不可逆性电穿孔介导HPV16 E6 shRNA干扰质粒对宫颈癌SiHa细胞增殖的影响

doi:10.13481/j.1671-587x.20150602

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (06): 1107-1112.doi: 10.13481/j.1671-587x.20150602

不可逆性电穿孔介导HPV16 E6 shRNA干扰质粒对宫颈癌SiHa细胞增殖的影响

王智亮¹, 余腾骅², 秦琴¹, 吴雨桐¹, 张文倩¹, 华媛媛¹, 熊正爱¹, 周玮³

1. 重庆医科大学附属第二医院妇产科, 重庆 400010;
2. 重庆医科大学附属第一医院内分泌乳腺外科, 重庆 400016;
3. 重庆市妇幼保健院产科, 重庆 400013

收稿日期:2015-04-03 出版日期:2015-11-28 发布日期:2016-01-11
通讯作者: 周玮,副主任医师(Tel:023-60333346,E-mail:dr.zhouwei@163.com) E-mail:dr.zhouwei@163.com
作者简介:王智亮(1987-),男,陕西省汉中市人,在读医学博士,主要从事妇科肿瘤方面的研究。
基金资助:
国家自然科学基金资助课题(81201745,81301928);重庆市卫生局医学科研项目资助课题(2011-2-155)

Influence of irreversible electroporation mediated HPV16 E6 shRNA interference plasmid in proliferation of cervical cancer SiHa cells

WANG Zhiliang¹, YU Tenghua², QIN Qin¹, WU Yutong¹, ZHANG Wenqian¹, HUA Yuanyuan¹, XIONG Zhengai¹, ZHOU Wei³

1. Department of Gynecology and Obstetrics, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China;
2. Department of Endocrine and Breast Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China;
3. Department of Obstetrics, Chongqing Health Center for Women and Children, Chongqing 400013, China

Received:2015-04-03 Online:2015-11-28 Published:2016-01-11

摘要/Abstract

摘要：

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100 μs、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。

关键词: 电穿孔, 干扰质粒, 宫颈肿瘤, 细胞增殖

Abstract:

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.

Key words: electroporation, interference plasmid, uterine cervical neoplasms, cell proliferation

中图分类号:

R737.33

王智亮, 余腾骅, 秦琴, 吴雨桐, 张文倩, 华媛媛, 熊正爱, 周玮. 不可逆性电穿孔介导HPV16 E6 shRNA干扰质粒对宫颈癌SiHa细胞增殖的影响[J]. 吉林大学学报(医学版), 2015, 41(06): 1107-1112.

WANG Zhiliang, YU Tenghua, QIN Qin, WU Yutong, ZHANG Wenqian, HUA Yuanyuan, XIONG Zhengai, ZHOU Wei. Influence of irreversible electroporation mediated HPV16 E6 shRNA interference plasmid in proliferation of cervical cancer SiHa cells[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1107-1112.

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不可逆性电穿孔介导HPV16 E6 shRNA干扰质粒对宫颈癌SiHa细胞增殖的影响

Influence of irreversible electroporation mediated HPV16 E6 shRNA interference plasmid in proliferation of cervical cancer SiHa cells

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