均匀的纺锤形细胞生长。 2、心肌细胞的形态学观察:心肌细胞一般在24 h后会自动从组织块周围迁出,刚开始从组织块中迁出时,心肌细胞呈现梭形,伴随着迁出细胞的增多,组织块周围的心肌细胞呈现出不规则的圆形、三角形。细胞间紧密连接,整体形如铺路石样,随着细胞生长密度的不断增加,细胞间伸出的伪足会交织成网状。有时一些细胞紧密结合形成细胞簇,呈现出放射状或同心圆状,经过2～3 d培养,这些细胞簇会自发地搏动。 3、GFP转染骨髓基质干细胞的结果:转染GFP后,细胞仍贴壁生长,呈梭形或多角形,仍分裂增殖,但增殖速度有所降低。荧光显微镜下, 24 h可见有绿色荧光表达,但数量较少,强度较低,48 h可见细胞发出强烈的荧光。 4、共培养后BMSCs形态学与生长行为变化:在共培养后,BMSCs增殖速度缓慢,并且容易变成多角形或扁平,随着共培养时间的延长,有相当数量的BMSCs发生细胞崩解等死亡现象。24h后发现有很多贴壁的未跳动的尖梭形细胞靠近搏动的心肌细胞;48h后,这些尖梭形细胞呈集落样生长,并且在荧光显微镜下观察到个别被标记的BMSCs呈肌管样结构,这些BMSCs通常靠近搏动的未标记的心肌细胞;在第4天,部分与心肌细胞接触的被标记的BMSCs开始呈微弱心肌样搏动。同时,也有些BMSCs开始死亡,至64h已有相当一部分BMSCs发生死亡。 结论 1.可以从大鼠骨髓中分离出骨髓基质干细胞,经体外培养可大量扩增。 2.GFP转染BMSC后可以长期高效表达,而且对宿主细胞毒性很小,是一个非常理想的组织工程种子细胞的示踪工具。 3.大鼠骨髓基质干细胞在不用诱导剂的情况下,与心肌细胞共培养后可以分化为心肌细胞。
[Abstract]:Purpose The bone marrow has a certain number of bone marrow stromal stem cells (BMSCs, bone marrow stem cells), which have self-replicating ability and great differentiation potential, and can be differentiated into the source of the mesoderm such as bone, cartilage, nerve, myocardium and fat under different conditions. The results of the study show that the 5-N-cell-like chemical can induce the differentiation of bone marrow stromal stem cells into the cardiac muscle cells, which makes the BMSCs become the most promising cells for the treatment of ischemic cardiomyopathy by cell transplantation. The purpose of this study was to observe the growth characteristics of BMSCs by in vitro separation, culture and dynamic observation of bone marrow stromal stem cells in rats. The ability of the cells to be enhanced and the expression of GFP in the labeled BMSCs was observed by the transfection of the bone marrow stromal cells with the GFP plasmid, with a view to further understanding the biological characteristics of the bone marrow stromal stem cells and to provide more reliable clinical application the theory of According to the method, the whole bone marrow adherent culture method is isolated and cultured in vitro to culture the bone marrow stromal stem cells of the rat, and the inverted phase difference microscope day-to-day observation of cell morphology and growth. 2. In vitro separation of the differential adhesion method, culture of the cardiac muscle cells and the inversion of the phase-difference microscope The morphology and growth of the cells were observed on a day-to-day basis. 3. The extracted GFP plasmid was transfected into a cultured third generation of bone marrow stromal stem cells, Olymp. the transfection efficiency of the cells is observed under the us fluorescence microscope. Olym The changes of the cells were observed on a day-to-day basis by the pus fluorescence microscope. The results of the experiment were as follows: 1. The morphological observation of the bone marrow stromal stem cells in the rat: The bone marrow stromal stem cells were inoculated into the culture flask and then the cells were observed with an inverted microscope. The grown cells proliferate in a dispersed and colony-like manner, with the majority of the cells in the form of a shuttle, similar to a fibroblast, even with a large, flat polygonal cell. 7-10d After the growth of the colonies, the colonies grew, and the cells were fused with the growth of the colonies. the cells were no longer grown in a colony-like manner, and the distribution of the spindle-shaped cells was uniform. The morphological observation of the cardiac muscle cells: the cardiac muscle cells usually move out of the tissue mass after 24 h, and when the cells move out of the tissue mass, the cardiac muscle cells exhibit a shuttle-shape, with a companion. with moving out The cell is increased, and the heart muscle surrounding the tissue mass presents an irregular circular, triangular shape. The cells are closely connected, and the whole body is like a stone-laying stone. and as the growth density of the cells is increasing, the pseudo-feet that extend between the cells are woven into a net. Sometimes, some cells are tightly combined to form a cell cluster, radial or concentric circles, after 2-3 days of culture, these cell clusters spontaneously beat. 3. Results of GFP transfection of bone marrow stromal cells: after transfection of GFP The cell is still adherent to grow, is in the form of a shuttle or a polygon, but the proliferation is still split, but the proliferation rate is reduced. Under the fluorescence microscope, 24 h can be seen The expression of green fluorescence was small, the intensity was low, and the cells of 48 h showed strong fluorescence. 4. The morphology and growth behavior of BMSCs after co-culture: after co-culture, The proliferation of BMSCs was slow, and it was easy to be polygonal or flat. With the extension of co-culture time, there was a significant number of BMSCs in cell disintegration and the like. The colony-like growth was observed and the individual labeled BMSCs were observed with a myotube-like structure under a fluorescence microscope, which were usually close to the unlabeled cardiomyocytes of the beat. At the fourth day, the labeled BMSCs, which were partially in contact with the cardiomyocytes, began to have a weak heart-like beat. in that case of the present invention, Some BMSCs started to die, and a significant portion of BMSCs died in 64h. Conclusion: 1. The bone marrow stromal cells can be isolated from the bone marrow of the rat. C can be expressed for a long period of time, and the toxicity of the host cells is very small, which is an ideal tissue engineering seed cell.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
本文编号：2360912
[Abstract]:Purpose The bone marrow has a certain number of bone marrow stromal stem cells (BMSCs, bone marrow stem cells), which have self-replicating ability and great differentiation potential, and can be differentiated into the source of the mesoderm such as bone, cartilage, nerve, myocardium and fat under different conditions. The results of the study show that the 5-N-cell-like chemical can induce the differentiation of bone marrow stromal stem cells into the cardiac muscle cells, which makes the BMSCs become the most promising cells for the treatment of ischemic cardiomyopathy by cell transplantation. The purpose of this study was to observe the growth characteristics of BMSCs by in vitro separation, culture and dynamic observation of bone marrow stromal stem cells in rats. The ability of the cells to be enhanced and the expression of GFP in the labeled BMSCs was observed by the transfection of the bone marrow stromal cells with the GFP plasmid, with a view to further understanding the biological characteristics of the bone marrow stromal stem cells and to provide more reliable clinical application the theory of According to the method, the whole bone marrow adherent culture method is isolated and cultured in vitro to culture the bone marrow stromal stem cells of the rat, and the inverted phase difference microscope day-to-day observation of cell morphology and growth. 2. In vitro separation of the differential adhesion method, culture of the cardiac muscle cells and the inversion of the phase-difference microscope The morphology and growth of the cells were observed on a day-to-day basis. 3. The extracted GFP plasmid was transfected into a cultured third generation of bone marrow stromal stem cells, Olymp. the transfection efficiency of the cells is observed under the us fluorescence microscope. Olym The changes of the cells were observed on a day-to-day basis by the pus fluorescence microscope. The results of the experiment were as follows: 1. The morphological observation of the bone marrow stromal stem cells in the rat: The bone marrow stromal stem cells were inoculated into the culture flask and then the cells were observed with an inverted microscope. The grown cells proliferate in a dispersed and colony-like manner, with the majority of the cells in the form of a shuttle, similar to a fibroblast, even with a large, flat polygonal cell. 7-10d After the growth of the colonies, the colonies grew, and the cells were fused with the growth of the colonies. the cells were no longer grown in a colony-like manner, and the distribution of the spindle-shaped cells was uniform. The morphological observation of the cardiac muscle cells: the cardiac muscle cells usually move out of the tissue mass after 24 h, and when the cells move out of the tissue mass, the cardiac muscle cells exhibit a shuttle-shape, with a companion. with moving out The cell is increased, and the heart muscle surrounding the tissue mass presents an irregular circular, triangular shape. The cells are closely connected, and the whole body is like a stone-laying stone. and as the growth density of the cells is increasing, the pseudo-feet that extend between the cells are woven into a net. Sometimes, some cells are tightly combined to form a cell cluster, radial or concentric circles, after 2-3 days of culture, these cell clusters spontaneously beat. 3. Results of GFP transfection of bone marrow stromal cells: after transfection of GFP The cell is still adherent to grow, is in the form of a shuttle or a polygon, but the proliferation is still split, but the proliferation rate is reduced. Under the fluorescence microscope, 24 h can be seen The expression of green fluorescence was small, the intensity was low, and the cells of 48 h showed strong fluorescence. 4. The morphology and growth behavior of BMSCs after co-culture: after co-culture, The proliferation of BMSCs was slow, and it was easy to be polygonal or flat. With the extension of co-culture time, there was a significant number of BMSCs in cell disintegration and the like. The colony-like growth was observed and the individual labeled BMSCs were observed with a myotube-like structure under a fluorescence microscope, which were usually close to the unlabeled cardiomyocytes of the beat. At the fourth day, the labeled BMSCs, which were partially in contact with the cardiomyocytes, began to have a weak heart-like beat. in that case of the present invention, Some BMSCs started to die, and a significant portion of BMSCs died in 64h. Conclusion: 1. The bone marrow stromal cells can be isolated from the bone marrow of the rat. C can be expressed for a long period of time, and the toxicity of the host cells is very small, which is an ideal tissue engineering seed cell.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
【引证文献】
中国硕士学位论文全文数据库 前1条
1 罗建红;人脐血间充质干细胞移植治疗DCM大鼠的实验研究[D];湖南师范大学;2012年
，本文编号：2360912
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