慢病毒介导沉默信息调节因子1基因表达增高的骨髓间充质干细胞模型建立  

Establishment of a bone marrow mesenchymal stem cells cell model with lentivirus-mediated silent information regulator sirtuin 1 gene overexpression

作　　者：张耕 曾晖[2] 于斐[2] 翁鉴[2] 秦浩添 朱元超 生卫北 ZHANG Geng;ZENG Hui;YU Fei;WENG Jian;QIN Haotian;ZHU Yuanchao;SHENG Weibei(Zhuhai Campus of Zunyi Medical University,Zhuhai 519041,Guangdong;Department of Orthopaedics,Peking University Shenzhen Hospital,Shenzhen 518036,Guangdong,China)

机构地区：[1]遵义医科大学珠海校区,广东珠海519041 [2]北京大学深圳医院骨科,广东深圳518036

出　　处：《中华骨与关节外科杂志》2023年第12期1117-1123,共7页Chinese Journal of Bone and Joint Surgery

基　　金：国家自然科学基金(82102568,82172432,82001319);深圳市科技研发资金(KCXFZ20201221173411031);广东省基础与应用基础研究基金(2021A1515012586);北京大学深圳医院科研基金(KYQD2021099)。

摘　　要：目的:利用携载大鼠沉默信息调节因子1(SIRT1)基因的慢病毒感染大鼠骨髓间充质干细胞(BMSCs),构建SIRT1基因表达增高的BMSCs模型。方法:设计合成大鼠SIRT1基因序列,包装成可感染的慢病毒,用不同感染复数(MOI)的慢病毒感染大鼠BMSCs,筛选出适宜的MOI值后,在经慢病毒感染的BMSCs中通过RT-PCR检测SIRT1基因mRNA含量,通过细胞免疫组化检测慢病毒感染的BMSCs中SIRT1蛋白的表达情况。结果:成功构建LV-SIRT1慢病毒,病毒滴度为1×10^(9)TU/mL,其感染大鼠BMSCs后,与对照组相比,SIRT1基因mRNA的相对表达量增高4.71倍,过表达效率为471%(P<0.05),且从细胞免疫组化结果中可见,慢病毒过表达组的染色较深,SIRT1蛋白表达高。结论:成功构建SIRT1基因表达增高的BMSCs模型,为骨科相关疾病的研究提供了细胞模型基础。Objective:To construct a bone marrow mesenchymal stem cells(BMSCs)model with increased expression of silent information regulator sirtuin 1(SIRT1)by lentiviral transduction of rat SIRT1 gene into the rat BMSCs.Methods:The sequence of rat SIRT1 gene was designed and synthesized,and it was then packaged into an infectable lentivirus.Rat BMSCs were infected with lentiviruses at different multiplicity of infections(MOIs)to identify the optimal MOI.Then the mRNA level of SIRT1 was detected by RT-PCR in BMSCs infected with lentiviruses,detection of SIRT1 protein expression in BMSCs infected with lentivirus through cellular immunohistochemistry.Results:The LV-SIRT1 lentivirus was successfully constructed with a viral titer of 1×10^(9)TU/mL.After infecting rat BMSCs,the relative expression level of SIRT1 mRNA was increased by 4.71 times,and the overexpression efficiency was 471%(P<0.05).The immunohistochemical results showed SIRT1-positive staining in the BMSCs,indicating a higher expression level of SIRT1 protein.Conclusions:We successfully constructed the BMSCs model with increased expression of SIRT1,which provides a cellular basis for the study of orthopedic-related diseases.

关 键 词：骨髓间充质干细胞 慢病毒感染 沉默信息调节因子1基因 

分 类 号：Q28[生物学—细胞生物学]