磷酸化cAMP反应元件结合蛋白对发育期人牙乳头细胞增殖和分化的影响

目的 构建CBP抑制表达慢病毒载体,探讨CBP有效表达沉默后对体外培养人牙乳头细胞(human dental papilla cells,HDPC)的增殖及分化影响.方法 利用基因重组技术构建重组慢病毒建立牙乳头细胞沉默稳转细胞,采用CCK-8检测细胞增殖的变化,采用检测分化相关蛋白的表达评价CBP对HDPC分化能力的影响.结果 测序结果提示成功构建四组人CBP基因的重组慢病毒;转染HDPC后,通过RT-PCR和蛋白质印迹法筛选获得有效靶点的重组慢病毒;CCK-8检测结果表明HDPC在CBP表达下调后增殖活性受到抑制;慢病毒介导CBP沉默后影响了HDPCs相关分化蛋白ColI、OCN、OPN的表达,较对照组显著下调(P＜0.05);牙乳头细胞的ALP活性在转染重组慢病毒后5,7,14 d后显著降低(P＜0.05);结论 成功构建了高效CBP抑制表达载体,CBP沉默可抑制HDPCs的增殖和分化.

Effect of CBP on human dental papilla cells in vitro

Objective To construct CBP suppression lentivirus vectors and determine its effects after infecting human dental papilla cells (HDPC) and analyze the biological of CBP effects on HDPCs.Methods Recombinant Lentivirus vector encoding CBP cDNA was constructed to infect HDPCs.The HDPCs were cultured in vitro and the experiments were carried out using third and fourth generation of HDPCs.Cell Counting Kit-8 (CCK-8) assay was used to evaluate the proliferation of HDPCs affected by CBP shRNA.The effect of CBP on HDPCs differentiation was determined by ALPase activity assay and real-time PCR and western blot method which were used to investigate HDPC-differentiation related and dentinogenesis proteins expressions.Results Recombinant lentivirus encoding CB-PcDNA was successfully constructed to investigate the biological role of CBP on HDPCs.The HDPCs by transduction of CBP shRNA were strongly inhibited proliferation by using the CCK8 assay.As well as,the effect of CBP on HDPCs differentiation was determined by western blotting was used to confirm with several mineralization-related proteins:ColI,OCN and OPN,which showed that CBP shRNA decreased differentiation of HDPCs.Conclusion CBP postively regulates the proliferation and the differentiation of HDPCs.