脂联素基因修饰对大鼠骨髓来源的内皮祖细胞活性的影响及机制探讨  

Effect of adiponectin gene modification on the activity of endothelial progenitor cells derived from rat bone marrow and its mechanism

作　　者：陈丽芳 戴颖仪 林宋斌[2,3] 卢海克 刘新通 Chen Lifang;Dai Yingyi;Lin Songbin;Lu Haike;Liu Xintong(Department of Ultrasonography,Hanjiang District Pingmin Hospital,Putian 351115,Fujian,China;Department of Neurology,Second Medical College of Southern Medical University,Guangzhou 510280,China;Guangdong Second Provincial People’s Hospital,Guangzhou 510317,China)

机构地区：[1]福建省莆田市涵江区平民医院超声科,福建莆田351115 [2]南方医科大学第二临床医学院,广东广州510280 [3]广东省第二人民医院神经内科,广东广州510317

出　　处：《广州医科大学学报》2020年第5期23-29,共7页Academic Journal of Guangzhou Medical University

基　　金：广州市科技计划项目(201707010436)。

摘　　要：目的:探究脂联素(APN)基因修饰对内皮祖细胞(EPC)活性的影响及其机制。方法:无菌条件采集3-4周龄SD大鼠胫骨和股骨的骨髓,利用Ficoll-Paque PLUS溶液进行密度梯度离心分离骨髓中的单个核细胞,用EGM-2 Bulletkit培养基定向诱导单个核细胞分化为EPC。通过观察形态、免疫荧光检测细胞表面抗原CD34鉴定EPC。以大鼠EPC细胞的RNA为模板,PCR扩增出携带NheⅠ、XmaⅠ酶切位点的大鼠APN基因,重组到pLKIRG载体中,得到pLKIRG-APN载体,通过NheⅠ、XmaⅠ双酶切检测和Sanger法测序鉴定pLKIRG-APN慢病毒真核表达载体。pLKIRG-APN和pLKIRG分别与慢病毒包装质粒瞬转293T细胞,分别获得对照慢病毒(LV-NC)和过表达APN慢病毒(LV-APN),利用qPCR Lentivirus Titration Kit检测慢病毒滴度。LV-NC和LV-APN分分别感染EPC细胞48 h,0.1μg/μl嘌呤霉素连续筛选3 d,分别收集LV-APN感染组细胞(EPC-APN)和LV-NC感染组细胞(EPC-NC)。分别通过qPCR和WB检测APN的mRNA和蛋白表达水平。利用CCK8和Transwell分别检测EPC-APN和EPC-NC细胞的增殖和迁移能力,qPCR检测EPC-APN和EPC-NC细胞中VEGF、eNOS基因mRNA表达水平,WB检测VEGF、磷酸化VEGFR2、eNOS和Ser1177位点磷酸化eNOS的丰度。结果:大鼠单个核细胞最初为悬浮状态,第3天开始细胞逐渐贴壁,随后出现细胞集落,第7天细胞呈梭形或者多边形,第14天细胞呈现铺路石样生长状态。免疫荧光检测结果显示诱导培养2周后EPC的CD34阳性率达到90%以上。利用PCR扩增出大鼠APN基因,成功地构建了pLKIRG-APN慢病毒真核表达载体。qPCR和WB结果表明EPC-APN组APN的mRNA和蛋白表达水平较EPC-NC组明显升高,表明成功构建了过表达APN细胞。细胞功能试验显示EPC-APN的增殖和迁移能力与EPC-NC相比显著增强。EPC-APN中VEGF和eNOS基因的表达水平以及磷酸化VEGFR2和Ser1177位点磷酸化eNOS蛋白丰度明显高于EPC-NC。结论:APN基因修饰很可能通过促进VEGF、eNOS表达以及eObjective:To determine the effect of adiponectin(APN)gene modification on the activity of endothelial progenitor cells(EPC)and its mechanism.Methods:The bone marrow of tibia and femur of 3-4 week old SD rats were harvested under aseptic condition.The mononuclear cells in bone marrow were isolated by density gradient centrifugation using Ficoll-Paque PIUS solution and were induced to differentiate into EPC by EGM-2 Bulletkit medium.EPCs were identified by morphology and immunofluorescence assay of cell surface antigen CD34.Using the RNA of rat EPC cells as template,the rat APN gene carrying Nhe I and Xma I enzyme cleavage sites was amplified by PCR and then inserted into the pLKIRG vector to form a pLKIRG-APN vector.The lentivirus eukaryotic expression vector pLKIRG-APN was validated by Nhe I and Xma I double enzyme cutting and Sanger sequencing.pLKIRG-APN and pLKIRG were transfected into 293T cells with lentivirus packaging plasmids,respectively,to obtain control lentivirus(LV-NC)and overexpressed APN lentivirus(LV-APN).The titer of lentivirus was measured by qPCR lentivirus titration kit.The EPC cells were infected with LV-NC and LV-APN for 48h,respectively,followed by continuous screening with 0.1μg/μL puromycin for 3 days,and then the LV-APN infected cells(EPC-APN)and LV-NC infected cells(EPC-NC)were collected respectively.APN mRNA and protein expression levels in EPC-APN and EPC-NC were detected by qPCR and Western blotting,respectively.CCK8 and transwell assays were performed to detect the proliferation and migration ability of EPC-APN and EPC-NC cell lines respectively.For EPC-APN and EPC-NC cell lines,the expression levels of eNOS and VEGF gene mRNA were detected by qPCR;the abundance of VEGF,phosphorylated VEGFR2,eNOS and Ser1177 phosphorylated eNOS proteins was measured by Western blotting.Results:During culture,the rat mononuclear cells were in suspension state initially,gradually adhered to the wall with colony formation starting from day 3.On day 7,the cells appeared fusiform or polygonal.On day 14,

关 键 词：脂联素 内皮组细胞 基因修饰 血管内皮生长因子 内皮一氧化氮合酶 

分 类 号：R394.2[医药卫生—医学遗传学]