沉默ERRα对Bak1、Bcl2腺病毒转染骨细胞的影响研究  

Effect of Silencing ERRɑon Osteoblast Transfected with Bak1 and Bcl2 Adenovirus Vectors

作　　者：黄佳纯 柴爽[1] 黄宏兴 汪悦东 Huang Jiachun;Chai Shuang;Huang Hongxing(Guangzhou University Of Chinese Medicine,Guangzhou 510006,China;the Third Affiliated Hospital of Guang zhou University of Chinese Medicine,Guangzhou 510240,China)

机构地区：[1]广州中医药大学,广东广州510006 [2]广州中医药大学第三附属医院,广东广州510240

出　　处：《实用骨科杂志》2018年第9期806-811,共6页Journal of Practical Orthopaedics

基　　金：国家自然科学基金(81674004;81373653)

摘　　要：目的研究沉默雌激素相关受体α(estrogen-related receptor alpha,ERRɑ)对沉默Bak1、Bcl2重组腺病毒载体转染的人成骨样细胞MG63的细胞活性以及相关蛋白的影响。方法构建Bak1、Bcl2腺病毒载体,将shBak1、shBcl2腺病毒载体转染的MG63细胞分成空载病毒组(NC对照组)、沉默Bak1组、沉默Bcl2组、沉默Bak1+Bcl2组四组,再用ERRɑ干预上述四组细胞,MTT法检测细胞增殖,考马斯亮蓝蛋白定量检测碱性磷酸酶(alkaline phosphates,ALP)活性,流式法测定钙离子浓度,Western blot法分析骨形态发生蛋白-4(bone morphogenetic protein-4,BMP-4)、结缔组织生长因子(connective tissue growth factor,CTGF)、骨桥蛋白(osteopontin,OPN)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达。结果 a)与NC对照组比较,(Ad-shERRɑ+Ad-shBak1)组细胞活性、ALP活性、CTGF、OPN、Runx2明显升高(P<0.01),BMP-4(P<0.05)、钙离子浓度、TNF-ɑ明显降低(P<0.01),(Ad-shERRɑ+Ad-shBcl2)组细胞活性、ALP活性、BMP-4、CTGF、OPN、Runx2均显著降低(P<0.01),钙离子浓度和TNF-ɑ明显升高(P<0.01),(Ad-shERRɑ+Ad-shBak1+shBcl2)组的钙离子浓度、BMP-4、CTGF、OPN、Runx2、TNF-ɑ均降低(P<0.05);b)与(Ad-shERRɑ+Ad-shBak1)组比较,(Ad-shERRɑ+Ad-shBcl2)组与(Ad-shERRɑ+Ad-shBak1+shBcl2)组细胞活性、ALP活性、CTGF、OPN、Runx2明显下降(P<0.01),钙离子浓度明显升高(P<0.05),(Ad-shERRɑ+Ad-shBcl2)组BMP-4、TNF-ɑ均显著降低(P<0.01),(Ad-shERRɑ+Ad-shBak1+shBcl2)组TNF-ɑ明显升高(P<0.01);c)与(Ad-shERRɑ+Ad-shBcl2)组比较,(Ad-shERRɑ+Ad-shBak1+shBcl2)组的BMP-4、CTGF、OPN均升高,细胞活性、钙离子浓度、TNF-ɑ均降低(P<0.01)。结论 ERRɑ沉默可提高Bak1沉默的细胞活性、BMP4、CTGF、OPN、Runx2表达量和降低钙离子浓度、TNF-α表达量;可降低Bcl2沉默的细胞活性、BMP4、CTGF、OPN、Runx2表达量和提高钙离子浓度、TNF-α表达量。Objective To investigate the effect of silencedEstrogen-related receptor alpha（ERRɑ）on the cell viability and related proteins of human osteoblast-like cells MG63 transfected with Bak1 and Bcl2 recombinant adenovirus vectors. Methods Bak1 and Bcl2 adenoviral vectors were constructed.MG63 cells were transfected with shBak1 and shBcl2 adenovirus vectors and were divided into empty virus group（NC control group）,silent Bak1 group,silent Bcl2 group,and silent Bak1＋Bcl2 group.ERRɑintervened in the above four groups of cells.We used MTT assay for cell proliferation,coomassie leucoprotein quantitative assay for alkaline phosphates（ALP）activity,flow cytometry for determination of calcium ion concentration,and Western blot analysis of bone morphogenetic protein-4（Bone morphogenetic protein-4,BMP-4）,connective tissue growth factor（CTGF）,osteopontin（OPN）,Runt-related transcription factor 2（Runx2）,tumor Expression of tumor necrosis factor-α（TNF-α）. Results a）Compared with NC control group,cell activity,ALP activity,CTGF,OPN and Runx2 were significantly increased in（Ad-shERRɑ＋Ad-shBak1）group（P〈0.01）,BMP-4（P〈0.05）,calcium ionconcentration and TNF-ɑdecreased significantly（P〈0.01）,（Ad-shERRɑ＋AdshBcl2）group＇s activity,ALP activity,BMP-4,CTGF,OPN,Runx2 were significantly decreased（P〈0.01）,calcium ion concentration and TNF-ɑincreased significantly（P〈0.01）,（Ad-shERRɑ＋Ad-shBak1＋shBcl2）group＇s calcium concentration,BMP-4,CTGF,OPN,Runx2,TNF-ɑdecreased（P〈0.05）;b）Compared with the（Ad-shERRɑ＋ AdshBak1）group,（Ad-shERRɑ＋Ad-shBcl2）group＇s and（Ad-shERRɑ＋Ad-shBak1＋shBcl2）group＇s cell activity,ALP activity,CTGF,OPN,Runx2 decreased significantly（P〈0.01）,calcium ion concentration increased significantly（P〈0.05）,and（Ad-shERRɑ＋Ad-shBcl2）group significantly decreased in BMP-4 and TNF-ɑ（P〈0.01）,（Ad-shERRɑ＋Ad-shBak1＋shBcl2）group＇s TNF-ɑwas significantly increased（P〈0.01）;c）C

关 键 词：雌激素相关受体α 凋亡蛋白 腺病毒 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]