兔骨髓基质细胞在条件培养液中向成骨细胞转化的特征

Characteristics of rabbit bone marrow stromal cells differentiating into osteoblast in conditioned medium

目的:观察不同类型的培养液环境对骨髓基质细胞向成骨细胞转化的诱导作用。方法:实验于2004-04/08在哈尔滨医科大学附属一院实验中心完成。实验动物为日本大耳白兔。制备标准培养液和条件培养液,体外分离兔长骨骨髓基质细胞,标准培养液和条件培养液体外培养骨髓基质原代细胞,3d后半量换液,其后每两三天换液1次。当细胞融合80%,用25g/L胰蛋白酶消化,以1∶2传代培养。应用倒置相差显微镜逐日观察体外培养的骨髓基质细胞的形态,采用常规24孔培养板计数法测定第3代细胞在标准培养液及条件培养液培养条件下的细胞生长曲线。以5×107L-1细胞接种到96培养板中,培养24h后,更换标准培养液、条件培养液,继续培养至第5天及第10天,经处理,每孔加100μL碱性磷酸酶底物溶液,37℃孵育40min,每孔加1mol/L氢氧化钠50μL终止反应,在490nm波长测定各孔吸光度值,以U/L表示细胞碱性磷酸酶相对活性。将传代培养至第10天的标准培养液和条件培养液细胞分作2组,去除培养液,磷酸盐缓冲液pH7.3清洗,10g/L多聚甲醛固定1h,行常规VonKossa染色观察矿化结节形成。实验数据进行配对资料t检验。结果:①骨髓基质细胞在标准培养液中生长良好,细胞接种后1d少量细胞贴壁,呈圆形,2d可见贴壁细胞增多,3d有少量细胞呈纺锤形,6d后见细胞呈多种形态,且形成分散的集落,其间混有少量圆形透光度较好的细胞。1周后细胞集落迅速增多,且集落中的细胞明显增多,近12～14d集落融合成片,细胞排列有一定的方向性,呈旋涡状排列,表现为类成纤维样细胞集落生长。②条件培养液有较明显的增加骨髓基质细胞碱性磷酸酶活性的作用,条件培养液组碱性磷酸酶活性在各时间点均显著高于标准培养液组(P<0.05),条件培养组液随培养时间延长其碱性磷酸酶活性显著增高(P<0.05),而标准培养液组碱性磷酸酶活性在各时间点无显著差异。③条件培养组VonKossa染色强阳性,细胞间连接处可见大量黑色沉积物,证实有大量矿化结节的存在。而在标准培养液组未见黑色沉积物,VonKossa染色阴性。

AIM： To observe the inductive effect of different medium environment on the differentiation of bone marrow stromal cells into osteoblasts. METHODS： The experiment was completed in the experimental center of the First Affiliated Hospital of Harbin Medical University from April to August 2004. Japanese big-ear rabbits were applied as animal models. The standard euhure medium and conditioned euhure medium were prepared, bone marrow stromal cells （BMSCs）, which were in vitro isolated from rabbit bone marrow, were cultured in standard culture medium and conditioned culture medium respectively. .The medium was changed for half after three days, and then once every two or three days. Cells were passaged with 25g/L trypsin upon reaching 80% confluency and expanded in 1：2. The shape of the in vitro cultured BMSCs was observed with the inverted phase contrast mieroscope everyday. The 24 well-plate counting method was determine the growth curve of cells of passage 4 cultured in standard medium and conditioned medium. The cells were inoculated into 96-well plate with 5×10^7 L^-1, the standard medium and conditioned medium were changed after 24 hours, and then the cells were continued to culture until the 5 th and 10 th days. After treatment, 100μL alkaline phosphatase substrate solution was added to each well, and then incubated under 37℃ for 40 minutes, and then 1mol/L sodium hydroxid was added to each well to stop the reaction, the absorbhnce （A） value in each well was determined with the wave length of 490 nm, and the relative activity of cell alkaline phosphatase was expressed as U/L, For the calcium assay, the cells passaged and cultured for 10 days in standard medium and conditioned medium were divided into two groups, the medium was removed, and then washed with sodium phosphate buffer （pH 7.3）,fastened with 10g/L formaldehyde for 1 hour, routine staining with VonKossa was applied to observe the formation of mineralization. The paired t test was used to analyze the experimental data. RESULTS：① BMSCs grew better in standard medium, after 1 day, a few of the cells attached to the wall and the shape was round; 2 days later adherent cells were increased significantly; after 3 days, a few of the cells developed into spindle shape; after 6 days, the cells showed great diversity of form, and developed some decentralized cells colony, there were some round and nonopaque cells between these colonies; 7 days later, cells colonies increased quickly, and the cells of these colonies also increased conspicuously; approaching 12-14 days, the colonies reached confluence, the cells arrayed according to .some kinds of direction and arranged as a whirlpool, showed colony-forming unit-fibroblastie. ② Conditioned medium could obviously promote the alkaline phosphatase activity of BMSCs, and the activity of alkaline phosphatase at each time point in the conditioned medium group were all significantly higher than those in the standard medium （P 〈 0.05）, and the activity of alkaline phosphatase in the eondi-tioned medium group was significantly increased with the prolongation of time （P 〈 0.05）, and no significant differences were observed in the stan-dard medium group at different time points （P 〉 0.05）. ③ In the eondi-tioned medium, VonKossa staining was positive apparently, a lot of black mineral was found at the conjunction of cells, which proved that plenty of mineralization existed. In the standard medium, no black mineral was found, and VonKossa staining was negative. CONCLUSION： Rabbit BMSCs can be cultured in primary and seeondary culture in vitro, and in the same time, the osteoblastic ability of BMSCs can be increased significantly in conditioned medium.