稳定表达人血红素加氧酶-1细胞系的建立及其功能鉴定

Establishment and Functional Identification of A Stable Cell Line Overexpression Heme Oxygenase-1

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。

Objective To establish a stable cell line overexpression heme oxygenase-1 （HO-1） mediated by a modified lentivirus system and identify its function.Methods The HO-1 gene was amplified by polymerase chain reaction and cloned into the modified pLentiLox3.7 expression vectors.The recombinant plasmids were transfected into HEK293T cells and the HO-1 was detected by Western blot.The recombinant plasmids were transfected into HEK293T cells to produce the viruses,with the helping plasmids including plp1,plp2,and VSVG.HEK293T cells were infected by the viruses and the cells that can express HO-1 were identified by Western blot.The reactive oxygen species were detected in the HO-1-overexpression HEK293T cells and the normal cells after the adding of hydrogen peroxide.The same experiment was performed with human umbilical vein endothelial cells.Results The stable cell line that can overexpress HO-1 was established,which was verified by Western blot.The reactive oxygen species in the HO-1-overexpression HEK293T cells and human umbilical vein endothelial cells decreased obviously after exposure to hydrogen peroxide.Conclusions The lentivirus-carrying HO-1 was successfully packaged and the stable cell line overexpression HO-1 was established.HO-1 can play a protective role in the course of oxidative damage.