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目的观察VP3基因、多西紫杉醇各浓度及VP3基因联合多西紫杉醇各浓度对PC-3细胞株的凋亡作用。方法构建重组真核表达载体PcDNA3-VP3,采用基因转染法转染人前列腺癌细胞株PC-3。利用RT-PCR技术检测VP3基因在PC-3细胞株中的表达状况。将多西紫杉醇浓度分为3组,分别为10~(-8) mol/L、10~(-7)mol/L、10~(-6)mol/L。应用光镜、HE染色、透射电镜形态学、MTT法、流式细胞仪TUNEL法观察转染重组质粒PcDNA3-VP3单独或联合多西紫杉醇各浓度对前列腺癌细胞株PC-3的影响。结果 VP3基因转染PC-3细胞后在细胞中得到了表达。HE染色和透射电镜下观察到PC-3细胞的典型凋亡形态学特征。MTT法检测结果显示转染质粒PcDNA3-VP3,单独应用10~(-7) mol/L以上浓度的多西紫杉醇及转染质粒PcDNA3-VP3联合应用10~(-8) mol/L以上浓度的多西紫杉醇均可使PC-3细胞株的增殖活性明显下降(P<0.01)。流式细胞仪TUNAL法检测单独转染质粒PcDNA3-VP3、质粒PcDNA3-VP3联合10~(-8) mol/L浓度组及质粒PcDNA3-VP3联合10~(-7)mol/L浓度组后24h、48h、72h各个时间点上的PC-3细胞株凋亡率分别为(20.31±1.96)%、(41.50±1.03)%、(50.03±3.00)%,(P<0.01);(31.10±0.59)%、(53.10±0.77)%、(68.90±2.66)%(P<0.01);(40.01±0.53)%、(62.23±0.74)%、(75.20±0.53)%(P<0.01)。结论联合10~(-8) mol/L以上浓度的多西紫杉醇能明显增加VP3基因诱导人前列腺癌PC-3细胞株凋亡。
Objective To observe the effect of VP3 gene, docetaxel concentration and the concentration of VP3 gene combined with docetaxel on PC-3 cell line. Methods The recombinant eukaryotic expression vector pcDNA3-VP3 was constructed and transfected into human prostate cancer cell line PC-3 by gene transfection. The expression of VP3 gene in PC-3 cell line was detected by RT-PCR. The concentrations of docetaxel were divided into three groups, which were 10 ~ (-8) mol / L, 10 ~ (-7) mol / L and 10 ~ (-6) mol / L, respectively. The effect of pcDNA3-VP3 alone or in combination with docetaxel on prostate cancer cell line PC-3 was observed by light microscopy, HE staining, transmission electron microscopy, MTT assay and flow cytometry (TUNEL). Results VP3 gene was transfected into PC-3 cells and expressed in the cells. The typical apoptotic morphological features of PC-3 cells were observed under HE staining and transmission electron microscopy. The results of MTT assay showed that pcDNA3-VP3 plasmid was transfected with docetaxel alone and pcDNA3-VP3 plasmid at a concentration of 10 -7 mol / L, Docetaxel could significantly reduce the proliferation activity of PC-3 cells (P <0.01). Flow cytometry TUNAL method was used to detect the expression of PcDNA3-VP3, pcDNA3-VP3 and pcDNA3-VP3 in the concentration of 10 ~ (-8) mol / L and pcDNA3-VP3 in the concentration of 10 ~ (-7) mol / (20.31 ± 1.96)%, (41.50 ± 1.03)%, (50.03 ± 3.00)%, respectively (P <0.01), (31.10 ± 0.59) at 48h, 72h, ), (53.10 ± 0.77)% and (68.90 ± 2.66)%, respectively (P <0.01); (40.01 ± 0.53)%, (62.23 ± 0.74)% and (75.20 ± 0.53)% respectively. Conclusions Combination of docetaxel at a concentration above 10 -8 mol / L can significantly increase the apoptosis of PC-3 cells induced by VP3 gene.