Nell-1型分子基因对大鼠骨髓基质细胞成骨分化的影响

Effect of Nell-1 gene on osteogenic differentiation of rat bMSCs:An in vitro study

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。

PURPOSE： To examine the ability of Nell-1 （Nel-like, type 1 molecule） gene in inducing osteogenic differentiation of bMSCs in vitro. METHODS： bMSCs obtained from Fischer 344 rats were transferred with Ad-Nell-1 or Ad-β-Galactosidase （Ad-LacZ） or left untransferred in vitro. Expression of Nell-1 gene was detected by reverse transcription-PCR （Rt-PCR） and Western blot. Alkaline phosphatase （ALP） activity and osteocalcin （OC） production were measured and Von Kossa test was conducted to examine Nell-1’s ability in inducing osteogenic differentiation of bMSCs. The data were analyzed for ANOVA with SNK method using SAS 6.04 software package （P〈0.05）. RESULTS： Nell-1 transgenic message and protein were detected in Nell-1 group but not in LacZ group and untransferred group. On day 3,6, 9 after gene transfer, ALP activity was significantly enhanced in the Nell-1 group. On day 14,21 after gene transfer, OC production in Nell-1 group was （1.23±0.05）ng/mL and （1.31±0.06）ng/mL, significantly higher than those in untransferred group（0.81±0.09）ng/mL and （1.00±0.05）ng/mL and LacZ group（0.92±0.08）ng/mL and （1.02±0.04）ng/mL].On day 21,28 after gene transfer, the nodules formed in Nell-1 group was 4.5±1.1 and 16.2±2.5 significantly, higher than those in untransferred group （0.8±0.7 and 3.2±1.2） and LacZ group（0.7±0.5 and 3.7±0.8）. CONCLUSION： Nell-1 gene induce osteogenic differentiation of bMSCs in vitro.