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目的 :将蜂毒素基因置于甲胎蛋白 (AFP)转录调控元件 (r AFP)驱动之下 ,构件重组腺病毒载体 ,观察蜂毒素基因转染对肝癌细胞生长及 AFP表达的影响。 方法 ::将蜂毒素基因置于 r AFP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 ,将腺病毒质粒用 Pac 酶切线性化后 ,用脂质体介导转染 2 93细胞进行腺病毒的包装。携有蜂毒素基因的腺病毒感染肝癌细胞后 ,RT- PCR实验观察蜂毒素基因是否发生转录 ;MTT法测定蜂毒素基因转染对肝癌细胞增殖的影响 ;EL ISA双抗体夹心法定量检测 AFP。 结果 :RT- PCR实验表明蜂毒素基因转染肝癌细胞后可以被转录 ;蜂毒素基因在 r AFP的控制下 ,可以特异性的抑制 AFP阳性肝癌细胞的增殖 ,并降低其 AFP的生成量。结论 :蜂毒素基因转染对肝癌细胞的生长及 AFP的表达具有抑制作用 ,可降低其恶性度
OBJECTIVE: To construct the melittin gene under the control of AFP transcriptional regulatory element (rAFP) and to construct the recombinant adenovirus vector to observe the effect of melittin gene transfection on the growth of hepatocellular carcinoma and the expression of AFP. Methods: After the melittin gene was placed on r AFP, the target gene was recombined into the adenovirus plasmid by homologous recombination in bacteria. After the adenovirus plasmid was linearized by Pac restriction enzyme digestion, Dye 2 93 cells were packaged for adenovirus. Adenovirus carrying the melittin gene was used to infect hepatoma cells. The transcription of the melittin gene was detected by RT-PCR. The effect of melittin gene transfection on the proliferation of hepatocellular carcinoma cells was detected by MTT assay. AFP was detected by ELISA double antibody sandwich method. Results: The results of RT-PCR indicated that the melittin gene could be transcribed into hepatocellular carcinoma cells. The melittin gene could specifically inhibit the proliferation of AFP-positive hepatocarcinoma cells and reduce the production of AFP under the control of r AFP. Conclusion: The melittin gene transfection can inhibit the growth of liver cancer cells and the expression of AFP, and can reduce the malignant degree