诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证

Generation and validation of inducible osteoblast-specific Stat3 knockout mice

目的:运用Cre-Loxp基因敲除系统,构建诱导型条件性Stat3基因敲除小鼠并验证其敲除效率。方法:通过Stat3fl/fl与Col1 creERT2基因型的C57小鼠多代杂交,构建诱导型成骨细胞特异Stat3敲除小鼠Stat3Col1ERT2。取其骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs),加入4-羟基他莫西芬(4-OTH)后,采用实时定量PCR技术和免疫印迹技术在m RNA与蛋白水平分别体外验证Stat3敲除效果。于小鼠腹腔注射他莫昔芬,采用免疫荧光染色技术,观察小鼠上颌牙槽骨区域STAT3的表达,以体内验证敲除效果。采用SPSS 24.0软件包对数据进行统计学分析。结果:实时定量PCR和免疫印迹结果显示,Stat3Col1ERT2小鼠BMSCs体外加药诱导敲除后,STAT3的m RNA水平显著下调(P<0.05)、蛋白表达降低(P<0.05)。免疫荧光染色结果显示,Stat3Col1ERT2小鼠体内上颌牙槽骨区域成骨细胞的STAT3表达显著降低(P<0.05)。结论:本研究成功构建了诱导型成骨细胞特异Stat3基因敲除小鼠,使基因敲除可受观察者时空调控,为今后正畸牙移动、颌骨牵引成骨、颌骨骨折等牙槽骨改建机制的研究提供了新的思路及依据。

PURPOSE:Based on the Cre-Loxp gene knockout system,this study intended to construct tamoxifen-inducible STAT3 conditional knockout mice and verify the knockout efficiency.METHODS:The inducible osteoblastsspecific Stat3 knockout mice Stat3 Col1 ERT2 were obtained by hybridization through C57 mice of Stat3 fl/fland Col1 creERT2.Bone mesenchymal stem cells(BMSCs)of these mice were isolated and cultured with or without 4-hydroxytamoxin(4-OTH),to verify the effect of Stat3 knockout in vitro by real-time quantitative PCR and Western blotting in the level of mRNA and protein.Meanwhile,wild type and Stat3 Col1 ERT2 mice were both intraperitoneally injected with tamoxifen,the expression of STAT3 in the maxillary alveolar bone was observed by immunofluorescent staining to confirm the knockout effect in vivo.Statistical analysis was conducted with SPSS 24.0 software package.RESULTS:Real-time quantitative PCR and Western blotting results demonstrated that mRNA(P<0.05)and protein levels of STAT3 were significantly decreased(P<0.05)in BMSCs derived from Stat3 Col1 ERT2 mice by 4-OHT induced knockout in vitro.Immunofluorescent staining indicated that STAT3 expression was significantly reduced(P<0.05)in osteoblasts of the maxillary alveolar bone in Stat3 Col1 ERT2 mice.CONCLUSIONS:This study successfully constructed the inducible osteoblasts-specific Stat3 gene knockout mice,which helped investigators control the time and space of gene knockout,therefore providing new insights and guidance for research fields of orthodontic tooth movement,distraction osteogenesis and jaw fractures in the future.