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目的 构建携带反义 bcl- 2的重组腺病毒并观察其对BT32 5细胞生长和凋亡的影响 .方法 利用细胞内同源重组的方法构建携带反义 bcl- 2的重组腺病毒 ,利用反转录 PCR和免疫组化法检测反义 bcl- 2在细胞内的表达及细胞 Bcl- 2蛋白表达的变化 ,用 MTT法和集落形成试验观察重组腺病毒对细胞生长和集落形成能力的影响 ,用电镜观察、DNA片段化分析和流式细胞仪技术检测细胞的凋亡 .结果 成功构建了携带反义 bcl- 2的重组腺病毒 Ad- abcl- 2 ,滴度达到 6 .5× 10 7nfu· L- 1 ;反转录 PCR表明反义 bcl- 2在 BT32 5细胞内有效转录 ,免疫组化检测到细胞 Bcl- 2蛋白表达水平明显下降 ;重组腺病毒感染 BT32 5细胞可以显著抑制细胞的生长 ,6 d生长抑制率可达 6 6 % ,同时细胞的集落形成能力明显下降 ;用电镜观察、DNA片段化分析和流式细胞仪技术均检测到细胞的凋亡 .结论 重组腺病毒 Ad- abcl- 2可以在细胞中有效表达反义 bcl- 2 ;反义 bcl- 2的表达抑制了 BT32 5细胞内 Bcl- 2蛋白的合成 ;反义 bcl- 2的表达可以显著抑制 BT32 5细胞的生长及其集落形成能力 ;反义 bcl- 2抑制 BT32 5细胞增殖是通过诱导细胞凋亡实现的
Objective To construct the recombinant adenovirus carrying antisense bcl-2 and observe its effect on the growth and apoptosis of BT32 5.Methods Recombinant adenovirus carrying antisense bcl-2 was constructed by homologous recombination in cells, The expression of antisense bcl-2 in cells and the expression of Bcl-2 protein were detected by PCR and immunohistochemistry. The effects of recombinant adenovirus on cell growth and colony formation were observed by MTT assay and colony formation assay. Electron microscopy, DNA fragmentation analysis and flow cytometry were used to detect the apoptosis of cells.Results The recombinant adenovirus Ad-abcl-2 carrying antisense bcl-2 was successfully constructed and its titer reached 6.5 × 10 7 nfu · L - 1; reverse transcriptase PCR indicated that antisense bcl-2 was efficiently transcribed in BT32 5 cells, and the expression of Bcl-2 protein was significantly decreased by immunohistochemistry. The results showed that BT32 5 cells were significantly inhibited by recombinant adenovirus, The growth inhibition rate reached 6 6% on the 6th day, and the colony forming ability of the cells decreased significantly.Apoptosis was detected by electron microscopy, DNA fragmentation analysis and flow cytometry.Conclusion The recombinant adenovirus Ad- The expression of antisense bcl-2 inhibited the Bcl-2 protein synthesis in BT32 5 cells. The antisense bcl-2 expression significantly inhibited the growth of BT32 5 cells And its colony forming ability; Antisense bcl-2 inhibits the proliferation of BT32 5 cells by inducing apoptosis