【摘要】 目的观察以甲胎蛋白转录调控序列(transcription regulatory element of-αfetoprotein,rAFP)驱动蜂毒素基因转录的重组腺病毒对肝癌细胞生物学性状的影响。方法将蜂毒素基因置于rAFP之后,用细菌内高效同源重组法将目的基因重组入腺病毒质粒中,重组质粒在293细胞中进行腺病毒的包装。携有蜂毒素基因的腺病毒感染肝癌细胞后,逆转录多聚酶链反应(reverse transcription polymerase chain reaction,RT-PCR)观察蜂毒素基因的转录;甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法测定蜂毒素基因转染对肝癌细胞增殖的影响;酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)双抗体夹心法定量检测甲胎蛋白(-αfetoprotein,AFP);流式细胞术检测基质金属蛋白酶-2(matrix metalloprotein-ase-2,MMP-2)分子表达情况。结果重组腺病毒载体构建成功,体外转染HepG2肝癌细胞后,蜂毒素基因在rAFP的控制下,可以抑制AFP阳性肝癌细胞的增殖,明显降低其AFP的生成量,但MMP-2分子的表达却是升高的。结论蜂毒素基因转染肝癌细胞后,可改变肿瘤细胞的部分生物学行为,使其表达谱发生改变。更多还原

【Abstract】 ObjectiveTo study effects of recombinant adenovirus harboring melittin gene,under the control of transcription regulatory element of α-fetoprotein(rAFP),on biological behavior of hepatocarcinoma cells.MethodsMelittin gene was synthesized chemically and put downstream of rAFP,afterwards,inserted into adenoviral backbone plasmid through a new simplified bacterial homologous recombinant system and the desired recombinant adenoviruses was packaged in 293 cells.Infected by the resultant adenovirus,proliferation inhibition of hepatocarcinoma cells was observed by methyl thiazolyl tetrazolium(MTT) assay,α-fetoprotein(AFP) expression in which was measured by enzyme linked immunosorbent assay(ELISA) double antibody sandwiched method and expression of matrix metalloproteinase-2(MMP-2) by flow cytometry.ResultsWhen the recombinant adenovirus was successfully constructed and infected hepatocarcinoma cells in vitro,melittin gene,under the control of rAFP,may inhibit proliferation of HepG2 cells and reduce the AFP expression,while stimulate the MMP-2 expression.ConclusionAfter being transducted into hepatocarcinoma cells,melittin gene may change some biological behavior of tumor cells,therefore influencing their expression spectrum.更多还原