成骨诱导因子Nell-1及Noggin短发卡RNA联合转染脂肪间充质干细胞的促成骨分化能力  

Co-transfection by Nell-1 and Noggin-shRNA promotes osteoblast differentiation of adipose derived mesenchymal stem cells

作　　者：刘浩 刘军 芮永军 汤峰林 陆淼 丁涛 Liu Hao;Liu Jun;Rui Yongjun;Tang Fenglin;Lu Miao;Ding Tao(Department of Orthopedics,Wuxi 9th People’s Hospital,Wuxi 214000,Jiangsu Province,China;Department of Orthopedics,Wuxi People’s Hospital Affiliated to Nanjing Medical University,Wuxi 214000,Jiangsu Province,China)

机构地区：[1]无锡市第九人民医院骨科,江苏省无锡市214000 [2]南京医科大学附属无锡人民医院骨科,江苏省无锡市214000

出　　处：《中国组织工程研究》2020年第31期4966-4970,共5页Chinese Journal of Tissue Engineering Research

基　　金：南京医科大学科技发展基金重点项目(2016NJMUZD069),项目负责人:丁涛。

摘　　要：背景:Nell-1在诱导成骨分化和新骨形成中的作用已有报道,但试图通过双基因联合转染起协同成骨作用的研究很少。目的:体外环境下抑制细胞中Noggin基因的同时上调Nell-1基因,观察其对脂肪间充质干细胞成骨分化能力的影响。方法:取健康成年大鼠脂肪组织获取脂肪间充质干细胞。将细胞分为3组,对照组转染空载体病毒Lv-EGFP(增强型绿色荧光蛋白),Nell-1组单纯转染Lv-Nell-1,Nell-1+Noggin shRNA组同时转染Lv-Nell-1和Lv-Noggin shRNA。转染后3,7,14 d,应用实时荧光定量PCR检测Nell-1及成骨相关标志物(Col-Ⅰ、ALP、OCN)的表达,转染后14 d行茜素红染色。结果与结论:①转染后3 d,对照组Nell-1 mRNA相对表达量与Nell-1组比较,差异无显著性意义(P>0.05)。与Nell-1+Noggin shRNA组比较,对照组和Nell-1组Nell-1 mRNA表达量偏低,差异均有显著性意义(P<0.05)。转染后7,14 d,Nell-1+Noggin shRNA组Nell-1 mRNA相对表达量仍高于对照组和Nell-1组,且Nell-1组高于对照组,两两比较差异均有显著性意义(P<0.05);②转染后3,7,14 d,Nell-1+Noggin shRNA组各成骨基因mRNA相对表达量均显著高于对照组和Nell-1组,且Nell-1组高于对照组,组间比较差异有显著性意义(P<0.05);③转染后14 d,Nell-1+Noggin shRNA组呈现较多细胞聚集形成的结节,Nell-1组钙结节数量少于Nell-1+Noggin shRNA组,对照组未见明显被染色的钙结节;④实验结果表明,Nell-1基因可以促进大鼠脂肪间充质干细胞成骨分化,而干扰Noggin基因会上调Nell-1基因的表达,形成显著的协同成骨分化作用。BACKGROUND:Nell-1 can induce osteogenic differentiation and accelerate bone regeneration.However,little is reported about co-transfection by Nell-1 and Noggin-shRNA.OBJECTIVE:To observe the effects of simultaneously down-regulating Noggin and up-regulating Nel-like molecule-1(Nell-1)on osteogenic differentiation of adipose derived mesenchymal stem cells(ADSCs)in vitro.METHODS:ADSCs were isolated and cultured in vitro from adipose tissue of healthy rats,and divided into three groups.ADSCs in control group were transfected with lentiviral(Lv)-enhanced green fluorescent protein.In Nell-1 group,the cells were transfected with Lv-Nell-1.And in co-transfection group,the cells were co-transfected with Lv-Nell-1 and Lv-Noggin shRNA.At 3,7 and 14 days after transfection,Nell-1 and osteogenesis-related genes(collagen type I,alkaline phosphatase and osteocalcin)were detected by real-time fluorescence quantitative PCR.At 14 days after transfection,ADSCs were detected by alizarin red staining.RESULTS AND CONCLUSION:At 3 days after transfection,there was no significant difference in the Nell-1 mRNA expression between the control and Nell-1 groups(P>0.05);and the Nell-1 mRNA expression in the control and Nell-1 groups was significantly lower than that in the co-transfection group(P<0.05).At 7 and 14 days after transfection,the Nell-1 mRNA expression was still lower in the control and Nell-1 groups than in the co-transfection group,and moreover,the expression level in the Nell-1 group was significantly higher than that in the control group(P<0.05).At 3,7,and 14 days after transfection,the levels of collagen type I,alkaline phosphatase and osteocalcin mRNA in the co-transfection group were significantly higher than those in the control and Nell-1 groups,and these expression levels were also significantly higher in the Nell-group than in the control group(P<0.05).At 14 days after transfection,there were more calcium nodules in the co-transfection group than in the Nell-1 group,while no calcium nodules were observed in the control

关 键 词：Nell-1 NOGGIN 共转染 脂肪间充质干细胞 成骨分化 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]