IL-1β反义RNA表达载体的构建及对肝癌细胞中IL-1β水平的影响  

Establishment of IL-1β Antisense RNA Expression Vector and Its Effect on Expression Level of IL-1β in Murine Hepatoma Cells

作　　者：梁淑娟[1] 吴慧娜[1] 肖伟玲[1] 牟东珍[1] 刘艳艳[1] 

机构地区：[1]潍坊医学院免疫学教研室山东省高校免疫学重点实验室,山东潍坊261042

出　　处：《潍坊医学院学报》2008年第3期193-195,共3页Acta Academiae Medicinae Weifang

基　　金：山东省优秀中青年科学家科研奖励基金(2004BSB14074);山东省卫生高层次人才1020工程专项基金;教育部科学技术研究项目重点项目(205090);山东省自然科学基金(Y2007C044)

摘　　要：目的构建小鼠IL-1β反义RNA表达载体,观察对肝癌细胞中IL-1β表达的阻断效果。方法提取细胞总RNA,RT-PCR扩增IL-1β1和IL-1β2,将其反向连接到肝癌组织特异性表达载体PafpIRES2-EGFP,构建PafpIRES2-antiIL-1β1和PafpIRES2-antiIL-1β2反义RNA表达载体,利用jetPEI转染H22肝癌细胞和YAC-1淋巴细胞,荧光显微镜下观察反义RNA载体的表达,RT-PCR方法分析H22细胞中IL-1β表达水平的变化。结果RT-PCR扩增到两条长度分别为264和284bp的片段,与IL-1β1和IL-1β2相符,将其反向与PafpIRES2-EGFP连接,经菌落PCR和DNA序列分析证实,获得了PafpIRES2-antiIL-1β1和PafpIRES2-antiIL-1β2反义RNA表达载体。上述载体转染H22细胞后,荧光显微镜下可见细胞表达出明亮绿色荧光,而YAC-1细胞则未见荧光。RT-PCR分析证实转染反义RNA后H22细胞中IL-1β表达水平明显下降,尤以IL-1β2反义RNA的阻断效果更为显著。结论成功构建了小鼠IL-1β肝癌细胞特异性反义RNA表达载体PafpIRES2-antiIL-1β1,PafpIRES2-antiIL-1β2,两载体能有效地阻断肝癌细胞中IL-1β的表达。Objective To construct murine interleukin 1β（ IL-1β） antisense RNA expression vectors and analysis its effect on expression level of IL-1β in murine hepatoma cells. Methods Murine IL-1β gene segments of IL-1β1 （264bp） and IL-1β2 （284bp） was amplified through RT-PCR, purified PCR products were inserted inversely into Pafp IRES2-EGFP to construct Pafp IRES2-antilL-1β1 and Pafp IRES2-antilL-1β2 antisense RNA expression vectors. Recombinant plasmid was verified through clolony PCR screening and DNA sequencing. The established vectors were transfected into H22 hepatoma cells by jetPEI. Specific expression the vectors were observed under fluorescence microscope and the level of the IL-1β were assessed through RT-PCR. Results Two gene segments of 264b and 284bp were prepared through RT-PCR as expected. After insertion into Pafp IRES2-EGFP,repeated PCR screening identified several positive clones, as proved by DNA sequencing that the two gene fragments inserted were identical to the reported murine IL-1β in GenBank（ M15131 ） ,and then the recombinant vectors were named as Pafp, IRES2-antilL-1β1 and Pafp IRES2-antilL-1β2. Following transfection into H22 and YAC-1 cells,48h later bright green fluorescence could be seen only on the cell surface of H22 but not YAC-1 cells when observed under fluorescent microscope,indicating that beth vectors could specifically express in hepatoma cells. RT-PCR indicated that the expression level of IL-1β was markedly inhibited by the antisense RNA compare with cells transfected with PafplRES2-EGFP, significant inhibition was observed in cells transfected with Pafp IRES2-antilL-1β2. Conclusion Two murine IL-1β antisense RNA expression vectors PafpIRES2-antilL-1β1 and PafpIRES2-antilL-1β2 were successfully constructed,and the recombinant vectors could decrease the expression level of IL-1β effectively in murine hepatoma cells.

关 键 词：IL-1Β 反义RNA 肝癌 

分 类 号：R392.12[医药卫生—免疫学]