The Impact To The Characteristics Of Human Umbilical Vein Endothelial Cell ECV304 By Transfecting CD74 Gene

CD74 is MHC-Ⅱclass molecules associated invariant chain(major histocompatibility complexⅡ-associated invariant chain,Ii), which is mainly expressed in dendritic cells, monocytes macrophages, B cells and other antigen-presenting cells(antigen presenting cells, APC), which is the auxiliary polymer molecule 9 formed by linked with the newly synthesized MHC-Ⅱclass molecules in the endoplasmic reticulum, which is a typeⅡmembrane molecule and which is associated with the function of the antigen-presenting. Recent studies also found that CD74 can be used as the receptor of macrophage migration inhibitory factor, by combinating with the appropriate cytokines to activate NF-κB and ERK1/ 2 signal transduction pathway, which can induce the secretion of inflammatory cytokines. The study also found that CD74, in addition to be expressed on antigen-presenting cells and having relation with the antigen-presenting, can be expressed on endothelial cells and tumor cells and associates with the incidence and development of the corresponding disease.Vascular endothelial cells are located in lining single-cells of the blood vessel, which can secret vasoactive substances, be involved in the migration of inflammatory cells and immune cells and maintain the vasomotor and many other biological functions. Human umbilical vein endothelial cells have the fundamental characteristics of human vascular endothelial cells, what's more, the umbilical cord of newborns have a adequate source which can be gained easily and operated conveniently, which make it become the main material for studying vascular endothelial cells in vitro. ECV304 is a commonly used human umbilical vein endothelial cell, which is an important means to study the biological and disease-related characteristics of endothelial cells. By transfecting the CD74 gene to ECV304 cells, this article is focused on the conditions of transfectin the CD74 gene to HUVEC, in order to have a in-depth research the biological treatment based on regulating the expression of CD74. Research methods1)Transforming pCMV-CD74 plasmid into E.coli, extracting and enzyme-cutting plasmid, identifing CD74 gene by Sequencing;2)Transfecting EGFP fluorescent plasmid to ECV304, detecting the efficiency of transfection and taking photo;3)Identificating the expression of CD74 mRNA and CD74 molecules by transfecting pCMV-CD74 plasmid to ECV304:Detecting the expression of CD74 and HLA-DR in before and after transfected ECV304 by immunohistochemical and Western blot; Detecting the expression of CD74mRNA in transfected ECV304 by Real-time RT-PCR;4)Detecting the expression of some function-associated genes(HLA-A,HLA-DR and IFNGR) in before and afer transfected ECV304 by Real-time;5)Using Real-time RT-PCR analysis software to analyzes the experimental data.Results1)By disgesting the plasmid extracted from amplified Escherichia coli by single EcorⅠ, the pCMV-CD74 plasmid linear size is approximately 4.7kb displayed by electrophoresis. The size of 1.35kb gene fragment can be seen by double-disgested with EcorⅠand XbalⅠ, which is in line with the full-length (1.327 kb) of the CD74 cDNA and correspond to the CD74 gene sequences by sequencing analysis;2)By observeing the expression of green fluorescent protein after transfection by fluorescence microscopy, we gain the rate of the liposome-mediated transfection in 65.8% by calculating;3) Immunohistochemistry results showing that after transfection CD74 expressed in ECV304 are increased after be transfected; Real-time PCR results showing that the value ofΔCt is 4.8599 in experimental group,9.2496 in control group and the value of 2-ΔΔCt is 20.9619 with statistical significance, which showing that CD74mRNA is significantly increased after transfection.;4) Before ang after transfecting CD74 gene to ECV304, Real-time PCR results showing that theΔCt values of HLA-A are 9.4881 and 12.1097, and 2-ΔΔCt is 6.1543, which proved that the expression of HLA-A has significant difference; the values of IFNGR and HLA-DR are 4.9828,5.5228,1.4539 and 14.1098,14.7924,1.3036 recpectively,which proved that the expressions of IFNGR and HLA-DR have no significant difference before and after transfection.ConclusionsHuman umbilical vein endothelial cell ECV304 can express HLA-A, HLA-DR, IFNGR and CD74 mRNA; ECV304 increases the expressions of CD74 gene and membrane molecules and it also increases the expressions of HLA-A gene, but it can't increase the expression of HLA-DR and IFNGR, which provide experimental data for exploring the function correlation between the CD74 gene and endothelial cell.