密度梯度离心并贴壁法分离成人骨髓间充质干细胞的成骨特性  

Osteogenic characteristics of adult human bone marrow mesenchymal stem cells isolated with combined density gradient centrifugation and attachment culture method

作　　者：吴国平[1] 滕利[2] 杨锴[2] 卢建建[2] 高寿松[2] 范新宇[2] 

机构地区：[1]泸州医学院附属医院整形外科,四川省泸州市646000 [2]中国协和医科大学中国医学科学院整形外科医院,北京市100041

出　　处：《中国组织工程研究与临床康复》2007年第7期1209-1212,I0001,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察密度梯度离心法结合贴壁法体外分离纯化培养人骨髓间充质干细胞的效果,以及诱导后的成人骨髓间充质干细胞的成骨特性。方法:实验于2005-05/09在中国协和医科大学中国医学科学院整形外科医院进行。用密度梯度离心法结合贴壁法分离纯化成人骨髓间充质干细胞。采用倒置显微镜观察,噻唑兰法测定细胞生长曲线,免疫细胞化学鉴定骨髓间充质干细胞膜抗原和细胞基质蛋白属性。取体外扩增的第3代骨髓间充质细胞,以3.0×103/cm2的浓度接种于放置有无菌处理的盖玻片的六孔板中,每孔内加2mL含10%胎牛血清的低糖型DMEM培养基培养液。当细胞贴壁生长达到60%～70%汇合时,将培养基更换为含5%胎牛血清的低糖型DMEM培养液,其中含骨诱导剂地塞米松、抗坏血酸和β-磷酸甘油,浓度分别为:100nmol/L、0.25mmol/L、10mmol/L。间充质细胞在这样的诱导体系中进行诱导培养,对照只加培养基。在第4、8、12、16天分别观察各孔中细胞的形态学和组织化学变化。体外加成骨诱导剂后分别作碱性磷酸酶活性测定、茜素红染色和VonKossa染色,观察骨髓间充质干细胞的成骨分化结果。结果:①分离的骨髓间充质干细胞培养48h后贴壁,72h后出现纺锤状细胞,贴壁的骨髓间充质细胞平均约12d后形成克隆。②第2代骨髓间充质干细胞99%表现为CD44+、Vim+、CD34-,CD29-。细胞表型稳定。③加成骨诱导剂后碱性磷酸酶活性明显增高,钙沉积在第8d就开始出现,茜素红染色、VonKossa染色阳性。结论:密度梯度离心法结合贴壁法分离纯化的成人骨髓间充质干细胞纯度高,表型稳定,体外加成骨诱导剂培养后,符合成骨细胞的形态特征和生物学特性,具有成骨活性,可为骨组织工程和基因治疗提供比较理想的种子细胞。AIM： To observe the effect of the in vitro isolation, purification and amplification of adult human bone marrow mesenchymal stem calls （hBMSCs） by density gradient cantrifugation method, and the biological characteristics of induced hBMSCs. METHODS： The experiment was conducted at the Plastic Surgery Hospital of Chinese Academy of Medical Sciences from May to September 2005. hBMSCs were isolated and purified by density gradient cantrifugation combined with attachment culture method. The growth of hBMSCs was observed under an inverted microscope; the growth curve was detected by the methyL thiazolyL tetrazolium; hBMSCs memberane antigen and cellular matrix protein property were identified with immunocytochemical method. The well-grown hBMSCs of the third generation were chosen to be plated at a density of 3.0×10^3 celLs/cm^2 in a six-hole plate with sterile coverglass, and each hole contained 2 mL DMEM solution with 10% fetal bovine serum. When the calls grew to 60%-70% adhesion to the bottom, the culture solution of medium were replaced by 5% fetal bovine serum, which contained 100 nmol/L dexamethasone, 0.25 mmol/L antiscorbic acid and 10 mmol/L β-glycarephosphate, hBMSCs were cultured in this medium, while the control group was only added culture solution, Morphological and histochemical changes of the hBMSCs were observed at the days of 4, 8, 12 and 16. After the osteogeneic inducer was added in vitro, the alkaline phosphatase activities were detected, and alizarin monosulfonate and Von Kossa staining were performed, respectively to observe the osteogenous differentiation of hBMSCs. RESULTS：（1）The isolated hBMSCs began to adhere after cultured for 48 hours, and appear spindle after 72 hours; the attached hBMSCs began to form clone at average 12 days. （2）Vimentin and CD44 antigen could be detected in 99% of the second filial generation of hBMSCs, but not CD34 and CD29 antigen, and the phenotype of hBMSCs was stabilization, （3）After osteogeneic inducer was added in vitro, the alkaline phos

关 键 词：骨髓细胞 干细胞 生物学 

分 类 号：R394.2[医药卫生—医学遗传学]