目的：探讨利用治疗剂量脉冲电场产生不可逆电穿孔（IRE）介导 HPV16 E6 shRNA 干扰质粒进入细胞的可行性，阐明二者共同作用对宫颈癌 SiHa 细胞增殖的影响及其作用机制。方法：将 HPV16 E6基因特异性干扰序列插入 pGenesil-1质粒，构建 HPV16 E6 shRNA 真核表达载体，将10个电压为800 V、脉宽100μs、频率1 Hz 的 IRE 作用于 SiHa 细胞与 HPV 16 E6 shRNA 干扰质粒的混悬液，根据处理因素组合，分为空白对照组、IRE 处理组、pGenesil-N 组、pGenesil-N＋IRE 组、pGenesil-E6组和 pGenesil-E6＋IRE 组。在荧光显微镜下观察 SiHa 细胞绿色荧光蛋白（GFP）的表达，计算 GFP 表达效率；RT-PCR 法检测 HPV 16 E6 mRNA 表达水平，Western blotting 法检测 HPV16 E6蛋白、P53及 PCNA 蛋白表达水平，CCK-8法检测各组 SiHa 细胞增殖能力的变化。结果：成功构建 HPV16 E6 shRNA 真核表达载体，IRE 处理后24 h 细胞即可见到绿色荧光；与IRE 组比较，pGenesil-E6＋IRE 组 E6 mRNA 表达水平下降（P ＜0.05），E6蛋白表达水平降低（P ＜0.05）， P53蛋白表达水平增高（P ＜0.05），PCNA 表达水平下降（P ＜0.05）；CCK-8法检测，与 pGenesil-E6组比较， pGenesil-E6＋IRE 组细胞增殖活性下降更明显（P ＜0.05）。结论：治疗剂量的 IRE 可介导外源基因进入细胞，二者联合作用能明显抑制宫颈癌 SiHa 细胞增殖。%Objective To explore the feasibility of using irreversible electroporation (IRE)mediating HPV16 E6 shRNA into cervical cancer cell line SiHa,and to clarify the influence of their co-effect on the proliferation of SiHa cells and its mechanism.Methods A HPV16 E6 gene specific interference sequence was inserted in pGenesil-1 to build a interference vector.10 pulses of IRE with 800 V,100 μs,and 1 Hz were applied to the suspension of SiHa cells and vectors.According to the treatment factors,control group,IRE group,pGenesil-N group,pGenesil-N+IRE group,pGenesil-E6 group and pGenesil-E6 + IRE group were set up.The expression of green fluorescent protein (GFP)and transfection efficiency were confirmed by inverted fluorescence microscope 24 h after the vector was transfected by IRE,and the expression efficancy of GFP was calculated.The expression levels of E6 mRNA and protein were detected by RT-PCR and Western blotting method which was also applied to detect the expressions of P53 and PCNA.The proliferative activity of SiHa cells was determined by CCK-8 assay.Results Enzyme digestion and DNA sequencing verified that the vectors were correctly constructed.GFP was seen under inverted fluorescence microscope 24 h after IRE transfection.Compared with IRE group,the expression levels of E6 mRNA and protein were decreased detected by RT-PCR and Western blotting method after the vectors were treated with IRE,the P53 protein expression level was increased (P < 0.05),and the PCNA expression level was decreased (P <0.05).The CCK-8 assay results showed the proliferative activity of SiHa cells in pGenesil-E6+IRE group was decreased more obviously than that in pGenesil E6 group (P <0.05).Conclusion IRE can play the role of gene transfection of mediating HPV16 E6 shRNA into SiHa cells, and their co-effect can significantly inhibit the proliferation of SiHa cells.
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