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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 a recombinant plasmid pEGFP-N1-IGF-Ⅰ by fusion expression of insulin-like growth factor-I (IGF-Ⅰ) and enhanced green fluorescent protein (EGFP). The effect of liposome-mediated rat oral follicle cells (rDFCs) transfected with pEGFP-N1-IGF-Ⅰ on proliferation and early osteogenic differentiation was analyzed in vitro. Methods: rDFCs were isolated and cultured in vitro and divided into blank group, pEGFPN1-IGF-Ⅰ group, pEGFP-N1 + liposome group and pEGFP-N1-IGF-Ⅰ + liposome group. The expression of green fluorescent protein (EGFP) was observed by fluorescence microscopy, the activity of cell proliferation was detected by MTT assay, the activity of ALP was detected by alkaline phosphatase (ALP) activity assay and the expression of collagen type 1 (Col1) was detected by PCR. Results: Fluorescence microscopy showed that pEGFP-N1-IGF-Ⅰ was successfully transfected into rDFCs. 48 h after transfection, the transfection efficiency of liposome-treated group (pEGFP-N1 + liposome group and pEGFP-N1-IGF-I + liposome group) was higher than that of non-liposome treated group -IGF-Ⅰ group). The result of MTT showed that pEGFP-N1-IGF-Ⅰ could enhance the cell activity of rDFCs, while the lipotoxicity could inhibit the cell viability. The results of ALP activity showed that pEGFP-N1-IGF-Ⅰ could enhance the ALP activity of rDFCs. PCR results showed that pEGFP-N1-IGF-I can increase the relative expression of Col1α1 and Col1α2. CONCLUSION: pEGFP-N1-IGF-I can promote the proliferation and early osteogenic differentiation of rDFCs and provide the experimental basis for the application of IGF-Ⅰ gene in periodontal tissue repair and regeneration.