【摘要】 目的观察人重组DKK1和pCMV-HA2/DKK1重组质粒对体外培养宫颈癌细胞株Hela细胞增殖的影响。方法体外培养Hela细胞,非放射性细胞增殖(MTS)法检测重组DKK1、抗DKK1抗体和人源DKK1真核表达质粒pCMV-HA2/DKK1对Hela细胞增殖作用的影响,western blot及ELISA法检测转染pCMV-HA2/DKK1重组质粒后Hela细胞中DKK1蛋白的表达情况。结果与对照组相比,10,50 ng/mL的DKK1对Hela细胞有明显抑制作用(F分别为162.31,184.65,P均<0.01);抗DKK1抗体对Hela细胞的增殖影响无统计学意义(P均>0.05);pCMV-HA2/DKK1重组质粒转染Hela细胞后对细胞增殖有明显抑制作用,加入抗DKK1抗体不能完全拮抗pCMV-HA2/DKK1对Hela细胞的抑制作用;pCMV-HA2/DKK1重组质粒转染Hela细胞后可检测到DKK1蛋白表达,经western blot证实为DKK1蛋白(Mr约为38 000),ELISA法结果表明,DKK1蛋白的分泌量随培养时间的延长而增加,第5天时达峰值(19.82 mg/L)。结论重组DKK1和人源DKK1真核表达质粒pCMV-HA2/DKK1可抑制Hela细胞的增殖,抗DKK1抗体可部分抵抗DKK1的抑制作用,提示DKK1在宫颈癌肿瘤发生中对肿瘤细胞具有抑制作用。更多还原

【Abstract】 Objective To examine the proliferative effect of recombinant DKK1 protein and recombinant pCMV-HA2/DKK1 plasmid on cervical cancer cell in vitro.Methods The cervical cancer Hela cell strain was cultured in vitro.The effects of recombinant DKK1 protein and recombinant pCMV-HA2/DKK1 plasmid on the proliferative response of Hela cells were detected by non-radioactive cell proliferation assay(MTS),and the expression patterns of recombinant plasmid pCMV-HA2/DKK1 in Hela cells were examined simultaneously.Results DKK1 in the concentration of 10 ng/mL and 50 ng/mL showed obvious inhibitory action on the proliferation of Hela cells,and the inhibitory action correlated with the concentration of DKK1 protein.There was statistical significance of the difference of the inhibitory action between DKK1 and the control(P<0.01).No statistically significant effect of anti-DKK1 antibody on the proliferation of Hela cells was shown(P>0.05).The DKK1 protein was detectable in the cultural supernatants of Hela cells which were transfected with recombinant plasmid pCMV-HA2/DKK1,and the recombinant protein was identified by western blot.The expression patterns of recombinant plasmid pCMV-HA2/DKK1 in Hela cells showed time-dependent manner and the peak yield was obtained on the fifth days after transfection.The proliferative response of Hela cells transfected with recombinant plasmid pCMV-HA2/DKK1 showed inhibitory action with statistical significance(P<0.01),while anti-DKK1 antibody could not reverse the inhibitory effect on Hela cells.Conclusion The expression of human DKK1 protein could be induced by recombinant plasmid pCMV-HA2/DKK1 in eukaryotic cells Hela in vitro.Recombinant DKK1 and eukaryotic expression of recombinant plasmid pCMV-HA2/DKK1 could inhibit the proliferation of Hela cells,while anti-DKK1 antibody could not reverse the inhibitory effect on Hela cells.The results suggested that DKK1 protein proved the inhibitory action on tumor cell in the cervical cancer tumorigenesis,so DKK1 may become a site of potential targeted therapy for cervical cancer.更多还原