Affection Of Lipocalin-2 Gene Expression Products On Growth Activity Of Human Embryonic Kidney Cell

Objective: To construct respectively eukaryotic expression plasmid pEGFP-C1 and prokaryotic expression plasmid PET32a(+) which both contain Lipocalin-2 gene, and to observe the affection of lipocalin-2 protein on human embryonic kidney cell in vitro.Methods : The gene Lipocalin-2 was amplified from the total RNA of murine macrophage RAW264.7 by RT-PCR. The gene was cloned respectively into the eukaryotic expression plasmid pEGFP-C1 and prokaryotic expression plasmid pET32a(+).The plasmid pET32a(+) was transformed into E.coli. to obtain the recombinant protein Lipocalin-2 . The recombinant plasmid pEGFP-C1 was transfected stably into human embryonic kidney cell by liposome transfection. The expression of Lipocalin-2 gene was detected by RT-PCR and florescence observation. The effects of lipocalin-2 on growth of human embryonic kidney cell was observed by using recombinant lipocalin-2 and pEGFP-C1 transfection. The cell proliferation was detected by MTT straining and Western blot.Results : After identification by endonuclease digesting and DNA sequencing , the recombinant plasmid pEGFP-C1 was constructed successfully;At the concentration of 0.5×10-6 mol/L ,recombinant protein lipocalin-2 and culture for 72 hours , the average optical density value of recombinant lipocalin-2 group was higher(1.891±0.165) than the untreated group(1.522±0.0467), ( P<0.01 ) ,in MTT straining .There was no significant difference between the recombinant plasmid PL-2 group and the plasmid pEGFP-C1 group(P>0.05).Under the same condition, Western blot was used to detect the expression of proliferating cell nuclear antigen(PCNA) in human embryonic kidney cell. The optical density value of recombinant Lcn-2 group was also higher (0.684±0.009)than the untreated group(0.563±0.001) ( P<0.05 ) .There was no significant difference between the two groups including recombinant plasmid PL-2 group and plasmid pEGFP-C1 group(P>0.05).Conclusion : The recombinant protein Lipocalin-2 has partly effects on proliferation of human embryonic kidney cell, but no effects is observed in groups transfected with plasmids PL-2 and pEGFP-C1.We speculate that the proliferation most likely depend on related receptors on human embryonic kidney cell.