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目的:研究Nel-like1型分子(Nell-1)基因对体外培养大鼠骨髓基质细胞(bone marrow stromal cells,bMSCs)成骨相关基因表达的影响。方法:取6周龄雄性Fischer344大鼠胫骨和股骨骨髓体外培养,以腺病毒为载体,进行基因转染,绘制生长曲线并测定转染效率。以转染β-半乳糖苷酶(β-Galactosidase,LacZ)基因的bMSCs为对照组,转染Nell-1的bMSCs为试验组,用实时定量PCR(real-time PCR)测定成骨相关基因碱性磷酸酶基因(alkaline phosphatase,ALP)、骨涎蛋白(bone sialoprotein,BSP)、骨钙素(osteocalcin,OC)、骨桥蛋白(osteopontin,OP)和Sox9的表达。以2-ΔΔCT法计算基因表达的相对比值。结果:随着Nell-1基因转染滴度的增高,细胞增殖速度减慢。基因转染效率随滴度增加而增高。Nell-1基因转染大鼠bMSCs,早期下调、中期和晚期上调ALP的表达,晚期上调OC的表达,整个成骨周期中均上调OP和BSP的表达。结论:Nell-1基因可能通过影响各成骨相关基因的表达而促进大鼠bMSCs的成骨分化。
Objective: To investigate the effect of Nel-like1 (Nell-1) gene on the osteogenesis-related genes in rat bone marrow stromal cells (bMSCs) in vitro. Methods: Six-week-old male Fischer344 rats were cultured in vitro and cultured in vitro. The adenovirus vector was used to transfect the gene and the growth curve was drawn. The transfection efficiency was determined. The bMSCs transfected with β-galactosidase (LacZ) gene were used as control group and Nell-1-bMSCs were transfected into the experimental group. Real-time PCR was used to detect the expression of osteogenesis-related genes The expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC), osteopontin (OP) and Sox9 were detected. The relative ratio of gene expression was calculated by the 2-ΔΔCT method. Results: With the increase of Nell-1 gene transfection, the cell proliferation rate slowed down. Transfection efficiency increased with increasing titer. Nell-1 gene was transfected into rat bMSCs for early down-regulation, mid-term and late up-regulation of ALP expression, late up-regulation of OC expression and up-regulation of OP and BSP expression throughout the osteogenic cycle. Conclusion: Nell-1 gene may promote the osteogenic differentiation of rat bMSCs by affecting the expression of osteogenesis-related genes.