β-catenin协同BMP9诱导骨肉瘤TE85细胞成骨分化  

The effect of combination of β-catenin and bone morphogenetic protein 9 on osteogenic differentiation of osteosarcoma cell line TE85

作　　者：罗光金 罗庆[1] 毕杨[1] 张璇[1] 迭小红[1] 仇超[1] 刘敏[1] 康权[1] 

机构地区：[1]重庆医科大学附属儿童医院 儿童发育疾病研究省部共建教育部重点实验室 儿科学重庆市重点实验室 重庆市儿童发育重大疾病诊治与预防国际科技合作基地 儿科研究所干细胞实验室,重庆400014

出　　处：《肿瘤》2015年第4期361-369,共9页Tumor

基　　金：国家自然科学基金资助项目(编号:81172545);国家临床重点专科建设项目[编号:国卫办医函(2013)544];重庆市科委项目(编号:cstc2012jjA10120,CSTC2013jcyj10026);重庆市卫生局重点项目(编号:2013-1-026)~~

摘　　要：目的:检测β-连环蛋白(β-catenin)联合骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对骨肉瘤TE85细胞成骨分化的影响。方法 :采用构建有β-catenin和BMP9基因的重组腺病毒Adβ-catenin和Ad BMP9,单独或联合感染人骨肉瘤TE85细胞,并用半定量RT-PCR法检测β-catenin和BMP9 m RNA在TE85细胞中表达水平的改变;荧光素酶报告基因系统检测β-catenin/TCF4相对活性的变化,碱性磷酸酶(alkaline phosphatase,ALP)染色和读数法分析TE85细胞的早期成骨能力,茜素红染色法检测细胞的晚期成骨能力;半定量RT-PCR法检测骨保护蛋白(osteoprotegerin,OPG)以及晚期成骨分化指标骨钙蛋白(osteocalcin,OC)和骨桥蛋白(osteopontin,OPN)m RNA的表达水平,蛋白质印迹法检测OC和OPN蛋白的表达水平。结果:重组腺病毒Adβ-catenin与AdBMP9感染TE85细胞后能显著提高外源性β-catenin与BMP9 mRNA的表达水平(P值均<0.05),Adβ-catenin能明显增强β-catenin/TCF4的相对活性(P<0.05)。Adβ-catenin与AdBMP9能分别诱导骨肉瘤细胞TE85早期成骨分化指标ALP活性增加(P<0.05),但增加的程度有限;对晚期成骨指标OC和OPN mRNA和蛋白表达以及钙盐沉积无明显上调的作用。Adβ-catenin与AdBMP9联合作用之后,上述各项早晚期成骨指标的表达均显著增强,差异均有统计学意义(P值均<0.05)。结论:β-catenin和BMP9单独诱导骨肉瘤细胞TE85成骨分化的能力有限,而联合作用后诱导成骨分化的能力显著增强,β-catenin能协同BMP9诱导骨肉瘤细胞TE85成骨分化。Objective： To investigate the effect of β-catenin and bone morphogenetic protein 9（BMP-9） on osteogenic differentiation of osteosarcoma TE85 cells.Methods： The TE85 cells were infected by recombinant adenovirus Adβ-catenin and AdBMP9 alone or in combination. The expression levels of β-catenin and BMP9 mRNAs in TE85 cells were detected by semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR）. The relative β-catenin/TCF4 activity was detected by Luciferase Reported System. Alkaline phosphatase（ALP） staining and numeration were used to detect the early osteogenic potential of TE85 cells, and the alizarin red staining was performed tod etect the late osteogenic potential. The expression levels of osteoprotegerin（OPG）, osteocalcin（OC） and osteopontin（OPN） mRNAs were detected by semi-quantitative RT-PCR, and the expression levels of OC and OPN proteins were detected by Western blotting.Results： The exogenous mRNAs of β-catenin and BMP9 in TE85 cells were increased by adenovirus-mediated Adβ-catenin and AdBMP9（both P 〈 0.05）, and the relative β-catenin/TCF4 activity was increased after infection with Adβ-catenin（P 〈 0.05）. Adβ-catenin or AdBMP9 infection could enhance the activity of ALP in a limited range in TE85 cells（P 〈 0.05）, but they could not promote the mRNA and protein expressions of late osteogenic biomarkers OPN and OC as well as the calcium deposition. However, both early and late potential osteogenic abilities were enhanced obviously after infection with combination of Adβ-catenin and AdBMP9（all P 〈 0.05）.Conclusion： Osteogenic differentiation can be limitedly induced byβ-catenin and BMP9 alone in TE85 cells, but this differentiation ability can be remarkably enhanced by combination of β-catenin and BMP9 with a synergistic effect.

关 键 词：骨肉瘤 Β-连环蛋白 骨形态发生蛋白9 腺病毒感染 协同作用 

分 类 号：R738.1[医药卫生—肿瘤]