脂质体介导反义LNA-DNA嵌合体抑制HBV S基因表达的体外实验研究
ntiviral Effects of Antisense LNA-DNA Chimera Targeted at HBV S Gene by Cationic Liposomes in HepG22.2.15 Cell
目的 探讨针对乙型肝炎病毒（HBV）S基因翻译起始区反义锁核酸-DNA嵌合体在HepG22.2.15细胞内抗病毒效果及有效LNA-DNA嵌合体序列筛选。方法 针对HBV S基因mRNA翻译起始区同一位点设计合成四段不同序列长度（10~25bp）的LNA-DNA嵌合体短序列及无关对照序列，以阳离子脂质体作为载药体系，共同作用于HepG22.2.15细胞，采用ELISA法和实时荧光定量聚合酶链技术（FQ-PCR）分别监测24h、48h和72h细胞培养上清液中HBsAg和HBV DNA的含量变化；四甲基偶氮唑蓝（MTT）法检测LNA对细胞代谢的影响。结果 四段不同序列长度（10bp、15bp、20bp和25bp）的反义LNA-DNA嵌合体对HBsAg的表达和HBV DNA的复制均有显著性抑制作用，72h后的抑制率分别为46.58%、54.38%、72.43%、69.92%和27.09%、28.77%、34.71%、32.68%，且抑制率随时间呈增高趋势。LNA-DNA嵌合体对细胞代谢无明显影响。结论 针对HBV S基因mRNA翻译起始区的反义LNA-DNA嵌合体短序列体外能显著抑制乙型肝炎病毒的表达，且最适LNA-DNA嵌合体序列长度在15~25bp之间。
Objective To investigate the antiviral effects of antisense locked nucleic acid DNA chimera targeted at hepatitis B virus(HBV) S gene by cationic liposomes in HepG22.2.15 cells ,and screen the effective short sequence of LNA. Methods Four different length short sequence of antisense LNA-DNA chimera which complementary to the initiator of HBV S gene were designed and synthesized, carried by cationic liposomes into HepG22.2.15 cells. The HBsAg and HBV DNA of supernatant was tested by Enzyme linked immunoadsorbent assay(ELISA) and Real-time fluorescent quantitative PCR(FQ-PCR) in 24th ,48th and 72th hour after treatment respectively. LNA-DNA chimera’s toxicity on cell was detected by MTT method. Results Four different length short sequence of LNA-DNA chimera could inhibit the expression of HBsAg and the replication of HBV DNA with the inhibition rates of 46.58%,54.38%,72.43%,69.92% and 27.09%,28.77%,34.71%,32.68% respectively after 72 hours. There’s no obviously toxicity on cell. Conclusion Antisense LNA-DNA chimera that targeted targeted at HBV S gene has strong inhibition effect on HBV in vitro, and the optimal length of LNA-DNA chimera sequence might be in the range of 15bp to 25bp.It has a therapeutic potential in the treatment of patients infected with HBV.
韦叶生、王燕菲、农乐根、邓益斌
基础医学生物科学研究方法、生物科学研究技术
乙型肝炎病毒锁核酸2.2.15细胞基因治疗脂质体
hepatitis B viruslocked nucleic acid2.2.15 cellgene therapycationic liposomes
韦叶生,王燕菲,农乐根,邓益斌.脂质体介导反义LNA-DNA嵌合体抑制HBV S基因表达的体外实验研究[EB/OL].(2009-05-27)[2025-08-04].http://www.paper.edu.cn/releasepaper/content/200905-769.点此复制
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