生物力学因素与BMP2单核苷酸多态性对颈椎后纵韧带骨化成骨作用的影响  

Mutual Effect of Biomechanical Factor and BMP2 Gene Nucleotide Polymorphisms on Osteogenic Differentiation in Ossification of the Posterior Longitudinal Ligament(OPLL)

作　　者：张耀[1] 刘宝戈[1] 林欣[1] 宋磊[1] 李家谋[1] 赵虎成[2] 

机构地区：[1]首都医科大学附属北京天坛医院骨科,100050 [2]清华大学工程力学系,北京100084

出　　处：《医学研究杂志》2014年第10期85-91,共7页Journal of Medical Research

基　　金：首都医科大学基础临床合作课题(12JL08)

摘　　要：目的探讨生物力学因素与骨形态发生蛋白-2(bone morphogenetic protein 2,BMP2)单核苷酸多态性对颈椎后纵韧带骨化(ossification of the posterior longitudinal ligament,OPLL)成骨作用的影响,为进一步发现OPLL多因素共同致病的具体机制奠定基础。方法采集颈椎OPLL病变组手术减压过程中骨化部位的后纵韧带和颈椎外伤等手术减压过程中的正常对照组后纵韧带。利用PCR和直接测序法分析BMP2上2个单核苷酸多态性位点109T>G(rs2273073),570A>T(rs235768)基因型及等位基因型的分布。Masson三色染色观察组织学结构改变。免疫组织化学染色和Western blot法检测BMP2的分布和表达。用C3H10T1/2细胞做转染构建细胞模型:正常组、空载体组、BMP2野生型组、BMP2(rs2273073)单突变组、BMP2(rs235768)单突变组、BMP2(rs2273073,rs235768)双突变组。采用机械应力装置对接种于Flexercell板的细胞施加10%、0.5Hz的机械应力,持续加载24h。以同样接种于Flexereell板未施加机械应力的细胞作为对照组。采用Western blot法检测机械应力加载前后各组BMP2表达情况。结果 Masson三色染色组织学观察显示病变组有成骨结构改变,免疫组织化学染色和Western blot法显示病变组BMP2的表达增加。机械应力加载后,细胞转染模型BMP2(rs2273073)单突变组和BMP2(rs2273073,rs235768)双突变组的BMP2表达较未施加应力组明显增加,差异具有统计学意义(P<0.05)。结论 BMP2单核苷酸多态性位点109T>G(rs2273073)的突变不仅能提高人们对OPLL的易感性,同时能提高OPLL患者对机械应力的敏感度,从而加速OPLL的进展。Objective To investigate the mutual effect of biomechanical factor and BMP2 gene nucleotide polymorphisms on osteogenic differentiation in ossification of the posterior longitudinal ligament （OPLL）,which may lay the foundation of future study of multi-factor pathogenesis of OPLL.Methods The ligament tissues were taken from 15 of OPLL and 18 of non-OPLL patients during anterior decompressive surgery.The complete coding sequence of the BMP2 gene was analyzed using direct sequencing and the two SNPs of rs2273073 and rs235768 were detected and genotyped.The histology of ligament tissues were assessed using Masson Trichrome staining and the distribution and expression of BMP2 were evaluated by immunohistochemistry and Western blot.The C3H10T1/2 cells with different BMP2 gene variants were constructed by transfecting with plasmid vector encoding SNP of rs2273073 or rs235768 and then subjected to mechanical stress （0.5 Hz,10% stretch） for 24h,the expression of BMP2 were detected by Western blot.Results Ligament tissues from OPLL patients showed enchondral ossification by Masson Trichrome staining.The distribution and expression of BMP2 were significantly higher by immunohistochemistry and Western blot analysis in OPLL patients.In the presence of mechanical stress,the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 （rs2273073） and BMP2 （rs2273073,rs235768） were significantly higher than the corresponding non-stretched groups （P 〈 0.05）.Conclusion The BMP2 SNP of rs2273073 can not only increase individual susceptibility to OPLL,but also the sensibility to mechanical stress which may play an important role during the pathological process of OPLL.

关 键 词：机械应力 单核苷酸多态性 骨形态发生蛋白-2 后纵韧带骨化 C3H10T1/2细胞 

分 类 号：R68[医药卫生—骨科学]