miR-31慢病毒载体质粒构建及其对宫颈癌HeLa细胞增殖和迁移能力的影响

发布时间：2018-06-21 23:09

本文选题：宫颈癌 + 微小核糖核酸　；参考：《山东医药》2017年28期

【摘要】：目的构建微小核糖核酸31(miR-31)慢病毒载体质粒,并观察其对宫颈癌HeLa细胞增殖和迁移能力的影响。方法以包含miR-31基因的序列为目的片段,设计末端含有相应酶切位点的引物,PCR扩增目的片段,产物双酶切后插入线性化慢病毒载体中,构建miR-31慢病毒载体质粒并鉴定。将miR-31慢病毒载体质粒与包装质粒pPACKH1-GAG、pPACKH1-REV、pVSV-G共转染宫颈癌HeLa细胞,并进行慢病毒包装,检测miR-31慢病毒载体质粒滴度。取对数生长期HeLa细胞,随机分为空白对照组、miR-31组、空载体组,空白对照组不做任何处理,miR-31组予miR-31慢病毒载体质粒处理,空载体组予空载体慢病毒质粒处理。采用实时荧光定量PCR法检测各组miR-31 mRNA表达;MTT法和细胞克隆实验检测各组细胞增殖能力,Transwell小室法检测各组细胞迁移能力。结果成功构建重组慢病毒表达质粒,其病毒滴度为3×107TU/m L。miR-31组miR-31 mRNA相对表达量明显高于空白对照组、空载体组(P均0.05),而空白对照组与空载体组比较P0.05。miR-31组细胞增殖和迁移能力均高于空白对照组、空载体组(P均0.05),而空白对照组与空载体组比较P均0.05。结论成功构建了miR-31慢病毒载体质粒,建立了稳定过表达miR-31的宫颈癌HeLa细胞。稳定过表达miR-31的宫颈癌HeLa细胞增殖和迁移能力明显增强。
[Abstract]:Objective to construct the vector plasmid of minimal ribonucleic acid 31 (miR-31) lentivirus and to observe the effect of miR-31 on the proliferation and migration of cervical cancer HeLa cells. Methods the sequence containing miR-31 gene was used as the target fragment. Primers with corresponding endonuclease sites were designed to amplify the target fragment. The product was digested by double enzyme and inserted into the linearized lentivirus vector to construct and identify the vector of miR-31 lentivirus. The miR-31 lentivirus vector plasmid and the packaging plasmid pPACKH1-GAGN pPACKH1-REVSV-G were co-transfected into cervical cancer HeLa cells and packaged with lentivirus to detect the titer of miR-31 lentivirus vector plasmid. HeLa cells in logarithmic growth phase were randomly divided into blank control group and empty vector group. The blank control group was treated with miR-31 lentivirus plasmid and empty vector group was treated with empty vector lentivirus plasmid. The expression of miR-31 mRNA in each group was detected by real-time fluorescence quantitative PCR and cell clone assay was used to detect the cell proliferation ability and the migration ability of each group was detected by Transwell chamber assay. Results Recombinant lentivirus expression plasmid was successfully constructed. The relative expression of miR-31 mRNA in the virus titer of 3 脳 10 7 TU / m L.miR-31 group was significantly higher than that in the control group. The cell proliferation and migration ability of P0.05.miR-31 group was higher than that of blank control group (P 0.05), while that of blank control group was 0.05 compared with empty carrier group (P 0.05). Conclusion the vector plasmid of miR-31 lentivirus was successfully constructed and HeLa cells stably expressed miR-31 were established. The proliferation and migration of HeLa cells with stable expression of miR-31 were significantly enhanced.
【作者单位】：广西壮族自治区人民医院;
【基金】：国家自然科学基金资助项目(81060219、81660434) 广西自然科学基金资助项目(2014GXNSFAA118266)
【分类号】：R737.33

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