人骨髓间充质干细胞与新生大鼠心肌细胞体外三维条件共培养的实验研究

发布时间：2018-04-14 20:32

本文选题：间充质干细胞 + 心肌细胞　；参考：《大连医科大学》2005年硕士论文

【摘要】：目的:心脏疾病的发病率和死亡率在各类疾病中始终处于重要地位,严重威胁人类生存和生活质量。由于成熟的心肌细胞受损后再生能力十分有限,因此,如何对受损心肌进行有效的修复治疗一直是医学界面临的最大难题之一。心肌重塑(cardiomyoplasty)是最近发展的治疗缺血性心脏病的一个新途径,即用细胞移植或组织工程的方法,将种子细胞直接移植或在体外培养成心肌组织后移植到受损的心肌中,用以修复受损心肌组织。以往的研究中,种子细胞的来源主要是新生大鼠心室肌细胞,但是,就临床应用而言,心肌细胞的来源极为有限,并且来源于异种的心肌细胞在移植过程中也不可避免地要受到免疫排斥反应的影响,因此,寻求其它来源的种子细胞也就成了目前亟待解决的问题。骨髓间充质干细胞(Mesenchymal stem cells, MSCs),已经被证实在一定的条件下可以转化为心肌细胞,并且因其具有取材容易、体外扩增能力强等优点,近年来受到了广泛的关注。本研究采用组织工程三维构建方法,将成人骨髓来源骨髓间充质干细胞(hMSCs)与新生大鼠心肌细胞在体外三维条件下共培养,探讨心肌细胞形成的心肌微环境对于人骨髓间充质干细胞向具有心肌细胞表型的细胞分化的影响。 ·2· 材料与方法: 1.经过反复实践,摸索出相对简便有效、实用的人骨髓间充质干细胞体外收集、分离、扩增培养技术 2.新生大鼠心肌细胞的原代分离培养:经过反复实践,摸索出相对简便有效、实用的新生大鼠原代心肌细胞体外收集、分离、培养技术,得到纯度和存活率都相对较高的原代心肌细胞。 3.采用组织工程三维构建方法,将成人骨髓来源骨髓间充质干细胞(hMSCs)与新生大鼠心肌细胞在体外三维条件下共培养,探讨心肌细胞形成的心肌微环境对于人骨髓间充质干细胞向具有心肌细胞表型的细胞分化的影响。将分离并培养的新生大鼠心肌细胞与 DIPI 标记的 hMSCs 以及液态Ⅰ型胶原混合,在环形槽中铸成环状细胞/胶原条带。构建的条带在环形槽中培养 14d后取出通过光学显微镜、荧光显微镜、免疫组织化学染色和激光共聚焦显微镜观察,对其中 DAPI 标记的 hMSCs 进行表型分析。结果:骨髓液密度梯度离心得到人骨髓间充质干细胞,所得细胞经流式细胞仪检测细胞表面抗原 CD34(-), CD45(-), CD29(+), CD44(+), HLA-DR(-),细胞纯度达到 99%以上。说明 hMSCs 的抗原表型不是单一的,具有间质细胞、上皮细胞等细胞的特征,但不表达造血干/祖细胞的标志如 CD34 和 CD45,因此证明我们所分离细胞是不同于造血干细胞的一类细胞群。组织学和免疫组织化学染色研究结果显示,在组织工程化三维构建物中,DAPI 标记的 hMSCs 分布广泛,呈椭圆形,似心肌细胞核,其排列方向与构建条带长轴排列方向一致。免疫组化检测标记的胞浆心肌特异蛋白肌钙蛋白 T 染色阳性。激光共聚焦显微镜观察显示,条带中标记的蓝染核 hMSCs 中具有类似周围心肌细胞的肌动蛋白和辅肌动蛋白的阳性表达;细胞的连接蛋白 Connexin43 也呈阳性表达。结论:hMSCs 在组织工程化心肌微环境的作用下可以向具有心肌细胞表型的细胞分化,并且可以和周围的细胞建立细胞连接。hMSCs 可以成为组织工程化心肌的另一种新型的种子细胞来源。在心肌细胞来源极为有限的情况下,我们可以考虑利用患者自身的 hMSCs 来部分、甚
[Abstract]:Objective: the incidence and mortality of heart disease always plays an important role in various diseases, serious threat to human survival and quality of life. Because of the maturity of the myocardial cell damage after regeneration ability is very limited, therefore, how to effectively repair the damaged myocardium has been one of the biggest problems facing the medical profession. Myocardial remodeling (cardiomyoplasty) is a new way for the treatment of ischemic heart disease in recent development. The method is used for cell transplantation or tissue engineering, seed cells transplanted directly or in vitro into myocardial tissue after transplantation into the damaged myocardium, to repair the damaged myocardium. In the previous study, the source of seed cells is the main cell ventricular myocytes of neonatal rats, however, clinical application, source of myocardial cells is extremely limited, and the origin of heterogeneous myocardial cells in transplantation process can not be To avoid to be affected, so the immune rejection, seed cells seeking other sources have become an urgent problem to be solved. Bone marrow mesenchymal stem cells (Mesenchymal stem, cells, MSCs) have been identified in certain conditions can be transformed into myocardial cells, and because of its easy. The advantages of in vitro amplification ability, has received widespread attention in recent years. This research adopts three-dimensional tissue engineering construction method, the source of adult bone marrow mesenchymal stem cells (hMSCs) and myocardial cells of neonatal rats were cultured in vitro in three-dimensional conditions, to investigate the myocardial cell formation of myocardial micro environment for human bone marrow mesenchymal stem cells cell differentiation with myocardial cell phenotype.
2
Materials and methods:
1. after repeated practice, a relatively simple, effective and practical human bone marrow mesenchymal stem was found.
In vitro collection, separation and amplification of cells in vitro
2. primary isolation and culture of neonatal rat cardiac myocytes: through repeated practice, fumble the phase
In vitro collection, separation and culture of a simple, effective and practical neonatal rat primary cardiomyocyte in vitro
Primary cardiomyocytes with relatively high purity and survival rate were obtained.
3. using a three-dimensional tissue engineering method, the bone marrow mesenchymal stem cells of adult bone marrow are dry and fine
Cell (hMSCs) and neonatal rat cardiomyocytes were co cultured in vitro in vitro to explore myocardial fine
The microenvironment formed by the cell to the phenotype of human marrow mesenchymal stem cells to the cardiomyocytes
The effect of cell differentiation. The isolated and cultured neonatal rat cardiomyocytes are labeled with DIPI
The mixture of hMSCs and liquid type I collagen is cast into a ring cell / collagen band in a ring groove.
The constructed strip is taken in a circular slot to be cultured for 14d and removed through an optical microscope, a fluorescence microscope,
Immunohistochemical staining and laser confocal microscopy were used to observe the DAPI markers.
HMSCs was used for phenotypic analysis.
Results: bone marrow mesenchymal stem cells were obtained from bone marrow liquid density gradient centrifugation, and the obtained cells were obtained.
Cell surface antigen CD34 (-), CD45 (-), CD29 (+) were detected by flow cytometry.
CD44 (+), HLA-DR (-), the cell purity of more than 99%. Indicating the antigen phenotype of hMSCs
Not single, having the characteristics of cells, such as interstitial cells, epithelial cells, but not expressing hematopoiesis
The markers of stem / progenitor cells, such as CD34 and CD45, prove that the cells we separate are different
A group of cell groups of hematopoietic stem cells.
Histological and immunohistochemical staining results show that the three-dimensional structure of tissue engineering
In the construction, the DAPI labeled hMSCs is widely distributed, oval, like the nucleus of the heart of the myocardium.
The direction is consistent with the orientation of the long axis of the constructed strip. The cytoplasm myocardium of the immunohistochemical detection marker
The isoprotein troponin T staining was positive. The laser confocal microscopy showed that the bands were marked in the strip.
The blue nuclear hMSCs with similar surrounding myocardial cell actin and the actin filament
Positive expression was expressed, and the connexin Connexin43 of the cell was also positive.
Conclusion: hMSCs can have fine myocardium under the action of tissue engineered myocardial microenvironment.
Cell phenotype cells differentiate and can establish cell connection.HMSCs with the surrounding cells.
Another new seed cell source of tissue engineered myocardium. In the source of cardiac myocytes
In a very limited case, we can consider the use of the patient's own hMSCs part, very much.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

【共引文献】