重组腺病毒介导miR-27b感染内皮祖细胞的实验研究

发布时间：2018-03-19 11:49

本文选题：内皮祖细胞　切入点：腺病毒载体　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景内皮祖细胞(Endothelial progenitor cells, EPCs)是血管内皮细胞的体细胞,参与血管再生和内皮损伤后的修复过程。EPCs衰老及其功能紊乱可致血管内皮功能失调,后者与动脉粥样硬化的发生、发展密切相关。餐后高甘油三酯血症患者血浆中增多的残粒脂蛋白(Remnant-like particles, RLPs)与血管内皮功能失调密切相关,可加速人外周血单个核细胞来源EPCs的衰老进程,但其机制尚不明确。微小RNA (microRNA, miRNA)是一类在进化过程中高度保守的内源性非编码单链RNA,通过在转录后水平抑制真核生物的基因表达而参与调控细胞分化、生长发育、凋亡、肿瘤形成等复杂的病理生理过程。近年来,许多研究发现miRNA参与调控细胞衰老。我们通过前期研究发现：RLPs诱导的衰老小鼠EPCs中miRNA-27b (miR-27b)表达显著下调。推测miR-27b可能参与调控RLPs诱导的EPCs衰老。目的利用细菌内同源重组法构建以绿色荧光蛋白(EGFP)为报告基因、含miR-27b基因的重组腺病毒,导入小鼠内皮祖细胞中表达,为进一步研究miR-27b在RLPs诱导的EPCs衰老中的作用奠定基础。方法利用密度梯度离心法分离小鼠骨髓中单个核细胞,差速贴壁结合EBM-2培养基扩增细胞,诱导单个核细胞分化为EPCs。流式细胞技术鉴定EPCs。将含有目的基因miR-27b的质粒与目的载体pDC316-siRNA分别进行酶切。酶切产物琼脂糖电泳回收后进行定向连接,连接产物转入细菌感受态细胞BJ5183。先对生长的克隆进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对分析正确的克隆即为构建成功的目的质粒。将重组质粒在人胚肾293细胞(HEK293)中包装、扩增出miR-27b重组腺病毒载体(Ad-miR-27b),检测其滴度；以腺病毒载体绿色荧光蛋白(Ad-EGFP)为阴性对照,分别感染EPCs,RT-PCR检测感染后miR-27b的表达情况。结果经密度梯度离心和差速贴壁法分离所得的细胞经EBM-2专用培养基培养后,培养到第12d行流式细胞仪检测其CD34、CD133、Flk-1、 CD31的阳性率分别为(65+4)%、(48±3)%、(37±3)%和(51±4)%。目的基因miR-27b的质粒与目的载体pDC316-siRNA双酶切后共转化BJ5183感受态菌,可获得阳性重组体细菌克隆。重组质粒在人胚肾293细胞(HEK293)中包装、扩增出miR-27b重组腺病毒载体(Ad-miR-27b),检测其滴度重组质粒病毒滴度1.5×109pfU/mL。 Ad-miR-27b感染EPCs后24h miR-27b表达较阴性对照组显著增加。结论成功制备的重组腺病毒Ad-miR-27b感染体外培养的EPCs后,在体外能有效表达目的基因产物。
[Abstract]:Background. Endothelial progenitor cells (EPCs) are vascular endothelial cells. Somatic cells, involved in the repair process after vascular regeneration and endothelial injury. Aging and dysfunction of EPCs may lead to vascular endothelial dysfunction, which is associated with atherosclerosis. The increase of residual lipoprotein in plasma of patients with postprandial hypertriglyceridemia is closely related to vascular endothelial dysfunction, which can accelerate the aging process of EPCs derived from human peripheral blood mononuclear cells. However, the mechanism of microRNAs is still unclear. MicroRNAs are a class of highly conserved endogenous non-coding single-stranded RNAs that regulate cell differentiation, growth, development and apoptosis by inhibiting gene expression in eukaryotes at the post-transcriptional level. Tumor formation and other complex pathophysiological processes. In recent years, Many studies have found that miRNA is involved in the regulation of cellular senescence. We have found that the expression of miRNA-27b miR-27b in EPCs induced by miRNA-27b RLPs is significantly down-regulated. It is suggested that miR-27b may be involved in the regulation of EPCs senescence induced by RLPs. Purpose. The recombinant adenovirus containing miR-27b gene was constructed by homologous recombination in bacteria. The recombinant adenovirus containing miR-27b gene was introduced into mouse endothelial progenitor cells and expressed in mouse endothelial progenitor cells, which laid a foundation for further study on the role of miR-27b in EPCs senescence induced by RLPs. Method. Mononuclear cells were isolated from mouse bone marrow by density gradient centrifugation. The cells were amplified by differential adhesion and EBM-2 medium. The plasmid containing the target gene miR-27b and the target vector pDC316-siRNA were digested respectively. The ligation product was transferred into the bacterial competent cell BJ5183.The clones were identified by colony PCR, then the positive clones identified by PCR were sequenced and compared with each other. The recombinant plasmid was packaged in human embryonic kidney 293 cell line (HEK293) to amplify the recombinant adenovirus vector Ad-miR-27bN and detect its titer, and the adenovirus vector Ad-EGFP was used as negative control. The expression of miR-27b was detected by RT-PCR. Results. The cells isolated by density gradient centrifugation and differential adhesion method were cultured on EBM-2 medium. On the 12th day of culture, flow cytometry was used to detect the positive rates of CD34, CD133, Flk-1 and CD31, respectively. The positive rates of CD31 were 48 卤3% and 51 卤4%, respectively. The plasmid of miR-27b was digested with the target vector pDC316-siRNA and then transformed into BJ5183 receptive bacteria. The recombinant plasmid was packaged in human embryonic kidney 293 cell line (HEK293) and the recombinant adenovirus vector Ad-miR-27b was amplified. The titer of the recombinant plasmid was 1.5 脳 10 9 pfU 路mL. Ad-miR-27b. 24 h after EPCs infection, the expression of miR-27b was significantly increased compared with the negative control group. Conclusion. The recombinant adenovirus Ad-miR-27b was successfully infected with EPCs in vitro, and the target gene product could be expressed effectively in vitro.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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