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目的构建针对survivin基因的shRNA真核表达载体,观察重组质粒pEGFP-survivin对胆囊癌细胞(GBC-SD)化疗敏感性的影响。方法设计、合成包含BbsI酶切位点的针对survivin的shRNA,与BbsI酶切后真核表达载体pEGFP-H1连接,将其定向克隆至H1启动子下,构建成重组载体pEGFP-survivin;采用脂质体法将pEGFP-H1和重组质粒导入GBC-SD细胞中;用G418对转染的细胞进行稳定筛选。用RT-PCR测各组细胞中survivinmRNA的表达;以合适浓度的顺铂(DDP)(3.0μg/mL)作用相同时间后,用MTT法检测GBC-SD,GBC-SD/EGFP,GBC-SD/survivin 3种细胞存活率,TUNEL法观察细胞调亡。结果 pEGFP-survivin成功构建。GBC-SD/survivin细胞中的survivin表达水平较其余2种细胞明显下降(分别下降74.7%和71.5%);经DDP作用后,GBC-SD/survivin细胞存活率较其他2组明显降低,3种细胞均可见棕色凋亡细胞核。结论成功构建了针对survivin基因的shRNA真核表达载体,并获得稳定表达survivin shRNA的细胞株GBC-SD/survivin。survivin shRNA能明显降低GBC-SD细胞中survivin的表达,提高其对化疗药物的敏感性。
Objective To construct a shRNA eukaryotic expression vector targeting survivin gene and observe the effect of recombinant plasmid pEGFP-survivin on the chemosensitivity of gallbladder carcinoma cells (GBC-SD). Methods shRNA targeting survivin containing BbsI restriction sites was synthesized and ligated with the eukaryotic expression vector pEGFP-H1 after digestion with BbsI. The recombinant plasmid pEGFP-survivin was constructed by direct cloning into the H1 promoter. The pEGFP-H1 and recombinant plasmids were introduced into GBC-SD cells by plastid method; the transfected cells were stably selected with G418. The expression of survivin mRNA in each group was detected by RT-PCR. After treated with DDP (3.0μg / mL) for the same time, MTT assay was used to detect the expression of GBC-SD, GBC-SD / EGFP, GBC-SD / survivin three kinds of cell survival rate, cell apoptosis observed by TUNEL method. Results pEGFP-survivin was successfully constructed. The survival rate of GBC-SD / survivin cells was significantly lower than that of the other two cells (74.7% and 71.5% respectively). After DDP treatment, the survival rate of GBC-SD / survivin cells was significantly lower than the other two groups The cells showed brown apoptotic nuclei. Conclusion The shRNA eukaryotic expression vector targeting survivin gene was successfully constructed and the cell line GBC-SD / survivin stably expressing survivin shRNA was obtained. Survivin shRNA can significantly reduce the expression of survivin in GBC-SD cells and increase its sensitivity to chemotherapeutic drugs.