Lipocalin-2基因表达产物对人胚肾细胞增殖的影响

目的构建Lipocalin-2(Lcn-2)基因真核表达质粒PL-2,并检测其及前期原核表达的重组Lcn-2蛋白对人胚肾细胞HEK293增殖的影响。方法利用RT-PCR从鼠巨噬细胞RAW264.7中扩增Lcn-2基因,克隆入真核表达质粒pEGFP-C1中,构建重组质粒PL-2。转染HEK293细胞,通过荧光观察和RT-PCR鉴定Lcn-2基因的表达。将重组质粒PL-2和前期原核表达的重组Lcn-2蛋白作用于HEK293细胞,通过MTT法检测细胞增殖情况,Westernblot法检测细胞增殖核抗原(PCNA)的表达。结果重组真核表达质粒PL-2经酶切和测序鉴定,证明构建正确。经荧光观察和RT-PCR鉴定,转染重组质粒PL-2的HEK293细胞中有Lcn-2基因的表达。加入重组Lcn-2蛋白后,HEK293细胞较对照组明显增殖,细胞中PCNA的表达也较对照组明显升高,而重组质粒PL-2对HEK293细胞增殖以及细胞中PCNA的表达均无影响。结论已成功构建了Lcn-2基因真核表达质粒,其对HEK293细胞的增殖无影响,而原核表达的重组Lcn-2蛋白对HEK293细胞的增殖有一定的促进作用,推测Lcn-2基因的表达产物可能通过与细胞膜受体结合促进HEK293细胞的增殖。

Effect of Expressed Product of Lipocalin-2 Gene on Proliferation of Human Embryonic Kidney Cells

Objective To construct the eukaryotic expression vector PL-2 for lipocalin-2(Lcn-2),and observe the effect of PL-2 as well as recombinant Lcn-2 protein previously expressed in prokaryotic cells on the proliferation of human embryonic kidney(HEK)cells.Methods Amplify Lcn-2 gene from murine macrophages RAW264.7 by RT-PCR and clone into eukaryotic expression vector pEGFP-C1.HEK293 cells were transfected with the constructed recombinant plasmid PL-2,and the expressed product was ob-served by fluorescent microscopy and identified by RT-PCR.Treat HEK293 cells with the recombinant plasmid PL-2 and the recom-binant Lcn-2 protein previously expressed in prokaryotic cells,and determine the proliferation of cells by MTT method,and the ex-pression of proliferation cell nuclear antigen(PCNA)by Western blot.Results Restriction analysis and sequencing proved that re-combinant plasmid PL-2 was constructed correctly.Fluorescent microscopy and RT-PCR proved expression of Lcn-2 gene in HEK293 cells transfected with PL-2.After treatment with recombinant Lcn-2 protein,the proliferation level of HEK293 cells and the expres-sion level of PCNA were significantly higher than those of control.However,recombinant plasmid PL-2 showed no significant effect on the proliferation of HEK293 cells and the expression of PCNA.Conclusion The eukaryotic expression vector PL-2 for Lcn-2 was successfully constructed,which showed no significant effect on proliferation of HEK293 cells.However,the recombinant Lcn-2 protein expressed in prokaryotic cells promoted effect on the proliferation of HEK293 cells.It is speculated that the expressed product of Lcn-2 gene may promote the proliferation of HEK293 cells by binding to cell membrane receptor.