人ODCmRNA反义抑制敏感位点的筛选

目的：为建立有效的肿瘤治疗方法，筛查ODCmRNA最佳反义抑制位点。方法：采用RT—PCR的方法从肝癌细胞中扩增出鸟氨酸脱羧酶（ODC），外显子2、外显子3、外显子6—10、外显子11—12 4段基因片段。通过TA克隆反向连接到pcDNA3．1上，酶切测序并鉴定插入方向。脂质体法转染重组载体及空载体到肝癌MMC7721细胞中，采用Western—blot法检测各载体对ODC蛋白表达的抑制作用，采用四唑磕比色试验验证并比较各载体对MMC7721细胞增殖活性的影响。结果：外显子2、外显子3、外显子6—103个位点的ODC反义RNA表达载体转染细胞后，细胞内ODC蛋白表达量明显降低，细胞增殖活性受到明显抑制，抑制率分别为59％、62％、64％。结论：ODC外显子2、外显子3、外显子6—10是较好的反义抑制敏感位点。

The screening of the sensitive antisense target in the ODCmRNA

Objective:To Screen the sensitive antisense targets in the ODCmRNA.Methods:Four parts of ODC gene which were extron2,extron3,extron6-10,extron11-12 were amplified from hepatic cancer cell by RT-PCR and then reversely subcloned into pcDNA3.1 vector.Their inserted directions were identified by sequence.After transfecting the hepatic cancer cell line MMC7721 with these recombinant vectors and empty vectors pcDNA3.1,we adapted Western-blot and MTT assay to respectively identify and compare the effect of these vectors in the expression of ODC and cell growth.Results:Three ODC recombinant vectors of the best inhibitive effect were successfully selected.Conclusion:extron2,extron3 and extron6-10 were found to be the best antisense sites of inhibition.