稳定表达人血红素加氧酶-1细胞系的建立及其功能鉴定

发布时间：2018-05-21 21:10

本文选题：血红素加氧酶- + 慢病毒　；参考：《中国医学科学院学报》2014年04期

【摘要】：目的构建稳定表达人血红素加氧酶-1(HO-1)的细胞系,探讨内源性过表达HO-1对抗细胞氧化损伤的作用。方法 PCR扩增HO-1基因并将其克隆至改造的慢病毒载体pLentiLox3.7中,构建携带目的基因的重组质粒。用重组质粒转染HEK293T细胞,并用Western blot检测HO-1的表达。将重组质粒与慢病毒辅助质粒(plp1、plp2、VSVG)共同转染HEK293T细胞,包装有活力的慢病毒。用携带HO-1的慢病毒感染HEK293T细胞,筛选出能稳定表达目的基因的单克隆,并行Western blot检测HO-1的表达。将过氧化氢加入正常的及过表达HO-1的HEK293T细胞和人脐静脉内皮细胞,检测细胞内活性氧的含量。结果成功筛选出能稳定表达HO-1的单克隆细胞系并扩大培养,Western blot检测其HO-1的表达明显升高。加入过氧化氢后,过表达HO-1的HEK293T细胞和人脐静脉内皮细胞内的活性氧明显减少。结论成功构建了能稳定表达人HO-1的细胞系,内源性过表达HO-1能够对抗细胞的氧化损伤。
[Abstract]:Aim to construct a cell line stably expressing human heme oxygenase-1 (HO-1) and to investigate the role of endogenous overexpression of HO-1 in the prevention of oxidative damage. Methods the HO-1 gene was amplified by PCR and cloned into the modified lentivirus vector pLentiLox3.7 to construct the recombinant plasmid carrying the target gene. HEK293T cells were transfected with recombinant plasmid and the expression of HO-1 was detected by Western blot. The recombinant plasmid was co-transfected with lentivirus-assisted plasmids (plp1 / plp2hVSVG) into HEK293T cells and the active lentivirus was packaged. After infection of HEK293T cells with lentivirus carrying HO-1, monoclonal clones were screened for stable expression of the target gene, and HO-1 expression was detected by Western blot. Hydrogen peroxide was added to normal and overexpressed HEK293T cells and human umbilical vein endothelial cells to detect the content of reactive oxygen species in the cells. Results Monoclonal cell lines with stable expression of HO-1 were successfully screened and their HO-1 expression was detected by Western blot. After adding hydrogen peroxide, reactive oxygen species (Ros) in HEK293T cells and human umbilical vein endothelial cells (HUVEC) overexpression of HO-1 decreased significantly. Conclusion A cell line with stable expression of human HO-1 was successfully constructed, and endogenous overexpression of HO-1 could antagonize oxidative damage.
【作者单位】：中国医学科学院北京协和医学院北京协和医院血管外科;中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室;
【基金】：北京市自然科学基金(7122145)~~
【分类号】：R346

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