【摘要】： CD74即MHC-Ⅱ類分子相關(guān)恒定鏈(major histocompatibility complexⅡ-associated invariant chain, Ii),主要表達(dá)于樹突狀細(xì)胞、單核巨噬細(xì)胞、B細(xì)胞等抗原提呈細(xì)胞(antigen presenting cells, APC),是在內(nèi)質(zhì)網(wǎng)中與新合成MHC-Ⅱ類分子連接在一起形成九聚體的輔助分子,屬Ⅱ型膜分子,與抗原提呈功能有關(guān)。近來研究還發(fā)現(xiàn)CD74可做為巨噬細(xì)胞移動(dòng)抑制因子的受體,與相應(yīng)細(xì)胞因子結(jié)合激活NF-κB和ERK1/2信號(hào)轉(zhuǎn)導(dǎo)途徑,進(jìn)而誘導(dǎo)炎性細(xì)胞因子的分泌；研究還發(fā)現(xiàn),CD74除了表達(dá)于抗原提呈細(xì)胞,與抗原提呈有關(guān)外,CD74分子還在內(nèi)皮細(xì)胞、腫瘤細(xì)胞表達(dá),與相應(yīng)疾病的發(fā)生、發(fā)展有關(guān)。 血管內(nèi)皮細(xì)胞是位于血管內(nèi)壁的單層細(xì)胞,具有分泌血管活性物質(zhì)、參與炎癥細(xì)胞和免疫細(xì)胞游走、維持血管舒縮等多方面生物學(xué)功能。臍靜脈內(nèi)皮細(xì)胞因具有人體血管內(nèi)皮細(xì)胞的基本特性,且新生兒臍帶來源充足、取材容易、操作方便,而成為血管內(nèi)皮細(xì)胞體外研究的主要材料。ECV304是常用的人臍靜脈內(nèi)皮細(xì)胞,也是研究內(nèi)皮細(xì)胞生物學(xué)特性與疾病相關(guān)性的重要手段。本文將CD74基因轉(zhuǎn)染ECV304細(xì)胞,旨在探討CD74基因在HUVEC的轉(zhuǎn)染條件和CD74基因轉(zhuǎn)染對細(xì)胞特性及功能的影響,為深入研究基于調(diào)節(jié)CD74表達(dá)的生物治療打下基礎(chǔ)。 研究方法 1)將含有CD74 cDNA的pCMV-CD74 EGFP熒光質(zhì)粒通過轉(zhuǎn)化大腸桿菌進(jìn)行擴(kuò)增、質(zhì)粒提取、并經(jīng)酶切和測序進(jìn)行鑒定； 2)將pCMV-CD74質(zhì)粒轉(zhuǎn)染臍靜脈內(nèi)皮細(xì)胞ECV304,通過EGFP熒光質(zhì)粒對ECV304細(xì)胞進(jìn)行轉(zhuǎn)染率的檢測； 3) pCMV-CD74質(zhì)粒轉(zhuǎn)染前后ECV304細(xì)胞CD74 mRNA和CD74分子表達(dá)的鑒定：采用免疫組化法和Western blot,檢測轉(zhuǎn)染前后ECV304細(xì)胞CD74分子的表達(dá)；采用Real-time RT-PCR法檢測轉(zhuǎn)染后CD74 mRNA水平的表達(dá)； 4) pCMV-CD74質(zhì)粒轉(zhuǎn)染前后ECV304細(xì)胞功能相關(guān)基因(HLA-A、HLA-DR、IFNGR)表達(dá)的檢測：采用Real-time RT-PCR比較轉(zhuǎn)染前后臍靜脈內(nèi)皮細(xì)胞各功能相關(guān)基因的表達(dá)； 5)應(yīng)用Real-time RT-PCR分析軟件分析實(shí)驗(yàn)數(shù)據(jù)。 結(jié)果 1)在大腸桿菌擴(kuò)增后提取的質(zhì)粒經(jīng)EcorⅠ單酶切,電泳顯示pCMV-CD74質(zhì)粒線性大小約4.7kb; EcorⅠ和XbalⅠ雙酶切可見大小約1.35kb的基因片段,與CD74 cDNA全長(1.327kb)大小符合,并經(jīng)測序分析符合CD74基因序列； 2)熒光顯微鏡觀察轉(zhuǎn)染后綠色熒光蛋白的表達(dá)情況,并且經(jīng)過計(jì)算得出脂質(zhì)體介導(dǎo)的轉(zhuǎn)染率是65.8%； 3)免疫組化結(jié)果顯示,轉(zhuǎn)染后,ECV304細(xì)胞CD74表達(dá)增強(qiáng)；Real-time RT-PCR結(jié)果顯示,實(shí)驗(yàn)組△Ct值為4.8599,對照組△Ct值為9.2496,2-△△Ct結(jié)果為20.9619大于2,有統(tǒng)計(jì)學(xué)意義,說明轉(zhuǎn)染后CD74 mRNA表達(dá)顯著升高； 4) Real-time RT-PCR結(jié)果顯示,轉(zhuǎn)染前后ECV304細(xì)胞HLA-AΔCt值分別為9.4881和12.1097,2-△△Ct為6.1543大于2,說明轉(zhuǎn)染前后HLA-A表達(dá)有顯著差異；IFNGRΔCt值分別為4.9828和5.5228,2-△△Ct為1.4539小于2,轉(zhuǎn)染前后無顯著差異；HLA-DRΔCt值分別為14.1098和14.7924,2-△△Ct為1.3036小于2,轉(zhuǎn)染前后無顯著差異。 小結(jié) 人臍靜脈內(nèi)皮細(xì)胞ECV304表達(dá)HLA-A、HLA-DR、IFNGR和CD74 mRNA; pCMV-CD74轉(zhuǎn)染可增加ECV304細(xì)胞CD74基因和膜分子的表達(dá),增加HLA-A基因的表達(dá),但不增加HLA-DR和IFNGR的表達(dá),為深入探討CD74基因與內(nèi)皮細(xì)胞功能的相關(guān)性提供實(shí)驗(yàn)數(shù)據(jù)。
[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，

本文編號(hào)：2451546