犬骨髓基质细胞向成骨细胞诱导分化的实验研究

目的:研究犬骨髓基质细胞(bone marrow stromal cells,BMSCs)在体外诱导分化为成骨细胞的方法和活性检测。方法:抽取犬骨髓3 mL,使用成骨条件培养液,贴壁法体外培养,将培养的第2、3代细胞进行透射电镜、流式细胞仪、生长曲线、碱性磷酸酶、Von Kossa和免疫组化等检测。结果:第2代细胞碱性磷酸酶染色呈阳性表达;第3代细胞Von Kossa染色、骨桥素、骨涎蛋白和骨钙蛋白呈阳性表达;第3代细胞透射电镜显示高尔基体和线粒体丰富;流式细胞仪检测显示第3代处于S期的细胞比例较第2代增加。结论:犬骨髓基质细胞在体外能定向诱导分化为成骨细胞。

Experimental Study of Dog Bone Marrow Stromal Cells Induced into Osteoblasts

Objective: To study the osteogenic methods and abilities of the dog's bone marrow stromal cells(BMSCs) in vitro to estimate its feasibility of being seed cells of bone tissue engineering.Methods: Under general anesthesia,3 mL bone marrow was aspirated from dog caput femoris.The cells were cultured in delbecco's modified eagle medium(DMEM) at 5×105 cells/cm2 and incubated under standard cell culture conditions.The 2nd and 3rd generated cells were collected for studies including transmission electron microscopy(TEM)?fluorescent-activated cell sorter(FACS)?growth curve with MTT method?alkaline phosphatase(ALP) staining?Von Kossa and immunohistologic examination.Result: The expression of ALP of the 2nd generated cell were detected.The 3rd generated cells showed abundant endoplasmic reticulum and mitochondria by TEM,and had more S-staged cells than the 2nd in FACS.The expression of calcic node,osteonctin(ON),bone sialoprotein(BSP) and osteocalsin(OCN) were proved by Von Kossa staining and immunohistochemical examination.Conclusion: BMSCs have the potentials of being induced into the osteoblasts when cultured in vitro.