过表达Notch配体Delta-like基因的MLO-Y4骨细胞株的建立及其对骨髓基质细胞成骨分化的作用  

Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells

作　　者：冯玮 谢正松 罗岑 涂小林[1] FENG Wei;XIE Zhengsong;LUO Cen;TU Xiaolin(Bone Development and Regeneration Platform,Life Science Institute,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学生命科学研究院骨发育与再生平台,重庆400016

出　　处：《第三军医大学学报》2020年第9期899-907,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金-广东联合基金项目(U1601220)。

摘　　要：目的建立稳定表达外源Dll1、Dll3、Dll4基因的MLO-Y4骨细胞株,研究骨细胞Notch信号Delta-like配体Dll1、Dll3、Dll4对骨髓基质细胞成骨分化的影响。方法用过表达Dll1、Dll3、Dll4基因的重组慢病毒液感染MLO-Y4样骨细胞,嘌呤霉素筛选分别稳定表达Dll1、Dll3及Dll4基因的MLO-Y4骨细胞株。采用实时荧光定量PCR(real time fluorescence quantitative,qPCR)对筛选出来的MLO-Y4骨细胞株中Dll1、Dll3及Dll4基因的表达进行鉴定。将3种细胞株分别与野生型小鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)进行共培养,采用碱性磷酸酶(ALP)染色及ALP定量检测共培养3 d ALP活性变化;qPCR检测共培养3 d成骨相关标志基因及Notch信号靶基因的表达水平,在使用Notch信号抑制剂DAPT后检测上述指标。结果与阴性对照组相比,Dll1、Dll3及Dll4转染组中Dll1、Dll3及Dll4 mRNA的水平显著增加(P<0.05)。共培养3 d后,骨细胞中Dll4基因的过表达促进了BMSC的ALP活性,与阴性对照组相比有统计学差异(P<0.05)。qPCR结果显示,MLO-Y4-Dll4组的ALP、Runx2、OPN及CollagenⅠ成骨相关标志基因的表达均有所升高(P<0.05),Notch信号靶基因Hey1、HeyL、Hes1及Hes7的表达也有所升高(P<0.05)。而在加入DAPT后,MLO-Y4-Dll4组的ALP活性下降,成骨相关标志基因及Notch信号靶基因的表达有所下降(P<0.05)。结论建立了稳定表达外源Dll1、Dll3或Dll4基因的MLO-Y4骨细胞株,在骨细胞中过表达Dll4能促进骨髓基质细胞的早期成骨分化,经典Notch信号通路在其中可能发挥了重要作用。Objective To construct the MLO-Y4 cell strains stably expressing exogenous genes of ligands for Notch receptors Delta like-1(Dll1), Dll3, and Dll4 and determine the effects of these ligands on osteogenic differentiation of bone marrow stromal cells. Methods MLO-Y4 cells were infected with recombinant lentiviral vectors carrying Dll1, Dll3 and Dll4 genes. Puromycin was used to choose the MLO-Y4 cells with stable expression of these genes. Then, real-time fluorescence quantitative(qPCR) was applied to identify the expression of above genes in the selected MLO-Y4 cells. The 3 cell strains were respectively co-cultured with wild-type mouse bone marrow stromal cells(BMSCs). In 3 d after co-culture, alkaline phosphatase(ALP) staining and ALP quantitative detection were used to detect changes in ALP activity, and qPCR was employed to detect the expression levels of osteogenesis-related markers and target genes of Notch signal. The above indicators were measured again after DAPT, a Notch signaling inhibitor was added in the culture medium. Results Compared with the blank group and the negative control, the mRNA levels of Dll1, Dll3, and Dll4 were significantly increased in the corresponding transfection cells(P<0.05). After 3 days of co-culture, the overexpression of Dll4 gene in MLO-Y4 cells promoted the ALP activity in the BMSCs, which was significantly stronger than that of the negative control group(P<0.05). qPCR results showed that the expression levels of osteogenesis-related marker genes, such as ALP, Runx2, OPN and CollagenⅠ in the MLO-Y4-Dll4 group were all risen(P<0.05), and so were those of Notch signaling target genes, Hey1, HeyL, Hes1 and Hes7(P<0.05). However, the addition of DAPT inhibited the ALP activity in the MLO-Y4-Dll4 group, and decreased the expression levels of above osteogenesis-related markers and Notch signaling target genes(P<0.05). Conclusion MLO-Y4 cell strains stably expressing the exogenous genes Dll1, Dll3 and Dll4 are established successfully. Overexpression of Dll4 in osteocytes can p

关 键 词：慢病毒 Delta-like基因 过表达 小鼠骨样细胞 骨髓基质细胞 成骨分化 DAPT 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]