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目的构建凋亡素基因(vp3)重组真核表达质粒(pcDvp3),观察vp3基因在人乳腺癌细胞435中的表达及诱导凋亡作用,探讨其诱导肿瘤细胞凋亡的作用机制。方法(1)克隆vp3基因并与pcDNA3.1质粒连接构建pcDvp3重组真核表达载体,测序鉴定;(2)用脂质体将pcDvp3和pcDNA3.1转染人乳腺癌细胞435,48h后分别用透射电镜、琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;(3)建立人乳腺癌细胞435的裸鼠模型,分组给药后测定抑瘤率,并用原位凋亡(TUNEL法)检测肿瘤细胞的凋亡情况。结果(1)核酸序列分析表明已构建成重组质粒pcDvp3;(2)pcDvp3转染肿瘤细胞48h后,电镜下可见明显形态改变及凋亡小体形成;琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,其凋亡百分率高达14.42%;(3)裸鼠实验可见pcDvp3组抑瘤率分别为65.52%和68.23%,明显高于pcDNA3.1组(t=4.06,P<0.01),TUNEL检测可见pcDvp3组肿瘤细胞凋亡。结论vp3基因在体内外均能够有效地诱导人乳腺癌435细胞凋亡。
Objective To construct recombinant eukaryotic expression plasmid pcDvp3 of vp3 gene and observe the expression of vp3 gene in human breast cancer cell line 435 and to explore its mechanism of apoptosis. Methods (1) Cloning vp3 gene and connecting with pcDNA3.1 plasmid to construct recombinant eukaryotic expression vector pcDvp3, sequencing identification; (2) pcDvp3 and pcDNA3.1 transfected into human breast cancer cells by liposome 435,48 h were Transmission electron microscopy, agarose gel electrophoresis and flow cytometry were used to detect the apoptosis of tumor cells. (3) The nude mouse model of human breast cancer cell line 435 was established. The inhibitory rate of tumor was determined after group administration. TUNEL France) detection of tumor cell apoptosis. Results (1) Nucleic acid sequence analysis showed that it had been constructed into recombinant plasmid pcDvp3. (2) After transfected with pcDvp3 for 48 hours, obvious morphological changes and apoptotic body formation were observed under electron microscope; typical trapezoidal bands appeared on agarose gel electrophoresis; (3) In nude mice, the inhibitory rates of pcDvp3 group were 65.52% and 68.23%, respectively, which were significantly higher than that of pcDNA3.1 group (t = 4.06, P <0.01), TUNEL assay showed that the apoptosis of pcDvp3 group of tumor cells. Conclusion The vp3 gene can effectively induce the apoptosis of human breast cancer 435 cells both in vitro and in vivo.