Effects Of Gene Of Lipocalin 2 Lcn₂ On The Process Of Rat Liver Regeneration

The gene of Lipocalin-2 lcn₂ encodes a small molecule secreted protease which is a member of lipocalin family. Lipocalin-2 regulate a variety of physiological processes, such as cell proliferation, differentiation and apoptosis. To research effects of lcn₂ on liver regeneration by excessive or weakened expression, I designed cloning primers and primers with restriction sites based on lcn₂ mRNA sequence. In this paper, I cloned lcn₂ as the material of regeneration liver by using molecular cloning technology. After confirming its sequence is correct, I further cloned lcn₂ into the eukaryotic expression vector pEGFP-N1 to construct expression vector pEGFP-N1-lcn2. In addition, after reading siRNA design-related papers, I synthesised oligonucleotides related to the two regions of lcn₂ mRNA by using online design tool such as Rui Bo, Genesil, Ambion, and QIAGEN sites. Then oligonucleotides were connected to the interference vector pGenesil-1.0 to construct two interference vectors pGenesil-1.0-l292 and pGenesil-1.0-l616. And the open reading frame (ORF) of lcn₂ were connected to the interference vectors pGenesil-1.0-l292 and pGenesil-1.0-l616 to construct test vectors corresponding interference vectors pGenesil-1.0-l292-lcn₂ and pGenesil-1.0-l616-lcn2, which is from expression vector pEGFP-N1-lcn2. The constructed expression vector pEGFP-N1-lcn2, interference vectors pGenesil-1.0-l292 and pGenesil-1.0-l616, test vector pGenesil-1.0-l292-lcn₂ and pGenesil-1.0-l616-lcn₂ were respectively injected into rats with hydrodynamics-based transgene (HDT) method. Green fluorescent positive cells (number) were detected by microscopy in the liver tissue. At the same time mortality,liver index,liver regeneration rate and changes in the structure and morphology were detected after the vectors were injected in rat. By observating the expression of green fluorescent protein of the expression vectors,interference vectors and the test vectors, I found that the expression peak of green fluorescent protein of pEGFP-N1-lcn2, pGenesil-1.0-l292 and pGenesil-1.0-l616 is 12h, the expression peak of green fluorescent protein of pGenesil-1.0-l292-lcn₂ and pGenesil-1.0-l616-lcn₂ is 6h. Compared with the control vectors pGenesil-1.0-hk-lcn2, green protein expression rate of pGenesil-1.0-l292-lcn₂ and pGenesil-1.0-l616 was significantly lower than that of control about 5%. This indicates two designed interference fragments have a certain interference effect. Compared with the control and partial hepatectomy group, liver index reduces of transfering vector pEGFP-N1-lcn₂ group at 120 and 168h. In contrast, liver index of transfering pGenesil-1.0-l616-lcn₂ group is slightly larger than the control and partial hepatectomy group at 72, 120 and 168h. Compared with the control and partial hepatectomy group, liver regeneration rate of transfering pEGFP-N1-lcn₂ group is slightly larger at 120h, but it is smaller at 168h. Liver regeneration rate of transferring pGenesil-1.0-l616 vector group is almost coincident with the control and PH groups, but it is larger than the control and PH groups. In short, I cloned lcn₂ and constructed the expression vector, interference vectors and test vectors. The physical analysis results indicate that lcn₂ is related to liver regeneration.