摘要
目的探究早期生长因子1(Egr-1)对骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。方法采集成年男性骨髓组织,分离培养原代BMSCs并在显微镜下观察其形态,流式细胞术鉴定BMSCs表面标志物;pcDNA3.1/Egr-1转染BMSCs,MTT检测BMSCs的增殖,茜素红钙染色试剂盒检测细胞基质内钙结节的形成,ALP活性测定试剂盒检测细胞内ALP的活性,实时定量qPCR和Western blot分别检测细胞内Egr-1、Runx2、NDRG1mRNA和蛋白表达;Egr-1siRNA转染BMSCs,检测细胞内ALP活性、Egr-1、Runx2和NDRG1mRNA及蛋白表达。结果离体培养的BMSCs高表达CD90和CD29,而CD34和CD45呈阴性表达;pcDNA3.1/Egr-1转染对BMSCs增殖无明显作用,却能促进细胞基质内钙结节的形成,上调ALP活性和Egr-1、Runx2、NDRG1的表达,而Egr-1siRNA则作用相反。结论 Egr-1通过上调NDRG1诱导BMSCs的成骨分化。
Objective To explore the effects of early growth response gene-1 （Egr-1） on bone marrow mesenchymal stem cells （BMSC） proliferation and osteogenie differentiation,which is aimed at providing new molecular targets for the treatment of osteoporosis. Methods Bone marrow was collected from adult men and the BMSCs were cultured primarily and observed by micro- scope. Meanwhile,flow cytometry was used for BMSCs phenotypic identification~ After transfection of pcDNA3.1/Egr-1 into BM- SCs,the level of BMSCs proliferation was determined by MTT respectively on the 2 d,4 d and 6 d^On the 7 d after transfection, the ALP activity assay was used for testing the ALP activity in BMSCs. And then,alizarin red S-calcium kit was used for measuring the calcified knots respectively on the 7 d, 14 d and 21 d^On the 21 d after transfection, real-time qPCI~ and Western blotting were used respectively for measuring the expression of mRNA and protein of Egr-1, Runx2 and NDRGl^Further, BMSCs were transfected with Egr-1 siRNA,and the content of calcium nodules,ALP aetivity,the expression of Egr-l,Runx2 and NDRG1 were detected as above methods. Results The cells cultured in vitro showed high level of CDg0 and CD29 and very low level of CD34 and CD45, which is accorded with the characteristic of BMSCs. The pcDNA3.1/Egr-1 transfection for BMSCs had no effect on cells prolifera- tion. However, the calcified knots, ALP activity and the expression of Egr-1 ,Runx2 and NDRG1 were increased after transfection of peDNA3.1/Egr-1 for BMSCs. In addition, Egr-1 siRNA showed the opposite effect with pcDNA3.1/Egr-1 transfection for BMSCs. Conclusion Egr-1 induces osteogenie differentiation of BMSCs by promoting NDRG1 but has no effects on proliferation of BMSCs.
出处
《重庆医学》
CAS
北大核心
2017年第4期442-445,共4页
Chongqing medicine
作者简介
史素琴（1977--），主治医师，博士，主要从事骨质疏松研究。
通信作者，E-mail：zhaolu．cn@163．com。
