冻存和传代后脂肪间充质干细胞向心肌样细胞的分化

Adipose tissue-derived mesenchymal stem cell differentiation into cardiomyocyte-like cells after cryopreservation and passage

背景:脂肪间充质干细胞是一种成体多能干细胞,将成为一种重要的用于心肌组织工程和心脏病细胞治疗研究的种子细胞。目的:观察脂肪间充质干细胞经液氮冻存和长期传代后向心肌样细胞分化的能力。设计、时间和地点:对比观察,于2007-09/2008-06在解放军军事医学科学院卫生装备研究所组织工程实验室完成。材料:大鼠脂肪间充质干细胞,来自2月龄Wistar大鼠附睾脂肪垫。方法:第3代大鼠脂肪间充质干细胞液氮冻存6个月,复苏后用10μmol/L5-氮胞苷诱导,2周后检测细胞分化状况。另外,将间充质干细胞长期传代培养,用10μmol/L5-氮杂胞苷处理,2周后检测细胞分化状况。以未经5-氮杂胞苷处理的脂肪组织间充质干细胞为对照。主要观察指标:免疫组织化学染色观察α-肌节型肌动蛋白、骨骼肌快肌肌浆球蛋白(骨骼肌细胞特有)和GATA-4的表达。结果:液氮冻存后的脂肪间充质干细胞,经诱导后α-肌节型肌动蛋白和GATA-4免疫组织化学染色均为阳性,骨骼肌快肌肌浆球蛋白免疫组织化学染色为阴性;第6代脂肪间充质干细胞,经诱导后免疫组织化学染色结果与冻存后相同;第9代脂肪间充质干细胞,经诱导后α-肌节型肌动蛋白、骨骼肌快肌肌浆球蛋白和GATA-4免疫组织化学染色均为阳性。对照组均为阴性表达。结论:脂肪间充质干细胞冻存后可向心肌样细胞分化;长期传代的脂肪间充质干细胞也能向心肌样细胞分化,但经5-氮杂胞苷诱导也可能同时向骨骼肌样细胞分化。

BACKGROUND： Adipose tissue-derived mesenchymal stem cell is a kind of adult multipotential stem cells, and will become seed cells for cardiac tissue engineering and cell therapy of heart disease. OBJECTIVE： To observe adipose tissue-derived mesenchymal stem cell differentiation into cardiomyocyte-like cells after cryopreservation or passage for a long time. DESIGN, TIME AND SETTING： The controlled observational experiment was performed at the Tissue Engineering Laboratory in the Institute of Medical Equipment, Academy of Military Medical Sciences of Chinese PLA from September 2007 to June 2008. MATERIALS： Rat adipose tissue-derived mesenchymal stem celts were obtained from epididymis adipose pad of 2-month-old Wistar rats. METHODS： The third passage rat adipose tissue-derived mesenchymal stem cells were preserved in liquid nitrogen for six months and thawed, then the cells were induced with 10 μ mol/L 5-azacytidine. After 2 weeks, the mesenchymal stem cell differentiation potential was detected. Additionally, the third passage adipose tissue-derived mesenchymal stem cells were passed for long time in vitro, then induced with 10μmol/L 5-azacytidine. After 2 weeks, the cell differentiation potential was detected. The stem cells not induced with 5-azacytidine served as control group. MAIN OUTCOME MEASURES： Immumohistochemistrical staining was used to observe expression of a -sacromeric actin, a -skeletal myosin （only detected in skeletal muscle ） and GATA-4. REAULTS： After cryopreservation, a -sacromeric actin and GATA-4 immumohistochemistrical staining were both positive in the induced adipose tissue-derived mesenchymal stem cells, the a-skeletal myosin staining was negative in the induced cells. After inducement, the immumohistochemistrical staining result in the passage 6 adipose tissue-derived mesenchymal stem cells was the same as the cells mentioned above. After inducement, in the passage 9 adipose tissue-derived mesenchymal stem cells, the immumohistochemistrical staining results of G -sacromeric actin, a -skeletal myosin, GATA-4 were all positive. Control groups were all negative. CONCLUSION： After cryopreservation, adipose tissue-derived mesenchymal stem cells could differentiate to cardiomyocyte-like cells. After passage for a long time, adipose tissue-derived mesenchymal stem cells could differentiate into cardiomyocyte-like cells, but could differentiate into skeletal muscle-like cells after induced with 5-azacytidine,