Lmo4基因对骨髓间充质干细胞成骨分化的调控作用

Regulation of Lmo4 on Osteogenic Differentiation of BMSCs

为了探讨Lmo4基因对骨髓间充质干细胞(BMSCs)成骨分化的调控作用。转录调节因子Lmo4与细胞的成骨分化有着密切的联系,本实验通过包装靶向Lmo4的sg RNA慢病毒转染表达Cas9蛋白的骨髓间充质干细胞,运用CRISPR/Cas9技术敲除Lmo4,同时设置sg RNA空载体病毒作为阴性对照;包装Lmo4的pMSCV逆转录病毒转染野生型骨髓间充质干细胞,过表达Lmo4,同时以pMSCV空载体病毒作为阴性对照。对病毒转染后的细胞进行成骨诱导分化,采用碱性磷酸酶染色和茜素红S染色两种方法对各组细胞进行染色,并运用实时荧光定量PCR技术对细胞的Lmo4基因,以及成骨分化相关基因Runx2、ALP、Col1a1、Osteocalcin的表达水平进行定量分析,比较各组细胞成骨能力的差异。结果显示,Lmo4敲除组与对照组相比,两种染色明显加深,Lmo4的表达量明显下降,成骨分化相关基因Runx2、ALP、Col1a1、Osteocalcin的表达量明显上升。相反,Lmo4过表达的细胞与对照组相比,两种染色明显变浅,Lmo4的表达量明显上升,成骨分化相关基因的表达量明显下降。说明Lmo4能够抑制骨髓间充质干细胞的成骨分化。本研究发现了一个新的骨发育与分化的调控因子。

Aim to investigate the role of Lmo4 gene in the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).The transcription regulator Lmo4 is potentially involved in osteogenic differentiation.In this experiment,Cas9-expressing BMSCs were transfected with Lmo4-targeted sg RNA by lentivirus,to knockout Lmo4 gene in cells by CRISPR/Cas9 system.Meanwhile,lentivirus containing empty vector was transfected as a negative control.On the other hand,Lmo4 expressing pMSCV retrovirus was transfected into wild type BMSCs,with pMSCV empty retrovirus as a control.After osteogenic differentiation,the transfected cells of each group were examined by alkaline phosphatase(ALP)staining and alizarin Red S staining.Quantitative PCR was used to assess the expressions of Lmo4 and osteogenic marker genes(Runx2,ALP,Col1 a1 and Osteocalcin)for each group of cells.The results are indicated as following:compared with the controls,both ALP and Alizarin Red S staining of Lmo4-knockout cells was significantly stronger,coupled with the decreased expression of Lmo4 and the augmented expressions of osteogenic differentiation genes.In contrast,the ALP and alizarin Red S staining of Lmo4-overexpression cells were relatively weaker,accompanied by the upregulation of Lmo4 and the decreased expression of osteogenic differentiation genes.The results indicate that Lmo4 inhibits the osteogenic differentiation of BMSCs,providing a new regulator of osteogenic differentiation.