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目的构建人BNIP3真核表达载体,观察BNIP3高表达对人结肠癌细胞HT-29化疗敏感性的影响。方法PCR法扩增BNIP3基因,酶切后插入质粒pEGFP-C3,构建重组真核表达载体pEGFP-C3/BNIP3。脂质体转染人结肠癌细胞HT-29,Western blot检测BNIP3蛋白表达。MTT法检测5-氟尿嘧啶(5-Fu)的化疗敏感性和细胞增殖,AnnexinV-APC/PI双染流式细胞术检测细胞凋亡。结果酶切电泳分析和DNA序列测定证实,重组质粒pEGFP-C3/BNIP3构建成功;转染重组质粒的HT-29细胞BNIP3蛋白明显高表达。与未转染组和转染空质粒pEGFP-C3组比较,转染pEGFP-C3/BNIP3组5-Fu的IC50值显著降低[(120.11±5.45)、(113.40±4.72)μg/mL vs(19.08±2.62)μg/mL,P<0.05],细胞凋亡率显著增加[(5.51±0.32)%、(7.19±0.47)%vs(41.72±1.48)%,P<0.05],细胞克隆形成显著减少[(52±6)、(49±5)vs(11±3),P<0.05],细胞增殖速度减慢。结论成功构建了人BNIP3真核表达载体,BNIP3高表达可增加HT-29细胞对5-Fu的化疗敏感性。
Objective To construct human BNIP3 eukaryotic expression vector and observe the effect of BNIP3 overexpression on chemosensitivity of human colon cancer cell line HT-29. Methods BNIP3 gene was amplified by PCR and inserted into pEGFP-C3 plasmid to construct recombinant eukaryotic expression vector pEGFP-C3 / BNIP3. Human colon cancer cell line HT-29 was transfected with liposome, and the protein expression of BNIP3 was detected by Western blot. The chemosensitivity and cell proliferation of 5-fluorouracil (5-Fu) were detected by MTT assay. Apoptosis was detected by Annexin V-APC / PI double staining flow cytometry. Results The results of restriction endonuclease analysis and DNA sequencing confirmed that recombinant plasmid pEGFP-C3 / BNIP3 was successfully constructed. BNIP3 protein was highly expressed in HT-29 cells transfected with recombinant plasmids. The IC50 values of 5-Fu transfected with pEGFP-C3 / BNIP3 group were significantly lower than those of untransfected and transfected empty vector pEGFP-C3 [(120.11 ± 5.45), (113.40 ± 4.72) μg / mL vs (P <0.05), and the cell apoptosis rate was significantly higher than that of control group (± 2.62) μg / mL, P <0.05] [(52 ± 6), (49 ± 5) vs (11 ± 3), P <0.05]. The rate of cell proliferation slowed down. Conclusion The eukaryotic expression vector of human BNIP3 was constructed successfully. The high expression of BNIP3 increased the chemosensitivity of HT-29 cells to 5-Fu.