论文部分内容阅读
背景:目前并没有特定的实验对骨髓间充质干细胞分离的两种基本方法进行系统评估,所以贴壁分离法和密度梯度离心法对细胞诱导分化是否存在不同影响,尚无定论。目的:拟验证两种基本分离方法对骨髓间充质干细胞成软骨分化影响的差别。设计、时间及地点:对照观察实验,2005-03/09在中国医科大学附属盛京医院儿外实验室完成。材料:2～3月龄体质量1.2～2.0kg的日本大耳兔20只用于抽取骨髓。方法:用贴壁分离法和密度梯度离心法分离骨髓间充质干细胞。选择两组同代的骨髓间充质干细胞,用转化生长因子β1诱导其向软骨方向分化。主要观察指标:①倒置显微镜观察骨髓间充质干细胞的生长情况,绘制两组细胞生长曲线。②免疫组化检测两组Ⅱ型胶原表达。③原位杂交检测两组细胞Ⅱ型胶原mRNA表达。结果:绘制生长曲线表明两组细胞生长速度趋于一致。免疫组化法检测两种方法分离的骨髓骨髓间充质干细胞的软骨细胞分化率为76.1%和77.7%,原位杂交法检测的软骨细胞分化率为70.3%和71.0%,差异无显著性意义。结论:两种分离方法对骨髓间充质干细胞的生长和成软骨分化结果的影响无差异。
BACKGROUND: There are currently no specific experiments that systematically evaluate two basic methods of bone marrow mesenchymal stem cell isolation. It is inconclusive whether adherent separation and density gradient centrifugation have different effects on cell differentiation. OBJECTIVE: To verify the difference between the two basic methods of separation of bone marrow mesenchymal stem cells into cartilage differentiation. DESIGN, TIME AND SETTING: The Controlled Observation Experiment was performed at the Foreign Laboratory of Shengjing Hospital, China Medical University from March 2005 to September 09, 2005. MATERIALS: 20 Japanese rabbits with body weight of 1.2-2.0 kg, 2 to 3 months old, were used for bone marrow extraction. Methods: Bone marrow mesenchymal stem cells were isolated by adherent separation and density gradient centrifugation. Two groups of the same generation of bone marrow mesenchymal stem cells were selected and induced to differentiate into cartilage by using transforming growth factor β1. MAIN OUTCOME MEASURES: ① Inverted microscope was used to observe the growth of bone marrow mesenchymal stem cells, and the cell growth curves of two groups were drawn. ② Immunohistochemical detection of type Ⅱ collagen expression in both groups. ③ In situ hybridization detection of two groups of cells collagen type Ⅱ mRNA expression. Results: The growth curve of the two groups showed that the cell growth rate tended to be consistent. The rates of chondrocytes differentiation by bone marrow mesenchymal stem cells isolated by two methods were 76.1% and 77.7% by immunohistochemistry and 70.3% and 71.0% by in situ hybridization, respectively, with no significant difference . Conclusion: There is no difference between the two methods on the growth of bone marrow mesenchymal stem cells and on the differentiation into cartilage.