硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

Experimental Study on Anti HBV by Antisense Phosphorothioate Oligodeoxynucleotides(S asODN)

针对ＨＢＶ的５个基因位点作为靶序列，设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段，观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达，及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用，且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性，在浓度为２０μｍｏｌ／Ｌ时，对细胞无明显杀伤作用，对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物，但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．

According to gene structre of HBV,5 kinds of antisense phosphorothioats oligodeoxy nucleotide(S asODN)were designed and synthesized.The expression of HBsAg and HBeAg,cytotoxicity,cell morphology and ultrastructure were observed by ELISA mehod,MTT colorimetrie assays and scanning electron microscope and transmission electron microscope.The result showed 5 kinds of S asODN could specificly inhibit the production of HBsAg and HBeAg of HepG 2 2215 cells.The inhibition showed sequence specific,dose related,combination cooperative and anti nuclease properties.Under 20 μmol/L,the killing activity of S asODN to HepG 2 2215 cells was all below 10%.The morphology and ultrastructure of the cells had no significant chage compared with those of the control groups.These results suggest that S asODN may be developed as anti HBV agents,but the target gene suquence,the uptake of S asODN by cell and cooperation of various S asODN should be further studied.