HBVPreS_2反义寡核苷酸对HepG2.2.15细胞系HLA-I类抗原表达及生物活性的影响

Effect on HLA-I expression and biological activity in the presence of antisense oligonucleotides

目的 :观察针对乙型肝炎病毒 (HBV)PreS2 基因特异性反义寡核苷酸 (asON)对转基因细胞HepG2 .2 .15表面的人类白细胞I类抗原 (HLA I)基因表达的影响。方法 :设计合成互补于HBVPreS2 翻译起始区的反义寡核苷酸即序列I ,并设非互补序列作对照即序列II ,免疫细胞化学染色观察asON对HepG2 .2 .15表面表达的HLA I类抗原的影响 ,用ELISA法动态检测细胞上清液中HBsAg和HBeAg的变化 ,放射免疫测定法检测asON对宿主细胞自身分泌蛋白 血清铁蛋白的影响。结果 :HepG2 .2 .15细胞表面HLA I类抗原表达降低 ,用药组和对照组相比有统计学意义 (P <0 .0 5 )。asON能够抑制HBV表达HBsAg和HBeAg ,对HBsAg和HBeAg的抑制率分别为 6 6 %和 91% ,而序列II对HBsAg和HBeAg的抑制率均为 11%。asON对宿主细胞自身分泌蛋白无影响 ,即对宿主细胞无毒性作用。结论 :asON以序列特异性方式抑制HBV基因表达而对宿主细胞无毒性作用 ,HLA I类抗原表达降低 ,这对减轻过度炎症反应 。

Objective: To investigate the effect of inhibited HLA-I expression on HepG2.2.15 cells by antisense oligonucleotides directed against HBV PreS 2 gene in vitro. Methods: Antisense phosphorothiote oligonucleotides (asON) complementary to hepatitis B virus (HBV) PreS 2 gene translational initiator were designed and synthesized ( Sequence I ).Noncomplementary sequence was also designed and synthesized as control (Sequence II). The effect of asON on human HLA-I expression by ABC was observed. HBsAg and HBeAg concentration in HepG2.2.15 cells cuture supernatant was assayed with ELISA. In addition , Serum ferritin concentration in supernatant of cells was measured by radioimmunoassay (RIA). Results: Expression of HLA-I on HepG2.2.15 cells reduced . asON could inhibit HBV gene from expressiong HBsAg and HBeAg . The inhibitory rates of HBsAg and HBeAg were 66% and 91% , respectively. Serum ferritin concentration in supernatant of cells showed not significant difference between sequence I group and the control group . Conclusion: The mechanism of asON inhibiting the expression HBV gene may be due to sequence specific inhibition .asON had not any cytoxicity for host cell .The reduc of HLA-I expression have significance to prevent chronic hepatitis and severe hepatitis.