HCCR-2反义核酸对肝癌HepG2细胞的作用

Effects of HCCR-2 antisense nucleic acid on HepG2 cells

目的研究反义HCCR-2真核表达载体对肝癌HepG2细胞增殖及凋亡的影响。方法构建反义HCCR-2真核表达载体（反义载体组），转染肝癌HepG2细胞，G418筛选阳性克隆，同样方法获得空载体pIRES2-EGFP稳定表达的细胞株（空载体组），取肝癌HepG2细胞为对照（肝癌HepG2组），用MTT法、流式细胞仪、透射电镜观察反义HCCR-2转染前后肝癌HepG2细胞生长曲线、细胞周期、细胞凋亡及细胞形态的变化。采用单因素方差分析和χ^2检验比较各组差异。结果反义载体组、空载体组、肝癌HepG2组HCCR-2 mRNA表达水平分别为0．39±0．04、0．62±0．06、0．72±0．03，3组比较差异有统计学意义（F=43．701，P〈0．05）；细胞凋亡率分别为13．30％、2．51％、2．07％，反义载体组与空载体组、肝癌HepG2组比较，差异有统计学意义（χ^2=6．793，8．721，P〈0．05）；反义载体组细胞生长减慢，阻滞于G0／G1期。结论HCCR-2反义核酸真核表达载体能抑制HCCR-2 mRNA的表达，促进细胞凋亡，HCCR-2蛋白可能参与肝癌细胞的周期调控，并与细胞的生长增殖有关。

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by C418 （antisense vector group）, pIRES2-EGFP vector was transfected into the HepG2 cells in the same way （pIRES2-EGFP group）. The conditions of the nontransfected HepG2 cells were used as control （HepG2 group）. Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfection were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39 ± 0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62 ± 0.06 and 0.72 ± 0.03, respectively, with significant difference among the 3 groups （ F =43. 701, P 〈 0.05）. The apoptotic rate of HepG2 cells in antisense vector group, pIRES2- EGFP grop and HepG2 group were 13.30% , 2.51% and 2.07% , respectively, with significant difference among the 3 group （ χ^2 =6. 793, 8. 721, P 〈0.05）. The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma ceils.