慢病毒介导核心结合因子α1基因感染对人牙龈成纤维细胞成骨特性的影响  

Effects of cbfα1 gene transfection on human gingival fibroblast by lentivirus in vitro

作　　者：陈晓霞 颜世果[2] 孙钦峰[2] 李玉兰[1] CHEN Xiaoxia;YAN Shiguo;SUN Qinfeng;LI Yulan(Department of Stomatology,Shaanxi Rehabilitation Hospital,Xi'an 710065,China)

机构地区：[1]陕西省康复医院口腔科,陕西西安710065 [2]山东大学口腔医学院牙周科,山东省口腔生物医学重点实验室,山东济南250012

出　　处：《口腔医学》2020年第8期682-687,共6页Stomatology

基　　金：国家自然科学基金(81200790);山东省重点研究开发计划(公益类)(2017GSF218063)。

摘　　要：目的研究慢病毒介导核心结合因子α1(cbfα1)基因感染对人牙龈成纤维细胞(hGFs)成骨特性的影响。方法构建携带目的基因的慢病毒表达载体pLenti6.3-cbfα1-Ires-EGFP(pL-cbfα1)(实验组),通过酶切、测序验证其构建成功,利用293T细胞包装病毒,并测定病毒浓度,将病毒液感染hGFs,pLenti6.3-Ires-EGFP(pL-E)为空载体组,未被感染的正常hGFs为空白对照组,通过荧光实时定量PCR检测cbfα1的mRNA的表达,通过细胞免疫化学染色检测cbfα1蛋白的表达;通过荧光实时定量PCR检测各组成骨相关基因骨涎蛋白(Bsp)、骨桥蛋白(Opn)、Ⅰ型胶原(ColⅠ)、骨钙蛋白(OC)的表达。结果慢病毒表达载体能够成功感染人牙龈成纤维细胞,并检测到cbfα1基因mRNA、蛋白的表达;实验组中,BSP、OPN、ColⅠ、OC的表达明显高于对照组和空载体组(P<0.05)。结论外源性基因cbfα1能够在人牙龈成纤维细胞中表达,并促进其向成骨细胞方向转化,为进一步研究该基因及种子细胞在牙周组织再生工程中的作用奠定了基础。Objective To study the effects of cbfα1 gene transfection on human gingival fibroblasts(hGFs).Methods Lentivirus expression vector with the target gene cbfα1 was constructed,and its construction was verified by enzyme digestion and sequencing.293T cells were used to package the virus and the virus concentration was determined.The cbfα1 gene was transfected into human gingival fibroblasts by lentivirus.The hGFs were divided into three groups:Lentivirus pLenti6.3-cbfα1-Ires-EGFP(pL-cbfα1)(experimental group),pLenti6.3-Ires-EGFP(pL-E)(empty vector group),and the untransfected normal hGFs group.The expressions of cbfα1 gene were determined by fluorescence real-time quantitative PCR and immunohistochemistry.Expressions of osteogenic genes Bsp,Opn,ColⅠand OC in each group were detected by fluorescence real-time quantitative PCR.Results The lentivirus with aim gene was able to infect human gingival fibroblasts successfully,and cbfα1 mRNA and protein were synthesized in the gingival fibroblasts.The expressions of osteogenic genes Bsp,Opn,ColⅠand OC in experimental group were significantly higher than those in the control groups(P<0.05).Conclusion The exogenous gene cbfα1 can be expressed in human gingival fibroblasts and promote its transformation to osteoblasts.This study is laying a foundation for further research on the role of this gene and seed cells in periodontal tissue regeneration engineering.

关 键 词：核心结合因子Α1 慢病毒 人牙龈成纤维细胞 

分 类 号：R349.64[医药卫生—基础医学]