重组pEGFP-N1-IGF-Ⅰ基因表达质粒促进SD大鼠牙囊细胞增殖及早期成骨分化效应的实验研究  

Recombinant plasmid pEGFP-N1-IGF-Ⅰtransfection enhances proliferation and early stage osteogenic differentiation of rat dental follicle cells in vitro

作　　者：李亚明[1] 田常生[2] 胡波[1] 曹礼[1] 宋锦璘[1] 

机构地区：[1]重庆医科大学附属口腔医院口腔疾病与生物医学重庆市重点实验室重庆市高校市级口腔生物医学工程重点实验室,重庆401147 [2]重庆医科大学附属第二医院口腔科,重庆400010

出　　处：《重庆医科大学学报》2017年第1期92-97,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81570981);重庆市科委自然科学基金项目(cstc2014jcyj A10039)

摘　　要：目的:通过胰岛素样生长因子-Ⅰ(insulin-like growth factor-Ⅰ,IGF-Ⅰ)与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合表达,构建重组质粒pEGFP-N1-IGF-Ⅰ。体外实验分析脂质体介导pEGFP-N1-IGF-Ⅰ转染SD大鼠牙囊细胞(rat dental follicle cells,rDFCs)对其增殖及早期成骨分化效应的影响。方法:体外分离培养rDFCs并分为空白组、pEGFPN1-IGF-Ⅰ组、pEGFP-N1+脂质体组和pEGFP-N1-IGF-Ⅰ+脂质体组。采用荧光显微镜观察绿色荧光表达情况,MTT法检测细胞增殖活性,碱性磷酸酶(alkaline phosphatase,ALP)活性检测法检测ALP活性,PCR检测Ⅰ型胶原(collagen type 1,Col1)表达情况。结果:荧光显微镜观察结果表明pEGFP-N1-IGF-Ⅰ成功转染入rDFCs。转染后48 h,脂质体处理组(pEGFP-N1+脂质体组和pEGFP-N1-IGF-Ⅰ+脂质体组)转染效率较非脂质体处理组(空白组和pEGFP-N1-IGF-Ⅰ组)高。MTT结果表明,pEGFP-N1-IGF-Ⅰ能增强rDFCs细胞活性,而脂质体毒性对细胞活性具有抑制作用。ALP活性检测结果显示pEGFP-N1-IGF-Ⅰ能增强rDFCs的ALP活性。PCR结果证明pEGFP-N1-IGF-Ⅰ能增加Col1α1及Col1α2的相对表达量。结论:pEGFP-N1-IGF-I能促进rDFCs增殖和早期成骨分化效应,为IGF-Ⅰ基因在牙周组织修复再生中的应用提供了实验依据。Objective：To construct recombinant plasmid p EGFP-N1-IGF-Ⅰ by fusing expression of insulin-like growth factor-Ⅰ（IGF-Ⅰ）and enhanced green fluorescent protein（EGFP）;to investigate accelerating effect of p EGFP-N1-IGF-Ⅰ transfection on cytoactive and early stage osteogenic differentiation of rat dental follicle cells（DFCs）in vitro. Methods：The rat dental follicle cells（r DFCs）were obtained and divided into four groups：blank group,p EGFP-N1-IGF-Ⅰ group,p EGFP-N1＋liposome group and p EGFPN1-IGF-Ⅰ＋liposome group. The proliferation and early stage osteogenic differentiation of DFCs were analyzed. The green fluores cence expression,MTT assay,ALP quantitative detection,Type Ⅰ collagen Col1α1 and Col1α2 gene expression analysis were used.Results：The green fluorescence expression results showed that the p EGFP-N1-IGF-Ⅰ was successfully transfected in DFCs and the transfection efficiency was higher in liposome treated group（p EGFP-N1＋liposome group and p EGFP-N1-IGF-Ⅰ＋liposome group）than in p EGFP-N1-IGF-Ⅰ group and blank group at 48 h. The MTT assay elucidated that the p EGFP-N1-IGF-Ⅰ may enhance the cytoactive of DFCs but the cytotoxicity of liposome may reduce the cell variability. The assay of alkaline phosphatase（ALP）activity indicated that p EGFP-N1-IGF-Ⅰ was able to increase the ALP activity. The PCR results illustrate that p EGFP-N1-IGF-Ⅰ could enhance the relative expression of Col1α1 and Col1α2. Conclusion：p EGFP-N1-IGF-Ⅰ could enhance the proliferation and early stage osteogenic differentiation of DFCs,and provide a reference for the usage of IGF-Ⅰ gene in periodontal tissue engineering.

关 键 词：胰岛素样生长因子-Ⅰ 牙囊细胞 增殖 早期成骨分化 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]