反义寡核苷酸对H1δ9细胞中了型肝炎病毒

Inhibitory effect of replication and expression of HDV by antisense oligodeoxynucleotides in H_1δ_9 cell

在Ｈ-１δ９细胞中观察反义寡核苷酸（ＡＳＯＤＮ）及其硫代修饰物（Ｓ－ＡＳＯＤＮ）对丁型肝炎病毒（ＨＤＶ）基因复制与表达的抑制作用。方法在分子水平证实互补于ＨＤＶ基因链核酶自裂位点和Ｓｔｅｍｌ区的ＡＳＯＤＮ可抑制核酶自裂的基础上，进一步台成针对该区６８４－６９８位核苷酸的１５聚ＡＳＯＤＮ及Ｓ－ＡＳＯＤＮ，在培养的ＨＩδ细胞中加人不同寡核苷酸，分别采用ＥＬＩＳＡ和斑点杂交法检测上清中ＨＤＡｇ及细胞中的ＨＤＶｃＤＮＡ。结果加人终浓度为６μｍｏｌ／Ｌ的Ｓ－ＡＳＯＤＮ后２４ｈＨ１δ９细胞中ＨＤＶＲＮＡ复制及ＨＤＡｇ分泌均受抑制，抑制率分别为８４５％和７６．１４％。Ｓ－ＡＳＯＤＮ的终浓度为２、４、６μｍｏｌ／Ｌ时，其抑制作用呈剂量依赖关系。在同等剂量时，ＡＳＯＤＮ与Ｓ－ＡＳＯＤＮ抑制作用相近。结论提示所用ＡＳＯＤＮ及Ｓ－ＡＳＯＤＮ均能有效抑制转基因细胞中ＨＤＶ基因的复制与表达，为进一步在动物体内研究Ｓ－ＡＳＯＤＮ抗ＨＤＶ的作用提供了实验依据。

Objective To Study inhibitory effect of an antisense oligodeoxynucleotide (ASODN) andits phosphorothioate (S-ASODN) on replication and expression of HDV in Hlδ9 cell. Methods in previousstudies, it was proved that the ASODNs which are complementary to genomic HDV ribozyme selfcleavage site and stem I regions can inhibit availably grnomic HDV ribozyme activity. In the presentstudy, a 15-iner ASODN and S-ASODS which are complementary to this region (nucleotide 684-698)were added to medium of cultured H169 cell. HDAg and HDV cDNA were detected by ELISA and Dotblot hybridization. Besults Twenty-fourrth hour aft6r 6pmol/L ASODN and S-ASODN were added, boththe secreting amount of HDAg in the supernatant and HDV RNA were inhibited. The inhibiting rateswere 76.14% and 84.50% respectively. When the concentration of S-ASODN was 2, 4. 6pmol/L, theinhibiting rate showed the feature of dosage dependence. Inhibitory effect of ASODN and S-ASODN weresimilar in the same dosage. Conclusion The results snarest that ASODN and S-ASODN can availablyinhibit replication and expression of HDV in H189 cell.