骨髓间充质干细胞的克隆培养及其向心肌细胞的诱导分化

Cloning Culture of the Marrow Mesenchymal Stem Cells and Their Differentiation Towards Cardiomyocytes

目的 建立骨髓间充质干细胞(MMSC)的单细胞克隆,筛选具有心肌特异性分化潜能的c-kit+MMSC克隆,进行体外心肌诱导分化研究。方法 取成年雄性SD大鼠骨髓细胞,进行原代培养和单克隆培养。通过c-kit免疫细胞化学标记和RT-PCR检测,筛选出向心肌特异性分化的MMSC克隆。然后用5-氮杂胞苷诱导分化,RT-PCR检测诱导前后心肌特异性基因表达的变化。结果 骨髓中含有多种MMSC克隆,以不同形态和大小的成纤维细胞样克隆数目最多。较大的成纤维细胞样克隆和扁平多角形细胞克隆均表达c-kit。用5-氮杂胞苷诱导后,表达Nkx2.5的c-kit+MMSC克隆的细胞形态和排列方式发生变化。诱导后2周,细胞变为短柱状,且平行排列;第3周,相邻细胞间形成明显的细胞连接;第4周,由多个细胞相互连接形成肌管样结构,并可观察到肌管的自发性搏动。心室肌特异性肌球蛋白轻链(MLC-2v)mRNA从第3周开始明显表达。结论 骨髓中含有多种干细胞克隆。筛选具有心肌特异性分化潜能的MMSC克隆并构建心肌干细胞库,将为临床干细胞移植治疗心肌梗死提供理想的干细胞来源。

Purpose: To establish single-cell clones of marrow mesenchymal stem cells (MMSC), select c-kit+ MMSC clones which have potential to differentiate specially towards cardiomyocytes and investigate their differentiation into cardiomyocytes in vitro. Methods: Primary MMSCs were isolated from the bone marrow of adult male SD rats and then single-cell cloning culture was performed. MMSC clones which may differentiate specially towards cardiomyocytes were selected through c-kit immunocytochemical staining and RT-PCR analysis. Differentiation of these MMSC clones was induced with 5-azacytidine. Expression of cardiac-specific genes of the MMSC clones was examined with RT-PCR analysis before and after induction. Results: There were multiple MMSC clones in bone marrow. The number of fibroblast-like clones which were different in shape and size was greatest. C-kit was expressed on large fibroblast-like clones and flat polygonal clones. After having been induced with 5-azacytidine, the shape and arrangement of the Nkx2.5-expressed c-kit + MMSC clones changed. The cells grew to puncheon-like and were parallel during the second week. Cell conjunctions formed during the third week. Myotube-like structures formed by adjacent cells and their spontaneous beats were observed around the fourth week. Myosin light chain-2v (MLC-2v) mRNA did not express obviously until the third week. Conclusions: There are many stem cell clones in the bone marrow. Selecting MMSC clones which have potential to differentiate specially towards cardiomyocytes and establishing a bank of cardiac stem cells will be useful for supplying autologous stem cells for clinical stem cell transplantation to treat infarction.