簇分化抗原74基因转染对人脐静脉内皮细胞ECV304特性的影响

Impact of CD74 Gene Transfection on Characteristics of Human Umbilical Vein Endothelial Cell ECV304

目的探讨簇分化抗原74(cluster of differentiation 74,CD74)基因在人脐静脉内皮细胞(human umbilicalvein endothelial cell,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。方法①将含有CD74 cDNA的pCMV-CD74增强型绿色荧光蛋白(enhanced green fluorescentprotein,EGFP)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;采用实时荧光定量反转录聚合酶链反应(real-time reverse transcriptase poly-merase chain reaction,RT-PCR)法检测转染后CD74 mRNA水平的表达;④pCMV-CD74质粒转染前后ECV304细胞功能相关基因人类白细胞抗原-A(human leukocyte antigens A,HLA-A)、人类白细胞抗原-DR(human leukocyte anti-gens DR,HLA-DR)、γ-干扰素受体(interferon-γreceptor,IFNGR)表达的检测:采用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RT-PCR分析软件分析实验数据。结果转染前后ECV304细胞HLA-A△Ct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表达有统计学差异;IFNGR△Ct值分别为4.9828和5.5228,2-△△Ct为1.4539小于2,转染前后无统计学差异;HLA-DR△Ct值分别为14.1098和14.7924,2-△△Ct为1.3036小于2,转染前后无统计学差异。结论人脐静脉内皮细胞ECV304表达HLA-A、HLA-DR、IFNGR和CD74mRNA;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。

Objective To explore the transfecting conditions of the cluster of differentiation（CD74） gene in human umbilical vein endothelial cells and impact of CD74 on cell characteristics and functions,and lay the basis for biotherapy based on CD74 expression modulation. Methods PCMV-CD74 enhanced green fluorescent protein（EGFP） plasmid was transformed into E.coli,extracted,enzyme-cut and sequenced.The EGFP fluorescent plasmid was transfected to ECV304 and the transfection efficiency was detected.The expressions of CD74 and HLA-DR before and after transfecting ECV304 were detected by immunohistochemical and Western blot.The expression of CD74 mRNA in transfected ECV304,and the expressions of function-associated genes including human leukocyte antigens A（HLA-A）,human leukocyte antigens DR（HLA-DR） and interferon-γ receptor（IFNGR） before and after transfecting ECV304 was detected by real-time reverse transcriptase polymerase chain reaction（RT-PCR）.Data was analysed by real-time RT-PCR software. Results The △Ct of HLA-A was 9.4881 and 12.1097 before and after CD74 transfection respectively,and 2-△△Ct was 6.1543,which indicated significant difference in the expression of HLA-A before and after CD74 transfection.The △Ct before and after CD74 transfection,and 2-△△Ct of IFNGR was 4.9828,5.5228 and 1.4539,and of HLA-DR was 14.1098,14.7924 and 1.3036,which implied no significant difference before and after transfection. Conclusion Human umbilical vein endothelial cell ECV304 can express HLA-A,HLA-DR,IFNGR and CD74 mRNA.PCMV-CD74 transfection can increase the expressions of CD74 gene,membrane molecules and HLA-A gene,but can＇t increase the expressions of HLA-DR and IFNGR.