骨形态发生蛋白2/7异二聚体对人脂肪间充质干细胞成骨分化的促进作用  

Promoted role of bone morphogenetic protein 2/7 heterodimer in the osteogenic differentiation of human adipose-derived stem cells

作　　者：张晓[1] 刘云松[1] 吕珑薇[1] 陈彤[1] 吴刚 周永胜[1,3] 

机构地区：[1]北京大学口腔医学院.口腔医院修复科,北京100081 [2]Department of Oral Implantology and Prosthetic Dentistry,Academic Centre for Dentistry Amsterdam(ACTA),Research Institute MOVE,VU University and University of Amsterdam [3]口腔数字化医疗技术和材料国家工程实验室,北京100081

出　　处：《北京大学学报（医学版）》2016年第1期37-44,共8页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81170937);教育部新世纪优秀人才支持计划(NCET-11-0026);国家临床重点专科建设项目资助~~

摘　　要：目的:探讨新型成骨诱导生长因子骨形态发生蛋白2/7异二聚体(bone morphogenetic protein 2/7heterodimer,BMP-2/7)在诱导人脂肪间充质干细胞(human adipose-derived stem cells,h ASCs)成骨分化中的作用。方法:体外培养h ASCs,以成骨诱导培养基(osteogenic medium,OM)中添加150μg/L BMP-2/7为实验组,以单纯OM为阳性对照组(OM组),以常规增殖培养基(proliferation medium,PM)为阴性对照组(PM组)。体外诱导的第1、4和7天检测细胞DNA含量,第7和14天进行碱性磷酸酶(alkaline phosphatase,ALP)染色及定量检测,第21和28天进行茜素红染色及定量检测,第1、4、7和14天分别进行成骨相关基因的检测。体内实验使用6只裸鼠,于其背部正中做皮肤切口,向两侧分离出4个皮下植入腔,分别植入:(1)单纯β-磷酸三钙(β-tricalcium phosphate,β-TCP)支架(支架对照组);(2)β-TCP支架+h ASCs(体外PM培养1周)(增殖细胞对照组);(3)β-TCP支架+h ASCs(体外OM培养1周)(诱导细胞对照组);(4)β-TCP支架+h ASCs(体外OM+150μg/L BMP-2/7培养1周)(实验组)。植入后4周进行取材,标本制成组织学切片,进行HE和Masson三色染色分析。结果:体外诱导后第1天,实验组细胞DNA含量与PM组差异没有统计学意义(P>0.05),第4天时实验组细胞DNA含量较PM组升高(P<0.05),第7天时组间DNA含量没有明显差异(P>0.05)。诱导7天和14天后,实验组的ALP染色及定量检测均高于OM组和PM组(P<0.05);第21和28天矿化结节染色及钙沉积定量检测均高于OM和PM组(P<0.05)。诱导第1天时实验组的成骨相关基因(Runx2、ALP、COL-1A1)的表达量即高于PM组(P<0.05),诱导第4天时骨钙素(osteocalcin,OC)基因表达量即开始升高,第4、7和14天的基因表达量较OM和PM组显著升高(P<0.05)。HE染色分析可见实验组和诱导细胞对照组的h ASCs能分泌出嗜酸性较强的均质细胞外基质,出现了强嗜酸性的类骨组织;与诱导细胞对照组相比,实验组的细胞外基质嗜酸性更强,Objective ： To investigate the role of bone morphogenetic protein 2/7 heterodimer （ BMP-2/ 7） in the osteogenesis of human adipose-derived stem cells （hASCs）. Methods： hASCs were exposed tothree different treatments in vitro： osteogenic medium with 150 μg/L BMP-2/7 （experimental group）, osteogenic medium alone （OM group） and proliferation medium （PM group）. After 1,4 and 7 days of osteogenic induction, the amount of cellular DNA was measured to investigate the cytotoxicity. After 7 and 14 days, alkaline phosphatase （ALP） staining and quantification were performed to test the activity of ALP. After 21 and 28 days, the calcification deposition was determined by Alizarin Red S （ARS） stai- ning and quantification. The expressions of the osteoblast-related genes were tested on days 1, 4, 7 and 14. In the in vivo study, 6 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice ： （ 1 ） β-TCP scaffold only （ scaffold control group） ; （ 2 ） β-TCP scaffold with hASCs cultured by PM in vitro for 1 week （PM control group） ; （3） β-TCP scaffold with hASCs cultured by OM in vitro for 1 week （OM control group） ; （4） β-TCP scaffold with hASCs cultured by OM with 150 μg/L BMP-2/7 in vitro for 1 week （test group）. After 4 weeks of implantation, histological staining was performed to evaluate the in vivo osteogenesis of hASCs. Results： After induction for 1 day, there was no significant difference between the experimental group and the PM group on the cellular DNA con- tent （P 〉 0.05 ）. After 4 days, the cellular DNA content increased under the stimulation of BMP-2/7 （ P 〈 0.05 ）. On day 7, there was no significant difference among the three groups （ P 〉 0.05 ）. ALP ac- tivity was higher by the induction of BMP-2/7 than in OM alone and PM （P 〈 0.05 ）. More mineraliza- tion deposition and more expressions of osteoblast-related genes such as Runx2, ALP, COL-1A1 and OC were determined in the expe

关 键 词：脂肪组织 间充质干细胞 骨形态发生蛋白质类 细胞分化 异二聚体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]