大鼠骨髓间充质干细胞定向成骨诱导的动态观察

背景:骨髓间充质干细胞由于其具有多向分化潜能和高增殖潜能而在组织工程领域具有广泛的应用前景.目的:观察大鼠骨髓间充质干细胞在向成骨细胞分化过程中细胞及生物活性变化.设计、时间和地点:随机对照实验,实验于2004-07/2006-06在解放军广州军区广州总医院医学实验科干细胞组织工程实验室完成.材料:成年清洁级SD大鼠20只,雌雄不拘用于取骨髓.方法:按随机数字表法将动物分为实验组和对照组,每组10只.通过改良的骨髓培养法分离成年大鼠骨髓间充质干细胞,对照组以基础培养液(10%胎牛血清 高糖DMEM 100U/L青霉素 100 U/L链霉素 2 mmol/L谷氨酰胺)培养,实验组用条件培养液(基础培养液,10 mmol/Lβ-甘油磷酸纳,10-7 mol/L地塞米松,50 mg/L维生素C)进行诱导培养.一段时间后制备细胞爬片.主要观察指标:①用倒置显微镜和透射电子显微镜进行细胞形态学观察.②采用Gomori改良钙钴法进行碱性磷酸酶染色,应用免疫细胞化学染色方法测定骨钙素的表达.结果:骨髓间充质干细胞接种48 h后,较多细胞贴壁,72 h后增殖,生长良好;7～9 d时细胞融合率可达到80%.透射电镜下骨髓间充质干细胞的细胞核较大,形态幼稚.经过体外成骨诱导的骨髓间充质干细胞表现出增殖、聚集、结节和矿化期的阶段性形态特征,诱导1周后表达碱性磷酸酶活性,2周后骨钙素表达呈阳性.结论:骨髓间充质干细胞经体外成骨定向诱导1周后进入成骨细胞的转化过程,并可以保持一定程度的增殖活性,可作为骨组织工程的种子细胞.

Directed differentiation of rat bone marrow mesenchymal stem cells into osteoblasts A dynamic observational study

BACKGROUND:With the potential of multi-directional differentiation and high proliferation, bone marrow mesenchymal stem cells (BMSCs) have wide application prospect in tissue engineering. OBJECTIVE:To investigate changes in cells and bioactivity in the differentiation from BMSCs into osteoblasts. DESIGN, TIME AND SETTING:A randomized control experiment was performed at the Laboratory of Stem Cells and Tissue Engineering, Department of Medical Experiment, General Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. MATERIALS:Twenty adult SD rats of both genders were used for bone marrow collection. METHODS:Rats were equally and randomly divided into an experimental group and a control group. BMSCs were isolated from adult rats by modified bone marrow culture method. BMSCs were inoculated in basic medium containing 10% fetal bovine serum, high glucose DMEM, 100 U/L penicillin, 100 U/L streptomycin and 2 mmol/L glutamine in the control group. BMSCs were inoculated in conditioned medium (basic medium, 10 mmol/L β-glycerophosphoric sodium, 10-7 mol/L dexamethasone, 50 mg/L vitamin C). Cell slide was prepared. MAIN OUTCOME MEASURES:Inverted microscope and transmission electron microscope were used to observe cell appearance. Gomori modified calcium-cobalt method was applied to do alkaline phosphatase staining. Immunocytochemistry was employed to measure osteocalcin expression. RESULTS:Forty-eight hours after inoculation, a mass of BMSCs adhered to a wall. Seventy-two hours later, BMSCs proliferated and well grew. Seven to nine days later, cells were grown to 80% confluency. Transmission electron microscope showed BMSCs with big cell nucleus and immature appearance. After in vitro osteoblast induction, BMSCs proliferated, aggregated, had node and mineralized. One week later, alkaline phosphatase activities were expressed in BMSCs. Two weeks later, osteocalcin expression was detected. CONCLUSION:After one week of in vitro osteogenic induction, BMSCs enter the process of osteoblast transformation, remain proliferative activities, and can be used as seed cells in bone tissue engineering