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-  2016 

BMP-14联合Noggin shRNA共转染对脂肪来源干细胞成骨分化能力的影响

DOI: doi:10.7507/1002-1892.20160259

Keywords: BMP-14, Noggin, 慢病毒, 共转染, 脂肪来源干细胞, 大鼠

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Abstract:

目的在体外环境下同时下调细胞中Noggin基因和增加BMP-14基因,观察对脂肪来源干细胞(adipose derived stem cells,ADSCs)成骨分化能力的影响。 方法取健康成年SD大鼠5只,体质量250~300 g,采用Ⅰ型胶原酶消化法获取原代ADSCs,体外培养、扩增。使用慢病毒载体将目的基因转染大鼠ADSCs,根据目的基因不同将实验分为3组,A组为空载体病毒Lv-增强型绿色荧光蛋白转染对照组,B组为Lv-BMP-14转染组,C组为BMP-14+Noggin shRNA转染组。分别于转染后3、7、14 d,采用实时荧光定量PCR检测BMP-14及成骨相关基因[Ⅰ型胶原、ALP、骨钙素(osteocalcin,OCN)]的表达,转染后14 d采用茜素红染色鉴定其成骨分化能力。 结果转染后3 d,各组BMP-14 mRNA相对表达量比较差异无统计学意义(P>0.05);7、14 d,C组BMP-14 mRNA相对表达量高于A、B组,B组高于A组,比较差异均有统计学意义(P<0.05)。转染后3 d,C组各成骨相关基因mRNA相对表达量显著高于A、B组(P<0.05);A、B组间比较差异无统计学意义(P>0.05)。转染后7、14 d,C组各成骨相关基因mRNA相对表达量显著高于A、B组,B组高于A组,除转染后7 d A、B组间Ⅰ型胶原mRNA相对表达量比较差异无统计学意义(P>0.05)外,其余各组间比较差异均有统计学意义(P<0.05)。转染后14 d茜素红染色示,A、B、C组钙结节量呈递增趋势。 结论BMP-14具有增强大鼠ADSCs向成骨细胞分化的能力;沉默细胞中的Noggin基因、联合BMP-14基因共同作用于大鼠ADSCs,其体外成骨分化能力明显强于BMP-14单基因转染,两者有显著的协同作用

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