BMP-2与TNF-α对成骨细胞增殖和分化作用的比较分析

›› 2014, Vol. 34 ›› Issue (9): 651-654.

BMP-2与TNF-α对成骨细胞增殖和分化作用的比较分析

孙玮¹,张君²,王乐秀¹,张茜³,王旭霞²

1. 山东济南市济钢总医院
2. 山东大学口腔医学院
3. 山东大学口腔医学院,山东济南(250012)

收稿日期:2014-01-14 修回日期:2014-04-16 出版日期:2014-09-26 发布日期:2014-10-07
通讯作者: 王旭霞 E-mail:wxx@sdu.edu.cn
基金资助:
山东省科学技术发展项目

A comparative analysis of the effect of BMP2 and TNFalpha (TNFα) on osteoblast proliferation and differentiation

Received:2014-01-14 Revised:2014-04-16 Online:2014-09-26 Published:2014-10-07

摘要/Abstract

摘要： 目的通过BMP-2及TNF-α分别刺激原代培养的成骨细胞，探讨BMP-2及TNF-α对成骨细胞增殖和分化不同作用并进行比较分析。方法在无菌条件下手术切取Wistar（1周龄）大鼠颅骨并提取成骨细胞，原代细胞培养、传代、纯化、扩增并进行鉴定。采用BMP-2及TNF-α进行诱导，观察成骨细胞增殖，用利用MTT法检测其变化，利用PNPP偶氮法（4-硝基苯基磷酸二钠盐）检测成骨细胞碱性磷酸酶（ALP）活性。结果通过ALP染色和钙化的细胞实验均为阳性，证明体外培养扩增的细胞具有典型的成熟成骨细胞的特征。采用BMP-2及TNF-α来诱导大鼠成骨细胞与对照组的差异有统计学意义（P＜0.05）。细胞增殖活性及ALP活性随BMP-2浓度的升高而增加，而随TNF-α浓度的升高而降低。结论 BMP-2转染后的成骨细胞增殖和分化功能都有所提高。而TNF-α对成骨细胞的增殖和分化均有抑制作用。对体外培养的成骨细胞，BMP-2和TNF-α分别起到促进和抑制作用。

关键词: 肿瘤坏死因子-α, BMP-2, 成骨细胞, 增殖, 分化

Abstract: Objective To investigate the BMP-2 and TNF alpha(TNF-α) on osteoblast proliferation and differentiation of different function and comparative analysis through the BMP – 2 and TNF alpha were stimulated the generation of osteoblast culture. Methods Wistar rat (1 week old) skull were surgery chipped under sterile conditions and osteoblasts were extracted, primary cell cultures, and passaged, purification, amplification. Using different concentrations of BMP-2 and TNF- α induced rat osteoblasts, Methyl thiazolyl tetrazolium (MTT) assay was used to detect the proliferation of osteoblasts, PP-nitrophenyl phosphate (PNPP) was used to detect the alkaline phosphatase (ALP) activity of osteoblasts.Results In vitro cultured cells by ALP staining and calcification were both positive, confirmed the cultured cells have the typical characteristics of mature osteoblasts. Rat osteoblast proliferation activity and ALP activity increases with BMP - 2 concentration, but decreased with increasing TNF-α, there was significant difference with control group (P < 0.05). Conclusion Transfection of BMP-2 can improve the proliferation and differentiation of osteoblasts. TNF- α has inhibitory effect on the proliferation and differentiation of osteoblasts. BMP -2 and TNF alpha respectively promote and inhibit in vitro culture of osteoblasts through different mechanisms.

Key words: tumor necrosis factorα, BMP2, osteoblast, proliferation, differentiation

中图分类号:

R329.29

孙玮张君王乐秀张茜王旭霞. BMP-2与TNF-α对成骨细胞增殖和分化作用的比较分析[J]. 口腔医学, 2014, 34(9): 651-654.

参考文献

[1]Sautier JM,Nefussi JR,Boulekbache H,et al.In vitro bone formation on coral granules[J].In Vitro Cell Dev Biol,1990,26(11):1079-1085
[2]Herbertson A,Aubin JE.Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures[J].Bone,1997,21(6):491-500
[3]Urist MR.The substratum for bone morphogenesis[J].Symp Soc Dev Biol,1970,29:125-163
[4]Alden TD,Varady P,Kallmes DF,et al.Bone morphogenetic protein gene therapy[J].Spine,2002,27(16):87-93
[5]Singhatanadgit W,Salih V,Olsen I.Bone morphogenetic protein receptors and bone morphogenetic protein signaling are controlled by tumor necrosis factoralpha in human bone cells[J].Int J Biochem Cell Biol,2006,38(10):1794-1807
[6]Kobayashi K,Takahashi N,Jimi E,et al.Tumor necrosis factor alpha stimulates osteoblast differentiation by a mechanism independent of the ODF/RANKLRANK interaction[J].Exp Med,2000,191(2):275-286
[7]Burgess TL,Qian Y,Kaufman S,et al.The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts[J].Cell Biol,1999,145(3):527-538
[8]Wu S,Flint JK,Rezvani G,et al.Nuclear factorkappaB p65 facilitates longitudinal bone growth by inducing growth plate chondrocyte proliferation and differentiation and by preventing apoptosis[J].J Biol Chem,2007,282(46):33698-33706
[9]薛立伟,张君,王旭霞.肿瘤坏死因子α对大鼠成骨细胞生长影响的实验研究[J].华西口腔医学杂志,2009,27(4):378-380
[10]王霖霞,李玉坤.BMP2信号通路与成骨细胞分化[J].国际骨科学杂志,2009,30(2):132-134
[11]Yao S,Prpic V,Pan F,et al.TNFalpha upregulates expression of BMP2 and BMP3 genes in the rat dental follicleimplications for tooth eruption[J].Connect T1 Res,2010,51(1):59-66
[12]Mukai T1,Otsuka F,Otani H,et al.TNFα inhibits BMPinduced osteoblast differentiation through activating SAPK/JNK signaling[J].Biochem Biophys Res Commun,2007,356(4):1004-1010
[13]Yamazaki M1,Fukushima H,Shin M,et al.Tumor Necrosis FactorαRepresses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF B[J].J Biol Chem,2009,284(51):35987-35995
[14]姜泊.T细胞凋亡基础与临床［M］.北京:人民军医出版社，1999:1-25.
[15]Boyle WJ,Simonet WS,Lacey DL.Osteoclast differentiation and activation[J].Nature,2003,423(6937):337-342
[16]Burgess TL,Qian Y,Kaufman S,et al.The ligand for osteoprotegerin（OPGL） directly activates mature osteoclasts[J].J Cell Biol,1999,145(3):527-538
[17]徐荣辉,朱雅萍,柴本甫.胚胎大鼠颅骨分离细胞原代早期体外培养的组织化学观察[J].中华骨科杂志,1990,10(1):45-47
[18]王华,黎海芪,杨锡强,等.大鼠长骨成骨细胞体外培养形态观察[J].第三军医大学学报,2002,24(5):603-605