转染核结合因子a1/成骨细胞特异性转录因子2基因在兔皮肤成纤维细胞成骨表达中的作用

目的探讨外源性小鼠核结合因子a1(Cbfa1)／成骨细胞特异性转录因子2(Osf2)基因在兔皮肤成纤维细胞中的瞬时表达对该细胞成骨表型表达的作用。方法在阳离子脂质体介导下,将含外源性小鼠Cbfa1／Osf2基因的真核表达载体pSG5一Cbfa1／Osf2导入新西兰兔皮肤成纤维细胞。用RT—PCR方法检测Cbfa1、骨钙素、碱性磷酸酶及Ⅰ型胶原前肽基因表达,以Western—Blot方法检测Cbfa1蛋白表达,以对硝基苯二磷酸底物法及放射免疫方法分别检测碱性磷酸酶活性及骨钙素分泌情况,并通过茜素红染色及扫描电子显微镜检测转染细胞形成骨性结节的能力。结果转染pSG5-Cbfa1／Osf2真核表达质粒的兔皮肤成纤维细胞内可见Cbfa1 mRNA及Cbfa1蛋白瞬时表达,骨钙素mRNA、碱性磷酸酶mRNA及Ⅰ型胶原前肽mRNA大量表达,碱性磷酸酶活性增强,骨钙素大量分泌,细胞表面形成骨性结节。结论转染．pSG5-Cbfa1／Osf2真核表达质粒的兔皮肤成纤维细胞能表达成骨相关基因及蛋白质,并在其表面形成骨性结节。

Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene

OBJECTIVE: To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2). METHODS: Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope. RESULTS: Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells. CONCLUSIONS: New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.