半乳糖基化聚-L-赖氨酸偶联反义寡脱氧核苷酸的分布及抗乙肝病毒作用
Distribution and anti-HBV effects of antisense oligodeoxynucleotides conjugated to galactosylated poly-L-lysine.
作者信息
Zheng Su-Jun, Zhong Sen, Zhang Jian-Jun, Chen Feng, Ren Hong, Deng Cun-Liang
机构信息
Institute of Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400016, China.
出版信息
World J Gastroenterol. 2003 Jun;9(6):1251-5. doi: 10.3748/wjg.v9.i6.1251.
AIM
To describe distribution of the phosphorothioated antisense oligodeoxynucleotides (PS-asODNs) conjugated to galactosylated poly-L-lysine (Gal-PLL) in mice, and to observe their effects on expression of HBV gene in the 2.2.15 cells and transgenic mice.
METHODS
According to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligodeoxynucleotides (PS-asODNs) directed against the HBV U(5)-like region was conjugated to the hepatotropic Gal-PLL molecules. Its distribution was demonstrated using asODNs labeled with (32)P at the 5' terminus with a T4-polynucleotide Kinase. Its inhibition effect on HBV expression was observed in the transfected 2.2.15 cells and transgenic mice.
RESULTS
The Gal-PLL and asODNs could form stable complex at a molar ratio of 2:1. As shown in the HBV-transfected 2.2.15 cells, the inhibition effects of asODNs alone and asODNs conjugated to Gal-PLL, at 10 micromol/L for both, on HBsAg and HBeAg production were different,the former being 70 % and 58 %, respectively, and the latter being 96 % and 82 %, respectively. A more pronounced reduction was also observed in viral DNA load in the culture supernatant for the test with Gal-PLL-asODNs. Among many mouse organs, livers retained more asODNs molecules after administration. The preferential concentration in liver was found to be 52.14 % for Gal-PLL-asODNs, as high as 2.38-fold of that for asODNs (21.9 %). Both elements decreased gradually in liver, with 2.9 % of the former, 5.99 % of the latter retained 24 hours after the administration. The injection interval, therefore, was recommended to be 24 hours. In the transgenic mice, serum HBsAg decreased significantly (P<0.01) at the 12th day after administrating Gal-PLL- asODNs, the serum HBV DNA turned negative in 4 of the 6 mice.
CONCLUSION
Antisense oligodeoxynucleotides conjugated to Gal-PLL can be concentrated in liver and intaked by hepatocytic cells. This may result in specific inhibition of expression and replication of HBV in vitro and in vivo.
目的
描述半乳糖基化聚-L-赖氨酸（Gal-PLL）偶联的硫代磷酸化反义寡脱氧核苷酸（PS-asODNs）在小鼠体内的分布情况，并观察其对2.2.15细胞及转基因小鼠中HBV基因表达的影响。
方法
根据PCR扩增产物的直接测序结果，将针对HBV U(5)-样区域的16聚体硫代磷酸化反义寡脱氧核苷酸（PS-asODNs）类似物与亲肝性Gal-PLL分子偶联。使用T4多核苷酸激酶对5'末端用（32）P标记的asODNs来证明其分布情况。在转染的2.2.15细胞和转基因小鼠中观察其对HBV表达的抑制作用。
结果
Gal-PLL与asODNs能以2:1的摩尔比形成稳定复合物。如在转染HBV的2.2.15细胞中所示，单独的asODNs和与Gal-PLL偶联的asODNs在浓度均为10 μmol/L时，对HBsAg和HBeAg产生的抑制效果不同，前者分别为70%和58%，后者分别为96%和82%。对于Gal-PLL-asODNs的检测，培养上清液中的病毒DNA载量也观察到更明显的降低。在给药后的多种小鼠器官中，肝脏保留了更多的asODNs分子。发现Gal-PLL-asODNs在肝脏中的优先浓度为52.14%，高达asODNs（21.9%）的2.38倍。两者在肝脏中的含量均逐渐降低，给药24小时后，前者保留2.9%，后者保留5.99%。因此，建议注射间隔为24小时。在转基因小鼠中，给予Gal-PLL-asODNs后第12天血清HBsAg显著降低（P<0.01），6只小鼠中有4只血清HBV DNA转阴。
结论
与Gal-PLL偶联的反义寡脱氧核苷酸可在肝脏中富集并被肝细胞摄取。这可能导致在体外和体内对HBV的表达和复制产生特异性抑制。
Zotero 插件