体外培养羊骨髓来源间充质干细胞的生物学特性观察  

Biological properties of goat bone marrow mesenchymal stem cells cultured in vitro

作　　者：向慧[1] 刘星[1] 覃佳强[1] 张德文[1] 

机构地区：[1]重庆医科大学附属儿童医院骨科,重庆市400014

出　　处：《中国组织工程研究与临床康复》2010年第10期1760-1763,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：重庆市卫生局基金(2006-2-007)~~

摘　　要：背景:目前关于鼠、兔等小动物骨髓来源间充质干细胞的研究较多,但涉及大动物骨髓来源间充质干细胞的报道甚少。目的:体外培养羊骨髓来源间充质干细胞,并了解其生物学特性。方法:取健康10月龄中国山羊1只,麻醉后于髂后上棘穿刺抽取骨髓5mL,采用全骨髓培养法接种于无菌塑料培养瓶中,加入含体积分数为10%胎牛血清的DMEM/F12培养基。待细胞达80%～90%融合后,胰蛋白酶消化传代。取P3代处于对数生长期的细胞予以冻存,然后进行复苏。倒置显微镜下观察细胞形态变化,MTT法测定细胞生长曲线,VonKossa’s染色检测成骨诱导分化潜能。结果与结论:原代培养羊骨髓来源间充质干细胞贴壁生长,呈梭形;P3代后细胞形态基本一致,均为长梭状;冻存复苏后细胞贴壁较传代细胞稍慢,细胞形态和活性与传代细胞没有明显差别。P3～P5代细胞生长周期基本一致,接种后两三天为生长迟滞期,3d后进入对数生长期,六七天为生长平台期,其后生长速度减缓。羊骨髓来源间充质干细胞成骨诱导3周后,VonKossa’s矿化结节染色呈阳性。证实所培养的羊骨髓来源间充质干细胞具有较强的遗传稳定性和增殖能力,可向成骨细胞定向分化。BACKGROUND： There are numerous studies on bone marrow mesenchymal stem cells （BMSCs） from small animals such as rats and rabbits, but no few reports addressing BMSCs from big animals. OBJECTIVE： To observe in vitro cultured goat BMSCs, and to understand its biological properties. METHODS： A healthy Chinese goat aged ten months was obtained to extract 5 mL fresh bone marrow from the posterior superior iliac spine by puncture following anesthesia. Using the whole bone marrow method, the samples were incubated in a sterile plastic culture flask and added with DMEM/F12 containing 10% fetal bovine serum. Following 80%-90% confluence, cells were digested by trypsin. Cells at passage 3 in logarithmic phase were collected and frozen, and then recovered. Changes in cell morphology were observed using an inverted microscope. Cell growth curve was measured using MTT assay. The potential of osteogenic differentiation was examined utilizing Von Kossa＇s staining. RESULTS AND CONCLUSION： The primary cultured BMSCs were cultured with adherent growth. The cells were spindle form. Cell morphology following passage 3 was similar, showing long spindle shape. Following freezing and recovery, cell adherence was slower compared with subculture cells, and no significant difference was detected in cell morphology and viability compared with subculture cells. Growth cycle was similar in passage 3-passage 5 cells. BMSCs entered lag phase at days 2 and 3, logarithmic phase at day 3, and platform phase at days 6 and 7, and then growth speed was slow. Goat BMSCs were positive for Von Kossa＇s stain at 3 weeks following osteogenic induction. Results verified that cultured goat BMSCs showed strong genetic stability and proliferation ability, and differentiated into osteoblasts.

关 键 词：体外培养 诱导 成骨 生物学特性 细胞形态 羊 骨髓 间充质干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]