兔外周血间充质干细胞的体外分离培养及诱导成骨  

Isolation, culture and differentiation of rabbit peripheral blood mesenchymal stem cells into osteoblasts in vitro

作　　者：孙良[1] 栾保华[1] 李中华[2] 王小霞[1] 刘萌萌[1] 

机构地区：[1]山东大学附属省立医院烧伤整形外科,山东省济南市250021 [2]济南市第四人民医院烧伤科,山东省济南市250021

出　　处：《中国组织工程研究与临床康复》2009年第27期5291-5295,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：山东省自然科学基金资助项目(Y2002C19)~~

摘　　要：背景:研究证明外周血中存在间充质干细胞,但含量极少。目的:拟从兔外周血中提取间充质干细胞,并诱导其向成骨细胞分化。设计、时间及地点:细胞学体外观察,于2008-06/12在山东省立医院中心实验室完成。材料:新西兰大白兔6只,购自山东省农科院。α-MEM培养基、L-DMEM培养基为Hyclone公司产品。方法:每隔2d从兔背部不同部位切去3cm×3cm的全层皮肤(保留背部肌层),所切皮肤做原位移植后包扎,每只兔连续创伤4次。分别于创伤前及末次创伤1周后从股静脉取外周血,采用密度梯度离心法获取间充质干细胞,设立2组,分别加入含体积分数为10%胎牛血清的α-MEM培养基或L-DMEM培养基。细胞传至第2代以1×105/cm2密度接种于12孔板,加入含成骨诱导剂的H-DMEM培养液进行成骨诱导。主要观察指标:创伤前后细胞形态观察,成纤维样细胞集落形成率,成骨诱导效果。结果:创伤前所获得的细胞首次换液后,未见有梭形细胞贴壁;创伤后所获得的细胞接种24h即可见有短梭形及多角形细胞贴壁,五六天后出现细胞集落。与L-DMEM培养基组比较,α-MEM培养基组外周血成纤维样细胞集落形成率明显升高(P<0.05)。外周血间充质干细胞成骨诱导后,呈梭形、三角形或多角形,有突起,可见两三个核仁和细胞分裂相,增殖速度缓慢,诱导7d后碱性磷酸酶阳性率达80%以上,诱导21d后茜素红染色形成钙结节。结论:皮肤损伤刺激后可成功从兔外周血中获取间充质干细胞,且具备成骨分化特性,α-MEM培养基较L-DMEM培养基更适合外周血间充质干细胞的体外生长。BACKGROUND： A few mesenchymal stem cells （MSCs） can be found in peripheral blood. OBJECTIVE： To isolate rabbit peripheral blood MSCs and induce its differentiation into osteoblasts. DESIGN, TIME AND SETTING： The cytological in vitro study was performed at the Central Laboratory of Shangdong Provincial Hospital from June to December 2008, MATERIALS： A total of 6 New Zealand rabbits were purchased from Shandong Academy of Agricultural Sciences. α -MEM and L-DMEM were bought from Hyclone. METHODS： Full-thickness skin （3 cm×3 cm） （dorsal muscular layer was left） was incised at various sites of rabbit back, every 3 days. Incised skin was dressed following orthotopic transplantation. Each rabbit received four consecutive wounds, Peripheral blood was collected from femoral vein before injury and 1 week after injury. MSCs were harvested from peripheral blood by density gradient centrifugation. MSCs were divided into 2 groups, which were respectively incubated in α -MEM supplemented with 10% fetal bovine serum and L-DMEM. Cells at passage 2 and 1 ×10^5/cm2 were incubated in a 12-well plate and induced with H-DMEM containing osteogenic inductor. MAIN OUTCOME MEASURES： The following parameters were measured： cell morphology before and after injury; colony forming efficiency of MSCs; outcome of osteogenic induction. RESULTS： Following primary medium change and before injury, obtained cells were normal. Twenty-four hours following incubation and after injury, MSCs were spindle or polygonal, and adhered to the wall. 5-6 days later, cell colonies appeared. Compared with the L-DMEM group, the number of primary culture colony formation in α -MEM group was significantly greater （P 〈 0.05）. Peripheral blood MSCs were spindle, tdangle or polygonal, with the presence of processes, 2 3 nuclei and cell division phase, and slowly proliferated following osteogenic induction. At day 7 after differentiation of MSCs into osteoblasts, positive rate of alkaline phosphates was above 80%. At day 21, calciu

关 键 词：外周血间充质干细胞 培养基 α-MEM L-DMEM 成骨诱导 

分 类 号：R394.2[医药卫生—医学遗传学]