Apoptin基因诱导视网膜母细胞瘤凋亡的研究

Acceleration of apoptosis by transfection of Apoptin gene in retinoblastoma cells

目的观察Apoptin基因对人视网膜母细胞瘤细胞株HXO-RB44的促凋亡作用,并探讨其可能的机制。方法用脂质体将Apoptin基因导入HXO-RB44细胞,通过RT-PCR法检测ApoptinmRNA的表达,同时用SABC免疫组织化学法分析Apoptin和p53的表达。采用细胞计数法检测细胞生长抑制率,流式细胞仪检测细胞周期的变化。结果转入Apoptin基因后,HXO-RB44细胞的生长明显受抑制(P<0.05)。细胞周期分析可见凋亡峰,凋亡率为38.5%。细胞中可见Apoptin阳性表达(P<0.05),p53表达差异无统计学意义(P>0.05)。凋亡细胞在荧光显微镜下形成凋亡小体。结论转入Apoptin基因可显著促进HXO-RB44细胞的凋亡,Apoptin诱导的凋亡不依赖功能性p53的生成。

Objective Present study aimed to observe the effects of Apoptin gene on killing retinoblastoma HXO-RB44 cells and illustrates its mechanisms. Methods Human retinoblastoma cells strain,/-/XO-RB44,was cultured and passaged in RPMI 1640 medium containing bovine serum. Apoptin genc was transfected into HXO-RB44 ceils by liposome into HXO-RBu/Apoptin, and pcDNA3 was transfected in HXO-RB44/pcDNA3 group. The expression of Apoptin mRNA was detected using Reverse Transcription Polymerase Chain Reaction （ RT-PCR）. The expression of protein of Apoptin and p53 were detected by SABC immunohistochemistry. The growth rate of HXO-RB4, cells was studied by constructing the growth curve and calculated as the formula：inhlbitory rate = 1-cell number in experiment group/cell number in control group × 100%. Cellular apoptosis was determined by flow cytometry. Results The RT-PCR result showed the 450 kb specific band in HXO-RB4a/Apoptin group and absent amplification result in HXO-RB44 group and HXO-RB44/pcDNA3 group. The difference in SABC-positive cell number between HXO-RB,4/Apoptin group and control group was statistically significant（ P 〈 0.05 ）. The growth of HXO-RB44 cells was significantly inhibited in HXO-RB4JApoptin group compared with eontrol group （P 〈 0. 05 ）. Apoptosis cells increased significantly. The apoptosis rate was 38.5%. Conclusion Apoptin gene could inhibit the growth of HXO-RBa, cells effectively. Up-regulation of expression of p53 gene might not be one of cell apoptosis mechanisms.