BMPR1b-siRNA对人骨髓间充质干细胞成骨分化及Smads通路的影响  

Effect of BMPR1b-siRNA on the osteogenic differentiation and Smads pathway of human bone marrow mesenchymal stem cells

作　　者：张博[1] 于振声[1] 周涛[1] 李建章 ZHANG Bo ＊;YU Zhen-sheng;ZHOU Tao;Li Jian-zhang(Department of Orthopedics,Henan General Hospital of Armed Police Force,Zhengzhou 450000,Chin)

机构地区：[1]武警河南总队医院骨科,郑州450000

出　　处：《实用药物与临床》2018年第7期755-760,共6页Practical Pharmacy and Clinical Remedies

基　　金：河南省医学科技攻关计划项目(201603049)

摘　　要：目的探讨BMPR1b对人骨髓间充质干细胞成骨分化及Smads通路的影响。方法构建BMPR1b特异性siRNA质粒,利用脂质体Lipo fectamineTM2000将BMPR1b-siRNA质粒转入hBMSCs,采用实时荧光定量PCR检测BMPR1b表达水平。利用四甲基偶氮唑蓝实验检测hBMSCs细胞增殖活性和碱性磷酸酶活性,茜素红染色鉴定hBMSCs成骨分化能力。利用实时荧光定量PCR及Western blot法检测成骨标志基因ALP、骨钙素、骨桥蛋白、Smads通路关键基因Smad1、Smad4水平。结果成功分离培养hBMSCs细胞。干扰组BMPR1b mRNA表达水平明显低于阴性对照组及空白组,差异有统计学意义(P<0.05),阴性对照组与空白组BMPR1b mRNA表达水平比较,差异无统计学意义(P>0.05)。MTT实验结果显示,沉默BMPR1b后,可明显抑制hBMSCs生长与增殖。沉默BMPR1b后可下调hBMSCs细胞ALP活性。茜素红染色结果显示,沉默BMPR1b后可明显降低hBMSCs细胞钙化程度。qRT-PCR及Western blot结果显示,沉默BMPR1b后可明显下调ALP、骨钙素、骨桥蛋白表达,下调Smad1、Smad4表达。结论沉默BMPR1b后可明显抑制hBMSCs细胞成骨分化,推测其机制可能与抑制Smads通路有关。Objective To explore the influence of BMPR1b-siRNA on the osteogenic differentiation and Smads pathway of human bone marrowmesenchymal stem cells（hBMSCs）. Methods BM PR1b specific siRNA plasmid was established,and transferred to hBMSCs by using liposome Lipo fectamineTM2000. BM PR1b expression level was detected by qRT-PCR,and the proliferation activity and alkaline phosphatase activity of HBMSCs cell was tested by using Four methyl blue tetrazolium test,and HBMSCs osteogenic differentiation ability was measured by using Alizarin red staining method. Osteogenic markers ALP,osteocalcin,osteopontin and expression levels of Smads pathway key genes Smad1 and Smad4 levels were detected by qRT-PCR and Western blot. Results HBMSCs cells were successfully isolated and cultured. The expression level of BM PR1b mRNA in the interference group was lower than that in the negative control group and blank group（P〈 0. 05）. There was no significant difference in BM PR1b mRNA expression between the negative control group and blank group（P〉 0. 05）. M TT test showed that BM PR1b-siRNA obviously inhibited the proliferation of hBMSCs. BM PR1b-siRNA down-regulated ALP activity of hBMSCs cells. Alizarin red staining showed that BM PR1b-siRNA significantly reduced the calcification degree of hBMSCs cells. The results of qRTPCR and Western blot showed that BM PR1b-siRNA down-regulated ALP,OCN and OPN expression,and Smad1 and Smad4 expression. Conclusion Silenced BM PR1b significantly inhibites the osteogenic differentiation of hBMSCs,which suggests that the mechanism may be related to the inhibition of Smads pathway.

关 键 词：RNA干扰 骨形态发生蛋白受体1b 人骨髓间充质干细胞 成骨分化 Smads通路 

分 类 号：R96[医药卫生—药理学]