摘要
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： At present, there has been no definite experiment systemically evaluating adherent separation and density gradient centrifugation to isolate bone marrow mesenchymal stem cells （BMSCs）. Whether the two methods produce different influences on BMSC induction and differentiation remains unclear. OBJECTIVE： This study was designed to verify difference of these two isolation methods in chondrogenic differentiation of BMSCs. DESIGN, TIME AND SETTING： A controlled observation was performed at the Shengjing Hospital Affiliated to China Medical University from March to September 2005. MATERIALS： Twenty Japanese big-ear rabbits, aged 2-3 months, weighing 1.2-2.0 kg, were included for this study. METHODS： BMSCs were isolated by adherent separation and density gradient centrifugation. Two groups of BMSCs were taken from the same passage and induced towards chondrogenic differentiation with transforming growth factor beta 1. MAIN OUTCOME MEASURES： Growth of BMSCs was observed under an inverted microscope to draw growth curves; Type Ⅱ collagen expression was detected by immunohistochemistry. Type Ⅱ collagen mRNA expression was determined by in situ hybridization. RESULTS： The growth curves demonstrated that cellular growth velocity of the two groups tended to be the same. Immunohistochemistry results showed that the efficiency of adherent separation and density gradient centrifugation for promoting chondrogenic differentiation of BMSCs was 76.1% and 77.7%, respectively, and in situ hybridization results showed that the efficiency was 70.3% and 71.0%, respectively. No significant difference in differentiation efficiency existed between the adherent separation and the density gradient centrifugation. CONCLUSION： Adherent separation and density gradient centrifugation had no different influences on BMSC growth and chondrogenic differentiation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7577-7581,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
the Grant from Project of Department of Education of Liaoning Province, No. 05L568~~
作者简介
王广斌★，男，1978年生，辽宁省丹东市人，汉族，2005年中国医科大学毕业，硕士，讲师，主要从事软骨组织工程方面的研究。
通讯作者：付勤，教授，中国医科大学附属盛京医院脊柱关节外科，辽宁省沈阳市110004。
