rhBMP-2诱导成肌细胞表达成骨表型的研究  

rhBMP-2 stimulates expression of osteogenic phenotype of myoblast in vitro

作　　者：曾融生[1] 束煌[2] 王剑宁[1] 

机构地区：[1]中山大学光华口腔医学院.附属口腔医院口腔颌面外科,广东广州510060 [2]深圳市儿童医院口腔科,广东深圳518000

出　　处：《中国口腔颌面外科杂志》2007年第1期50-55,共6页China Journal of Oral and Maxillofacial Surgery

摘　　要：目的:检测不同浓度rhBMP-2对诱导成肌细胞成骨表型表达的影响,探索诱导成肌细胞表达成骨表型的rhBMP-2的最适浓度。方法:采用双重酶消化法获取SD大鼠乳鼠的成肌细胞,纯化后,在含15%胎牛血清的DMEM培养液中培养。取第3代培养细胞,按培养液中不同rhBMP-2浓度分组,分为0、0.1、0.5、1.0、1.5和5.0μg/ml共6组(0μg/ml组为对照组),培养1、2和4周。收集不同时期细胞培养上清液和细胞爬片进行检测,检测指标为:细胞形态和数量、碱性磷酸酶(ALP)活性、骨钙素(OCN)含量、Ⅰ型胶原合成量和钙化结节染色。采用SPSS10.0统计软件包进行统计分析。各组间比较采用方差分析(ANOVA)。结果:应用rhBMP-2后,梭形成肌细胞减少,圆形或多突形成肌细胞明显增加,细胞呈集结性生长,培养4周可形成不透光结节。低、中浓度(0.1￣1.5μg/ml)rhBMP-2组细胞数量和对照组之间无显著性差异(P>0.05),但高浓度(5.0μg/ml)rhBMP-2组细胞数量显著下降(P<0.05)。0.1￣1.5μg/ml4组rhBMP-2均能促进成肌细胞合成分泌ALP、OCN和Ⅰ型胶原(所有组与对照组比较,P<0.05),其中0.5和1.0μg/ml2组促进作用更显著。细胞培养4周后,茜素红S染色法示结节呈红色钙阳性反应。结论:低、中浓度(0.1￣1.5μg/ml)rhBMP-2对成肌细胞增殖无显著影响,但高浓度(5.0μg/ml)rhBMP-2可显著抑制成肌细胞增殖。rhBMP-2可有效诱导成肌细胞表达成骨细胞各项表型并能体外成骨,其中0.5￣1.0μg/ml是一个较为适宜的诱导浓度。PURPOSE： To investigate the inducing effects of rhBMP-2 on osteogenic phenotype of myoblasts and determine the appropriate concentration of rhBMP-2 which exerts its strong inducing effect. METHODS： Myoblasts were isolated from the muscle tissue of SD rats and the dissociated cells were cultured.The third passage of the cells was used for the experiment. After subeuhivation, the DMEM medium was supplemented with 6 concentrations of rhBMP-2,i.e.,0, 0.1,0.5,1.0,1.5 and 5.01μg/ml （0μg/ml group as control）.The cells were cultured for 1,2 and 4 weeks respectively.The cell number,ALP activity,the amount of OCN and type Ⅰ collagen were measured and new calcifying nodule was stained by alizarin red S staining. The data were represented by x^-±s and analysed with SPSS10.0 software package for one-way ANOVA. RESULTS： Cultured with rhBMP-2, cells began to have muhangular, concentric growth and produced secretory granule, rhBMP-2 at concentration of 0.1-1.5μg/ml had not significant effect on cell growth（P〉0.05 ）, but when increased to 5.0μg/ml,rhBMP-2 significantly inhibited proliferation of myoblasts （P〈0.05）. At concentration of 0.1-1.5μg/ml,all four groups of rhBMP-2 increased the ALP activity, OCN and type I collagen （P〈0.05）, except group of 0.5μg/ml or 1.0μg/ml. Alizarin red S staining showed that the reaction of calcifying nodules was positive when cultured for 4 weeks. CONCLUSIONS： Low and moderate concentration （0.1-1.5μg/ml） of rhBMP-2 have little effect on the growth of myoblasts, but high concentration （5.0μg/ml） of rhBMP-2 strongly inhibit proliferation of myoblasts, rhBMP-2 can effectively induce the expression of osteogenic phenotype of myoblasts, and produce new calcifying nodule subsequently. The inducing effect of rhBMP-2 on myoblasts is dose-dependent.This study shows, that 0.5 -1.0μg/ml concentration of rhBMP-2 is optimal range of concentration.

关 键 词：成肌细胞 重组人骨形成蛋白-2 骨钙素 成骨 

分 类 号：R78[医药卫生—口腔医学]