构建靶向Runx2基因成骨样细胞MC3T3-E1慢病毒载体

Lentivirus-mediated Runx2 gene transfection into MC3T3-E1 cells

背景:研究表明成骨细胞特异性转录因子(Runx2)的杂合突变、基因插入、缺失等是造成颅骨锁骨发育不全的重要原因。目前关于Runx2基因沉默后成骨细胞功能会发生怎样的变化,及如何沉默成骨细胞Runx2基因表达的相关研究未见报道。目的:构建靶向Runx2基因的小鼠成骨样细胞MC3T3-E1慢病毒载体,为颅骨锁骨发育不全的研究提供实验依据。方法:根据预实验结果在含5 mg/L Polybrene的ENi.S培养液中,病毒按MOI=40进行感染,按病毒序号分为4组:NC组:pFU-GW-016PSC53349-1;KD1组:LVpFU-GW-016PSC60107-1;KD2组:LVpFU-GW-016PSC60108-1;KD3组:LVpFU-GW-016PSC60109-1。转染后16 h换液,72 h后,Celigo全视野细胞扫描分析仪观察检测基因的表达情况;两步法进行Real-Time PCR检测Runx2基因的沉默效果。结果与结论:(1)病毒转染后72 h,NC组未见荧光表达;KD1组可见少量荧光表达;KD2组荧光表达增强;KD3组可见强绿色荧光;(2)Real-Time PCR结果提示KD3组获得较好Runx2基因沉默效果;(3)实验成功构建靶向Runx2基因的MC3T3-E1细胞慢病毒载体,筛选出特异性抑制成骨细胞Runx2基因的病毒,并初步确定其作用的时间、相应的计量。

BACKGROUND： Heterozygous mutationgene insertion and gene deletion of Runt-related transcription factor 2（Runx2） have been found to be important causes of cleidocranial dysostosis. Howeverthere is a lack of studies on the changes of osteoblasts after Runx2 silencing and how to perform gene silencing.OBJECTIVE： To construct the lentiviral vector targeting Runx2 gene in mouse osteoblast-like cells（MC3T3-E1）so as to provide experimental basis for the research of cleidocranial dysostosis. METHODS： According to the preliminary experimental resultsthe lentiviral vector was infected by MOI=40 in the ENi.S medium containing 5 mg/L Polybrene for 10 hoursand divided into four groups according to the viral number： group NC： pFU-GW-016 PSC53349-1; group KD1： LVpFU-GW-016 PSC60107-1; group KD2： LVpFU-GW-016 PSC60108-1 and group KD3： LVp FU-GW-016 PSC60109-1then replaced with conventional medium at 16 hours after infection. The target gene expression was detected by Celigo~？ Image Cytometer at 72 hours after transfection. The two step method of real-time PCR was utilized to detect the silencing effect of Runx2 gene. RESULTS AND CONCLUSION： At 72 hours after transfectionthere was no green fluorescence in the group NC; a small amount of fluorescent expression in the group KD1; enhanced fluorescence expression in the group KD2 and obvious green fluorescence in the group KD3. Real-time PCR curves showed the better silencing effect of Runx2 gene in the group KD3. In summarywe successfully constructed the lentiviral vector targeting Runx2 gene into MC3T3-E1 cellsscreened the virus inhibiting Runx2 geneand clarified the action time and related measurement.