骨形态发生蛋白-4和-7对人牙周膜干细胞增殖和成骨分化的影响  

Effect of BMP-4 and BMP-7 on proliferation and osteogenic differentiation of human periodontal ligament stem cells

作　　者：李珏丹 王敏 代泉[1,2] 姚天华 饶国洲[1] 程政 LI Juedan;WANG Min;DAI Quan;YAO Tianhua;RAO Guozhou;CHENG Zheng(Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, Xi’ an 710004, China;Department of General Dentistry, College of Stomatology, Xi’ an Jiaotong University;Department of Periodontology, College of Stomatology, Xi’ an Jiaotong University)

机构地区：[1]陕西省牙颌疾病临床研究中心,西安710004 [2]西安交通大学口腔医院综合科 [3]西安交通大学口腔医院牙周科

出　　处：《山西医科大学学报》2019年第5期614-620,共7页Journal of Shanxi Medical University

基　　金：陕西省科学技术研究发展计划项目(2014K11-03-08-05)

摘　　要：目的探究骨形态发生蛋白-4(bone morphogenetic protein-4,BMP-4)和BMP-7对人牙周膜干细胞(human periodontal ligament stem cells,h PDLSCs)增殖和成骨分化的影响。方法免疫磁珠法分离培养人PDLSCs,观察细胞生长情况,鉴定细胞表型(CD146、CD44和CD34)和多项分化潜能(成骨、成脂)。将获得的PDLSCs分为BMP-4处理组、BMP-7处理组、BMP-4+BMP-7处理组,分别进行浓度梯度实验(每组均加入浓度梯度为0,5,10,20,40 ng/ml的相应BMP培养基培养192 h)和时间梯度实验(每组加入浓度为40 ng/ml的对应BMP培养基,均以0,48,96,144,192 h的时间梯度进行培养),收集细胞。MTT法和碱性磷酸酶(ALP)试剂盒检测PDLSCs增殖和ALP活性,免疫化学染色分析成骨分化相关基因骨钙蛋白(osteocalcin,OCN)、骨涎蛋白(bone sialoprotein,BSP)、Ⅰ型胶原蛋白(collagen typeⅠ,COLⅠ)和Ⅲ型胶原蛋白(collagen typeⅢ,COLⅢ)表达改变。结果分离培养的PDLSCs高表达表面抗原CD146(93%)、CD44(91. 2%),低表达CD34(1. 8%),经成骨、成脂诱导后,分别形成红色矿化结节和橘红色颗粒状脂滴。BMP-4、BMP-7、BMP-4+BMP-7均可呈时间和剂量依赖方式促进PDLSCs增殖和ALP活性表达(P <0. 05),且可增加成骨分化相关基因OCN、BSP、COLⅠ和COLⅢ表达(P <0. 05)。与BMP-4或BMP-7相比,BMP-4+BMP-7联合作用时,PDLSCs增殖和ALP活性更强(P <0. 05),然而OCN、BSP、COLⅠ和COLⅢ表达差异无统计学意义(P> 0. 05)。结论 BMP-4、BMP-7、BMP-4+BMP-7均可促进PDLSCs增殖和成骨分化,BMP-4+BMP-7联合作用时细胞增殖和ALP活性更强,具有协同作用。Objective To investigate the effect of BMP-4 and BMP-7 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells( PDLSCs). Methods PDLSCs were isolated and purified by immune magnetic method. The cell growth,surface antigen expressions( CD146,CD44 and CD34) and pluripotency( osteogenic and audiogenic) were detected and identified. Cultured PDLSCs were divided into three groups: BMP-4 group,BMP-7 group,BMP-4 + BMP-7 group. For concentration-dependent experiments,cultured PDLSCs were incubated with medium containing different concentrations of BMP( 0,5,10,20,40 ng/ml) for 192 h. For time-dependent experiments,PDLSCs were treated with medium containing 40 ng/ml BMP for 0,48,96,144,192 h. The proliferation and alkaline phosphatase( ALP) activity were measured by MTT and ALP kits. Some well-known osteogenic differentiation related-genes,including osteocalcin( OCN),bone sialoprotein( BSP),collagen type Ⅰ( COL Ⅰ) and collagen type Ⅲ( COL Ⅲ),were detected by immunochemistry staining. Results PDLSCs showed high expression of CD146( 93%) and CD44( 91. 2%),and low expression of CD34( 1. 8%),and had the osteogenic and audiogenic potential. The proliferation and ALP activity in PDLSCs treated by BMP-4,BMP-7,BMP-4 + BMP-7 were all increased in a dose-and time-dependent manner( P < 0. 05). The cell proliferation and ALP activity were stronger in BMP-4 + BMP-7 group than in that of BMP-4 group and BMP-7 group( P < 0. 05),while the protein levels of OCN,BSP,COL I and COL III in PDLSCs showed no significant difference among the three groups( P > 0. 05). Conclusion BMP-4,BMP-7,BMP-4 + BMP-7 all could promote the proliferation and osteogenesis differentiation of PDLSCs,and the cell proliferation and ALP activity are stronger under BMP-4 + BMP-7 combination conditions.

关 键 词：牙周膜干细胞 骨形态发生蛋白-4 骨形态发生蛋白-7 细胞增殖 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]