阳离子脂质体介导双靶区反义锁核酸抗病毒疗效研究

Anfiviral effects of dual-target antisense locked nucleic acid by cationic liposomes

目的探讨HBVS、C双靶区反义锁核酸（LNA）的抗病毒疗效。方法设计合成互补于HBVS、C基因翻译起始区的反义LNA与脂质体混合，经尾静脉注入小鼠体内，用时间分辨免疫荧光分析法定量检测小鼠血清HBsAg；荧光PCR定量检测血清HBVDNA含量；RT-PCR检测肝组织HBVC．mRNA的表达；免疫组化法检测肝细胞HBsAg、HBcAg的表达；自动生化分析仪检测血清白蛋白（Alb）、脚、尿素氮（BUN）、肌酐（Cr）等指标；小鼠肝脏、肾脏进行常规病理切片并HE染色，观察反义LNA对小鼠脏器的影响。结果血清HBsAg的表达，注射后第1、3、5天，单靶区S组的抑制率分别为23．3％、37．9％和48．7％；单靶区C组分别为21．2％、32．6％和40．7％；双靶区SC组分别为30．6％、61．2％和72．8％。血清HBVDNA，注射后第1天开始下降，双靶区SC组的DNA下降最明显，其第1、3、5天分别下降了18．5％、36．1％和52．9％。5d后，单靶区S组、单靶区C组和双靶区SC组小鼠肝细胞HBsAg、HBcAg的表达显著低于对照组。小鼠肝肾功能及组织学未见异常。结论HBVs、C双靶区反义LNA对HBV转基因鼠抗病毒有显著作用，且抑制作用优于单靶区反义LNA。

Objective To investigate the curative effects of dnal-target antisense locked nucleic acid（LNA） targeting the S and C regions. Method Antisense LNA complementary to the S and C regions in the genome of HBV was synthesized respectively. Cationic liposomes was used as drug carrier which targeted antisense LNA to mice liver. LNA was incubated with cationic liposomes for 60 minutes to produce the targeted Lipo-LNA mixture.30 HBV transgenic mice were randomly divided into 5 equal groups： single-target S group, single-target C group, dnal-target SC group, blank liposomes control group, and 5%GLU control group,to be injected into the caudal vein with corresponding Lipo-LNA mixture once for every other day. While in the control group,each mouse received the san~ volume（5% GLU） solution in the same way. Venous blood samples were collected before, and 1,3,5 days after the injection. Serum HBsAg was detected by time resolved fluoroisnmunoassy（TRFIA ）. HBV DNA was detected by real time PCR, while serum Alb, ALT, BUN and Cr were measured by automatic biochemistry analyzer. 5 days later, the mice were killed and immunohistochemistry was used to examine the HBsAg and HBcAg in the liver tissues. Pathological examination of the tissues was performed. Results The serum HBsAg concentrations in 1,3 and 5 days after injection were significantly lower than that before injection in the single-target S group,single-target C group and dual-target SC group（ P 〈 0.05）. The inhibition rates in single-target S group were 23.3% ,37.9% and 48.7% respectively, the rates in single-target C group were 21.2% ,32.6% and 40.7%, and the rates in dual-target group were 30.6% ,61.2% and 72.8% .In comparison with that before injection,the HBV DNA expression levels in 1,3 and 5 days after injection were significantly lower than in the single-target S group,single-target C group and dual-target SC group（all P 〈 0.05）. The inhibition rates in single-target S group were 12.6% ,26.5% and 32.8% respectively, in single-target C group were 11.6% ,24.5% and 27.3%, and in dnal-target group were 18.5%, 36.1% and 52.9%. HBsAg and HBcAg expressed in liver were significantly low in the single-target S group,single-target C group and dnal-target SC group in 5 days later. No significant difference was found in the tissues in all groups. Conclusions Dual-target antisense LNA targeting the S and C regions in the genome of HBV inhibits the replication and expression of HBV significantly, and the inhibition is stronger than single-target antisense LNA.