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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)成骨分化的影响。方法:实验分为未转染组、转染β-半乳糖苷酶(β-Galactosidase,LacZ)基因的LacZ组以及转染Nell-1基因的Nell-1组。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。以反转录PCR和Western印迹法验证bMSCs中Nell-1基因的表达。以碱性磷酸酶(alkaline phosphatase,ALP)活性定量测定、骨钙素(osteocalcin,OC)含量测定和Von Kossa法检测Nell-1基因转染对bMSCs成骨分化的影响。采用SAS6.04软件包对数据进行随机设计的方差分析(ANOVA-SNK)。结果:Nell-1组可检测到Nell-1基因转录水平和蛋白水平的表达,未转染组和LacZ组未见明显Nell-1基因表达。Nell-1组ALP活性在转染后3d、6d和9d均高于LacZ组和未转染组。Nell-1组在转染后14d、28d时,OC含量分别为(1.23±0.05)ng/mL和(1.31±0.06)ng/mL,高于未转染组的(0.81±0.09)ng/mL和(1.00±0.05)ng/mL,以及LacZ组的(0.92±0.08)ng/mL和(1.02±0.04)ng/mL。钙结节数Nell-1组在转染后21d和28d分别为4.5±1.1和16.2±2.5,高于未转染组的0.8±0.7和3.2±1.2以及LacZ组的0.7±0.5和3.7±0.8,其差异均有显著性(P<0.05)。结论:Nell-1基因可促进体外培养bMSCs的成骨分化。
Objective: To investigate the effect of Nel-like 1 (Nell-1) gene on osteogenic differentiation of rat bone marrow stromal cells (bMSCs) in vitro. Methods: The experiment was divided into untransfected group, LacZ group transfected with β-galactosidase (LacZ) gene and Nell-1 group transfected with Nell-1 gene. The 6-week-old male Fischer344 rat tibia and femur marrow were cultured in vitro and adenovirus vector was used for gene transfection. Nell-1 gene expression in bMSCs was verified by reverse transcription PCR and Western blotting. The effects of Nell-1 gene transfection on the osteogenic differentiation of bMSCs were determined by alkaline phosphatase (ALP) activity assay, osteocalcin (OC) assay and Von Kossa assay. ANOVA-SNK, a randomized design of the data using the SAS 6.04 software package. RESULTS: Nell-1 mRNA and protein levels were detected in Nell-1 group. Nell-1 gene expression was not found in untransfected and LacZ groups. ALP activity in Nell-1 group was higher than LacZ group and non-transfected group at 3d, 6d and 9d after transfection. The levels of OC in Nell-1 group were (1.23 ± 0.05) ng / mL and (1.31 ± 0.06) ng / mL at 14d and 28d after transfection, respectively, which were significantly higher than those in untransfected group (0.81 ± 0.09ng / mL) And (1.00 ± 0.05) ng / mL, respectively, and (0.92 ± 0.08) ng / mL and (1.02 ± 0.04) ng / mL in the LacZ group. Calcium nodules in the Nell-1 group were 4.5 ± 1.1 and 16.2 ± 2.5 at 21 and 28 days after transfection, respectively, which were significantly higher than those in the untransfected group (0.8 ± 0.7 and 3.2 ± 1.2) and in the LacZ group (0.7 ± 0.5 and 3.7 ± 0.8) , The difference was significant (P <0.05). Conclusion: Nell-1 gene can promote osteogenic differentiation of bMSCs cultured in vitro.