体外分离培养小鼠骨髓间充质干细胞Ⅰ、Ⅲ型前胶原的表达

Expression of type Ⅰ and Ⅲ procollagen in mouse bone marrow mesenchymal stem cells following in vitro isolation and culture

背景：尽管目前研究将骨髓间充质干细胞的Ⅰ、Ⅲ型胶原蛋白高表达作为细胞向某一方向分化的重要标志,但未分化的骨髓间充质干细胞是否表达该类蛋白目前尚无定论。目的：观察分离培养的小鼠骨髓间充质干细胞内Ⅰ、Ⅲ型前胶原的表达情况。设计、时间及地点：细胞学体外对照观察,于2007-10/2008-05在广东药学院完成。材料：ICR小鼠3只,1d龄ICR乳鼠1只,由广东省医学实验动物中心提供。方法：利用差速贴壁法体外分离纯化小鼠骨髓间充质干细胞,待细胞至70%-80%融合时传代。取1d龄ICR乳鼠皮肤组织,自行分离传代并冻存,经复苏后获得乳鼠皮肤成纤维细胞。实验组将稳定传代3次的小鼠骨髓间充质干细胞接种到6孔培养板中,按109L-1密度接种,1mL/孔;对照组同法接种乳鼠皮肤成纤维细胞,培养72h。主要观察指标：骨髓间充质干细胞的形态及生长情况,RT-PCR法检测细胞内Ⅰ、Ⅲ型前胶原的表达。结果：原代培养的小鼠骨髓间充质干细胞集落形成初期,细胞核较大,胞浆较少,细胞呈三角形、多角形和长梭形等多种形态;传代后细胞生长旺盛,多数呈长梭型,细胞表面有大量微绒毛。培养48h时,实验组无Ⅰ、Ⅲ型前胶原表达,对照组表达Ⅰ、Ⅲ型前胶原;培养72h时,实验组Ⅰ、Ⅲ型前胶原表达量显著低于对照组（t=42.692,85.349,P〈0.01）。结论：传代培养48h时,骨髓间充质干细胞内Ⅰ、Ⅲ型前胶原基因尚未启动,至72h后Ⅰ、Ⅲ型前胶原基因出现低表达,其并非像成纤维细胞那样在生长过程中持续的表达Ⅰ、Ⅲ型前胶原基因。

BACKGROUND： High expression of types Ⅰ and Ⅲ collagen protein of bone marrow mesenchymal stem ceils （BMSCs） is as a key marker of cell differentiation. However, whether undifferentiated BMSCs express these proteins or not is uncertain. OBJECTIVE： To observe the expression of types Ⅰ and Ⅲ procollagen in mouse BMSCs. DESIGN, TIME AND SETTING： The cytology in vitro controlled study was performed at the Guangdong Pharmaceutical University from October 2007 to May 2008. MATERIALS： A total of 3 ICR mice and 1 ICR neonatal mouse aged 1 day were supplied by Guangdong Medical Experimental Animal Center. METHODS： BMSCs of mice were isolated by using the differential adherence method in vitro. Ceils were incubated for passage when 70% -80% cells were confluent. Skin tissue of 1-day ICR neonatal mouse was obtained, incubated and frozen for use. Skin fibroblasts of the neonatal mouse were obtained following resuscitation. Mouse BMSCs in the experimental group following passage for three times were incubated in a 6-well culture plate at 10^9/L, 1 mL per well. Mouse skin fibroblasts were incubated in the control group for 72 hours. MAIN OUTCOME MEASURES： Morphology and growth of BMSCs were measured. Expression of types Ⅰ and Ⅲprocollagen in cells were quantified using reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： In the early colony forming period, primary cultured mouse BMSCs presented large nuclei, less cytoplasm, and were triangle, polygonal and spindle. Following passage, long spoindie-shape ceils productively grew, with a large amount of microvillus on the surface. At 48 hours, types Ⅰ and Ⅲ procollagen expression was found in the control group, but no these expression in the experimental group. At 72 hours, expression of types Ⅰ and Ⅲ procollagen was significantly lower in the experimental group than in the control group （t=42.692, 85.349, P 〈 0.01）. CONCLUSION： The expression of the products of type Ⅰ and type Ⅲ procollagen from BMSCs were not persistent at 48 hours, and low expression appeared at 72 hours. This is not the same as that of fibroblasts, which express type Ⅰ and Ⅲ procollagen during growth.