改良小鼠骨髓间充质干细胞培养方法及长效荧光标记的可行性

Modified culture method and prolonged action fluorescent labeling of bone marrow mesenchymal stem cells from mice

背景:小鼠骨髓间充质干细胞培养不同于人及大鼠,培养扩展难度高,传代后干细胞活性保持时间短;又鉴于干细胞本身特点,使现有干细胞示踪方法作用时间短,这些因素均影响小鼠骨髓间充质干细胞的实验研究。目的:观察改良小鼠骨髓间充质干细胞分离培养方法及长效荧光标记干细胞的可行性。设计、时间及地点:观察性实验,于2008-06/12在解放军军事医学科学院完成。材料:C57BL/6小鼠4～6周龄,体质量20g,雌雄不拘。方法:通过贴壁筛选和Percoll分离法,优化干细胞培养液血清和换液方式,培养扩增小鼠骨髓间充质干细胞。参照以往人和猕猴间充质干细胞培养经验,对小鼠间充质干细胞培养血清选择Hyclone的顶级胎牛血清,控制血清为培养液的10%。用LG-DMEM培养液冲出骨髓细胞后,滤去骨渣和小肌肉碎块。然后加在相对密度1.082的percoll分离液上,以1.5×106/cm2浓度接种75cm2培养瓶。对第2代骨髓间充质干细胞进行长效CM-DiI标记。主要观察指标:原代及传代小鼠骨髓间充质干细胞形态变化;对第2代小鼠骨髓间充质干细胞表面抗原,CD105,CD44,CD25,CD34进行流式细胞仪检测,评价改良方法获取干细胞纯度;通过成脂成骨分化,鉴定小鼠骨髓间充质干细胞的活性;观察多次传代后荧光细胞强度。结果:①小鼠骨髓间充质干细胞原代培养在接种48h后可见贴壁细胞,培养第7天细胞多数伸展呈梭形,也有三角形、多角形和扁平形。3代后形态趋于统一,融合状态时细胞排列呈束状、漩涡状或放射状。②骨髓间充质干细胞特异性抗原高表达CD105,CD44;造血系抗原低表达CD34和CD45。③骨髓间充质干细胞向成骨分化过程中,纺锤形的突起逐渐消失,胞体增大,培养10d后,部分细胞呈聚集生长,随细胞生长密度的增长形成多层的结节结构。在脂肪诱导体系中,可见细胞由成纤维样逐渐缩短,9d后胞浆中出现脂肪滴,油红O染色可见红色的脂肪颗粒。④首次标记,荧光显微镜观察可见长效CM-DiI标记在细胞膜发橙色光,标记率在80%以上。流式细胞仪检测CM-DiI细胞标记率可到47%以上,第4代培养细胞仍可见较多标记细胞。结论:改良方法早期第2代就可获得高浓度干细胞,同时长效CM-DiI标记方法稳定,标记率高,可作体内细胞示踪。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） of mice are different from human and rats. The difficulties of culture sustaining impermanency activity of BMSCs after passage and short-term effect of stem cells tracking limited the study of mice. OBJECTIVE： To obverse modified isolated culture method of BMSCs of mice and the feasibility of long-term fluorescent labeling stem cells. DESIGN, TIME AND SETTING： The experiments were conducted at the Affiliated Hospital of Academy of Military Medicine Science from June to December in 2008. MATERIALS： C57BL/6 mice, males and females, 4-6 weeks of age, mean weighing 20 g, were used. METHODS： Stem cell culture fluid serum and liquid change manner were optimized using adherent screening and Percoll separation method. Rat BMSCs were incubated. In accordance with previous experiences of MSCs between human and macaque, Hyclone high-grade fetal bovine serum was selected for mouse MSC incubation. Serum was 10% of the medium. Bone marrow cells were washed out using LG-DMEM to filter bone dregs and small muscle blocks. Subsequently, samples were added on the PercoU separating medium at relative density of 1.082, and then incubated in 75 cm2 culture flask at the concentration of 1.5×1 0^6/cm^2. BMSCs at the second passage were labeled with CM-Dil. MAIN OUTCOME MEASURES： Morphological changes in primary cultured and subcultured mouse BMSCs were measured. BMSC surface antigen of the second passage of mice （CD105, CD44, CD25, CD34） were determined using flow cytometry. The modified method was assessed to harvest the purity of stem cells. Activity of mouse BMSCs was identified using adipogenic and osteoplastic differentiation. The strength of fluorescent cells following multiple passage was observed. RESULTS： ①The attached cells were observed 48 hours after primary cells culture and changed shuttles, triangles and flats at 7 days after culture. The figure become bunches and radial pattern at 3 passages. ②MSCs highly expressed CD105, CD44 phenotypes and seldom expressed CD34 and CD45.③Spindle shape of cells gradually disappeared, with increased cell body. Some cells grew in cluster. MSCs changed figures to multilayer knots 10 days after inducing osteoplastic differentiation. MSCs became roundness and appeared fat drops in cells 9 days after inducing adipogenic differentiation. Red fat particles were shown following oil O staining. ④The labeling cells gave out oranges light, and marked rates were over 80% in MSCs under the fluorescent microscope. The labeling cells were over 47% in 4 passages MSCs using flow cytometry. CONCLUSION： The modified method gained high-dosage cells in shorten culture time at passage 2 and made CM-Dil long time labeling cells, which made more convenient for MSCs experiment on mice and stem cells tracer experiment in vivo.