神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白对牙髓细胞增殖的影响

目的探索神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白(NRAGE)对人牙髓细胞(h DPCs)和小鼠成牙本质细胞(MDPC-23)细胞增殖的影响。方法重组慢病毒转染细胞稳定敲除h DPCs和MDPC-23的NRAGE表达,体外组织块法原代培养h DPCs和MDPC-23,进而检测NRAGE对h DPCs和MDPC-23的增殖影响。采用CCK-8法分析NRAGE对h DPCs和MDPC-2细胞增殖的影响,流式细胞术分析NRAGE对h DPCs和MDPC-23的细胞周期分布和细胞凋亡影响。免疫荧光法检测NRAGE和NF-κB的表达和定位,分析NF-κB蛋白表达水平,并用IKK抑制剂处理细胞后,分析细胞周期和细胞凋亡。结果重组慢病毒转染后NRAGE的mRNA和蛋白水平下降显着。NRAGE敲减后抑制了h DPCs和MDPC-23的增殖活性和凋亡。NRAGE敲减后显示h DPCs的G0G1期滞留显著,而对MDPC-23没有影响。同时,NRAGE敲减后激活NF-κB信号通路。IKK抑制剂可以抑制NRAGE敲除后对h DPCs和MDPC-23的细胞凋亡的抑制作用。结论 NRAGE敲减后抑制牙髓细胞的增殖活性。NRAGE通过NF-κB信号通路调控h DPCs的细胞周期和凋亡。

Effect of neurotrophin receptor-mediated MAGE homology on proliferation of human dental pulp cells and MDPC-23

Objective To investigate the effects of neurotrophin receptor-mediated melanoma antigen-encoding gene homology ( NRAGE) on proliferation of human dental pulp cells ( hDPCs) and MDPC-23. Methods Cells were infected by recombinant lenti-virus to stably knock down the expression of NRAGE, then in vitro tissue explant method was used to conduct the primary culture of the hDPCs and MDPC-23, and the biological effects of NRAGE on the hDPCs and MDPC-23 were detected. Cell Counting Kit-8 was used to analyze effects of NRAGE on hDPCs and MDPC-23 proliferation, then the cell cycle distributions and apoptosis of hDPCs and MD-PC-23 were performed by flow cytometric analysis. Simultaneously, the cell cycle and apoptosis were also detected after cells treated with IKK inhibitor.Then, mRNA and protein levels of NRAGE and nuclear factor-κB ( NF-κB) protein expression were detected. Im-munofluorescence assay was used to detect expression and location of NRAGE and NF-κB. Results The mRNA and protein levels of NRAGE decreased significantly after infected by recombinant lentivirus. Knockdown of NRAGE inhibited the proliferation and apoptosis of hDPCs and MDPC-23. Knockdown of NRAGE showed significant G0G1 retention in hDPCs, while no significant difference in MD-PC- 23. Meanwhile, knockdown of NRAGE activated the NF-κB signaling pathway. After treated with IKK inhibitor, the effect of NRAGE knockdown on apoptosis was reversed in both hDPCs and MDPC-23. Conclusion Knockdown of NRAGE can inhibit prolifer-ation of hDPCs and MDPC-23. NRAGE is a potent regulator for cell cycle and apoptosis of hDPCs. Knockdown of NRAGE can inhibit apoptosis of hDPCs and MDPC-23 through the NF-κB signaling pathway.