CD74基因转染对人脐静脉内皮细胞ECV304特性的影响

发布时间：2019-04-01 12:10

【摘要】： CD74即MHC-Ⅱ类分子相关恒定链(major histocompatibility complexⅡ-associated invariant chain, Ii),主要表达于树突状细胞、单核巨噬细胞、B细胞等抗原提呈细胞(antigen presenting cells, APC),是在内质网中与新合成MHC-Ⅱ类分子连接在一起形成九聚体的辅助分子,属Ⅱ型膜分子,与抗原提呈功能有关。近来研究还发现CD74可做为巨噬细胞移动抑制因子的受体,与相应细胞因子结合激活NF-κB和ERK1/2信号转导途径,进而诱导炎性细胞因子的分泌；研究还发现,CD74除了表达于抗原提呈细胞,与抗原提呈有关外,CD74分子还在内皮细胞、肿瘤细胞表达,与相应疾病的发生、发展有关。 血管内皮细胞是位于血管内壁的单层细胞,具有分泌血管活性物质、参与炎症细胞和免疫细胞游走、维持血管舒缩等多方面生物学功能。脐静脉内皮细胞因具有人体血管内皮细胞的基本特性,且新生儿脐带来源充足、取材容易、操作方便,而成为血管内皮细胞体外研究的主要材料。ECV304是常用的人脐静脉内皮细胞,也是研究内皮细胞生物学特性与疾病相关性的重要手段。本文将CD74基因转染ECV304细胞,旨在探讨CD74基因在HUVEC的转染条件和CD74基因转染对细胞特性及功能的影响,为深入研究基于调节CD74表达的生物治疗打下基础。 研究方法 1)将含有CD74 cDNA的pCMV-CD74 EGFP荧光质粒通过转化大肠杆菌进行扩增、质粒提取、并经酶切和测序进行鉴定； 2)将pCMV-CD74质粒转染脐静脉内皮细胞ECV304,通过EGFP荧光质粒对ECV304细胞进行转染率的检测； 3) pCMV-CD74质粒转染前后ECV304细胞CD74 mRNA和CD74分子表达的鉴定：采用免疫组化法和Western blot,检测转染前后ECV304细胞CD74分子的表达；采用Real-time RT-PCR法检测转染后CD74 mRNA水平的表达； 4) pCMV-CD74质粒转染前后ECV304细胞功能相关基因(HLA-A、HLA-DR、IFNGR)表达的检测：采用Real-time RT-PCR比较转染前后脐静脉内皮细胞各功能相关基因的表达； 5)应用Real-time RT-PCR分析软件分析实验数据。 结果 1)在大肠杆菌扩增后提取的质粒经EcorⅠ单酶切,电泳显示pCMV-CD74质粒线性大小约4.7kb; EcorⅠ和XbalⅠ双酶切可见大小约1.35kb的基因片段,与CD74 cDNA全长(1.327kb)大小符合,并经测序分析符合CD74基因序列； 2)荧光显微镜观察转染后绿色荧光蛋白的表达情况,并且经过计算得出脂质体介导的转染率是65.8%； 3)免疫组化结果显示,转染后,ECV304细胞CD74表达增强；Real-time RT-PCR结果显示,实验组△Ct值为4.8599,对照组△Ct值为9.2496,2-△△Ct结果为20.9619大于2,有统计学意义,说明转染后CD74 mRNA表达显著升高； 4) Real-time RT-PCR结果显示,转染前后ECV304细胞HLA-AΔCt值分别为9.4881和12.1097,2-△△Ct为6.1543大于2,说明转染前后HLA-A表达有显著差异；IFNGRΔCt值分别为4.9828和5.5228,2-△△Ct为1.4539小于2,转染前后无显著差异；HLA-DRΔCt值分别为14.1098和14.7924,2-△△Ct为1.3036小于2,转染前后无显著差异。 小结 人脐静脉内皮细胞ECV304表达HLA-A、HLA-DR、IFNGR和CD74 mRNA; pCMV-CD74转染可增加ECV304细胞CD74基因和膜分子的表达,增加HLA-A基因的表达,但不增加HLA-DR和IFNGR的表达,为深入探讨CD74基因与内皮细胞功能的相关性提供实验数据。
[Abstract]:CD74 is an MHC-II-related constant chain (Ii), which is mainly expressed in dendritic cells, mononuclear macrophages, B cells and other antigen-presenting cells (APC). It is an auxiliary molecule which is linked with newly synthesized MHC-II molecules in the endoplasmic reticulum to form a nine-mer, and belongs to type II membrane molecules and is related to the function of antigen extraction. Recent studies have also found that CD74 can be used as a receptor for macrophage migration inhibitory factor, and the NF-B and ERK1/2 signal transduction pathways are activated in combination with corresponding cytokines to induce the secretion of inflammatory cytokines; it has also been found that CD74 is in addition to an antigen-presenting cell, The expression of CD74 in endothelial cells and tumor cells is related to the occurrence and development of the corresponding diseases. The vascular endothelial cell is a single-layer cell located on the inner wall of the blood vessel, and has various biological functions, such as the secretion of the vasoactive substance, the participation in the inflammatory cells and the immune cell migration, the maintenance of the vasomotor, and the like. The umbilical vein endothelial cells have the basic characteristics of human blood vessel endothelial cells, and the umbilical cord source of the newborn is sufficient, the materials are easy to obtain, the operation is convenient, and the umbilical vein endothelial cell is the main body of the in vitro study of the vascular endothelial cells. ECV304 is a commonly used human umbilical vein endothelial cell, and it is also the weight of the study of the relationship between the biological characteristics of the endothelial cells and the disease. In this paper, the CD74 gene was transfected into ECV304 cells. The purpose of this study was to study the effect of the transfection conditions of the CD74 gene on HUVEC and the effect of the transfection of CD74 gene on the characteristics and function of the cells, and to further study the biological treatment based on the regulation of CD74 expression. The underlying foundation. Method 1) The pCMV-CD74 EGFP fluorescent plasmid containing CD74 cDNA was amplified by transformation of E. coli, and the plasmid was extracted. and carrying out enzyme digestion and sequencing for identification;2) transfecting the pCMV-CD74 plasmid into an umbilical vein endothelial cell ECV304, Detection of the transfection rate of CV304 cells;3) identification of the expression of CD74 mRNA and CD74 in the ECV304 cells before and after the transfection of the pCMV-CD74 plasmid: the expression of the CD74 molecules in the ECV304 cells before and after transfection was detected by immunohistochemistry and Western blot; and the real-time RT-PCR was used. Detection of the expression of CD74 mRNA after transfection;4) pCMV-CD74 plasmid transfer Detection of the expression of ECV304 cell-related gene (HLA-A, HLA-DR, IFNGR) before and after dyeing: Real-time RT-PCR was used. Comparison of the expression of various function related genes of umbilical vein endothelial cells before and after transfection application Real-time RT-PCR was used to analyze the experimental data. (1) The plasmid extracted after the amplification of E. coli was digested with Ecor I, and the linear size of the pCMV-CD74 plasmid was about 4.7 kb. The full length of the DNA (1.327 kb) is in accordance with, and the sequence of the CD74 gene is in accordance with the sequencing analysis;2) the view of the fluorescence microscope The expression of the green fluorescent protein after transfection was examined, and the transfection rate of the liposome-mediated transfection was 65.8%. The results showed that the ECV304 cells were transfected after transfection. The expression of CD74 was enhanced; the results of Real-time RT-PCR showed that the Ct value of the experimental group was 4.8599, and that of the control group was 9.2496, and that of the control group was 9.2496. (4) The results of Real-time RT-PCR showed that ECV3 before and after transfection The values of HLA-A-Ct were 9.4881 and 12.1097, respectively. The results showed that the expression of HLA-A was significantly different before and after transfection. The value of IFNGR-Ct was 4.9828 and 5.5228. The results showed that the value of IFNGR-Ct was 1.4539, which was less than 2, and there was no significant difference before and after transfection. HLA-DR-C t鍊，

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