大鼠脂肪间充质干细胞分离培养及成骨潜能的试验研究  

Experimental Study of the Isolation,Cultivation and Osteogenic Potential of Rat Adipose-Derived Mesenchymal Stem Cells

作　　者：邓展生[1] 张胜利[1] 李宝军[1] 郭晓柠[1] 高嵩涛[1] 鞠洪斌[1] 

机构地区：[1]中南大学湘雅医院骨科,湖南长沙410008

出　　处：《医学临床研究》2006年第12期1891-1894,共4页Journal of Clinical Research

摘　　要：【目的】研究大鼠脂肪间充质干细胞分离培养的方法和成骨分化的潜能。【方法】取大鼠腹股沟脂肪垫,酶消化法分离细胞,接种入含新生牛血清的DMEM培养基进行原代培养,用含地塞米松、抗坏血酸、β-甘油磷酸钠的DMEM培养基诱导其成骨分化。倒置显微镜、光学显微镜、透射电镜观察细胞形态,测定细胞倍增时间,检测碱性磷酸酶染色、钙结节染色。【结果】脂肪间充质干细胞呈成纤维细胞样贴壁生长,倍增时间为35 h,传代稳定;经成骨诱导,细胞呈多角形,体积明显增大,倍增时间为68 h。Gomori改良钙钴法碱性磷酸酶染色显示其胞浆内含碱性磷酸酶颗粒,阳性率为80%;茜素红S染色见钙结节形成。【结论】成功地建立了一种分离、培养大鼠脂肪间充质干细胞的方法;经成骨诱导,其具有成骨的潜能,可作为骨组织工程的种子细胞。[Objective]To isolate and cultivate rat adipose tissue-derived mesenchymal stem cells（ADMSC） and study their osteogenic potentials as seeding cells in bone tissue engineering. [Methods]ADMSC were isolated from rat inguinal fat pads by digestion ,then placed in Dulbeco Modified Eagle Medium（DMEM） with fetal bovine serum in primary cultivation, followed by osteogenic cultivation in DMEM supplemented with dexamethasone, ascorbic acid, βglycerophosphate . ADMSC were observed under inverted microscope, light microscope and transmission electron microscope. The population doubling time was studied. The osteogenic differentiation was assessed with alkaline phosphatase activity （ALP） and alizarin red S staining. [Results]ADMSC were fibroblast-shaped and could passage stably, and the population doubling time was 35 hours. After osteogenic cultivation, ADMSC appeared more cuboidal and larger. The population doubling time was 68 hours. Gomori ALP staining demonstrated positive expression of ALP , and the positive ratio was 80%. Alizarin red S staining verified the formation of mineralized nodules. [Conclusion]ADMSC successfully isolated and cultivated by this method in this study can stably passage and rapidly proliferate in vitro. After osteogenic cultivation, ADMSC show the osteogenic potentials and could be used as seeding cells in bone tissue engineering.

关 键 词：干细胞 细胞分离 细胞培养 骨生成 脂肪类 大鼠 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学]