Lipocalin-2基因表达产物对人胚肾细胞增殖的影响

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

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。

Objective To construct the eukaryotie 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 elone into eukaryotie expression vector pEGFP-C1. HEK293 cells were transfected with the constructed recombinant plasmid PL-2, and the expressed produet was observed by fluorescent microscopy and identified by RT-PCR. Treat HEK293 cells with the recombinant plasmid PL-2 and the recombinant Lcn-2 protein previously expressed in prokaryotic cells, and determine the proliferation of cells by MTT method, and the expression of proliferation cell nuclear antigen （PCNA） by Western blot. Results Restriction analysis and sequencing proved that recombinant 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 expression 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 eonstructed, 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.