大鼠真皮多能间充质干细胞的分离培养  

Isolation and culture of multipotent mesenchymal stem cells from neonatal rat dermis

作　　者：史春梦[1] 程天民[1] 

机构地区：[1]第三军医大学预防医学系防原医学教研室,全军复合伤研究所,重庆400038

出　　处：《第三军医大学学报》2001年第9期1068-1079,共12页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划资助项目 (G19990 54 2 0 5)

摘　　要：目的　探讨发育成熟高等动物真皮组织中是否存在多能间充质干细胞 ,对其进行分离培养并初步研究其基本生物学性状。方法　以新生大鼠为研究对象 ,依据间充质干细胞的黏附特性采用酶消化法对其分离 ,通过传代培养进行筛选 ;流式细胞技术检测细胞周期 ,MTT法检测细胞对生长因子的反应性 ,免疫组织化学法检测细胞表面分子的表达。结果　早期贴壁的大鼠真皮细胞经过连续传代培养至第 3代 ,细胞形态较为均一 ,超过 88%的细胞处于G0 /G1期 ,细胞表面波形蛋白表达阳性 ,但Ⅷ因子和角蛋白表达阴性 ;体外培养细胞增殖旺盛 ,对bFGF有良好反应性 ,但对EGF和KGF反应不明显 ;地塞米松诱导分化实验证实该细胞可向成骨细胞、软骨细胞和脂肪细胞分化 ,具有多向分化潜能。结论　本实验证实发育成熟大鼠真皮组织中存在多能干细胞 ,成功地分离得到真皮来源的间充质干细胞并通过其基本生物学特性进行了鉴定。上述结果提示真皮有可能是间充质干细胞的又一重要来源途径。这为深入研究真皮间充质干细胞在组织修复与再生中的意义与应用提供了基础。Objective To isolate multipotent mesenchymal stem cells (MSCs) from adult rat dermis and study their basic phenotypical and functional properties. Methods Dermis of neonatal rat were digested with 0.25% trypsin and cultured for 6 h in vitro , the adherent cells were harvested and subcultured. After the third subculture,the cells were used for experiments. Cell cycle was analyzed by measuring DNA content with FACScan flow cytometer and cell proliferation activity was tested by MTT method. Phenotypical analysis of MSCs was made with immunohistochemical ABC technique and differentiated cells were identified by specific staining. Results The adherent cells in 6 h cultured neonatal rat dermis showed high activity of proliferation in ex vivo culture and more than 88% of cells were in the G 0/G 1 phases, expression of vimentin on cell surface was positive, Factor Ⅷ and cytokeratin were negative;bFGF can promote cell proliferation ,but EGF and KGF failed. Dexamethasone treatment induced cell differentiation into osteocytes, chondrocytes and adipocytes. BMP 2 treatment induced cells to form calcium nodules. Conclusion Mesenchymal stem cells exist in rat dermis and can be isolated by their adherent ability.Dermis may be another important source of mesenchymal stem cells.

关 键 词：真皮间充质干细胞 细胞分离 大鼠 细胞培养 皮肤再生 

分 类 号：R622.1[医药卫生—整形外科] R329.2[医药卫生—外科学]