人低氧诱导因子1-α（HIF1-α）转染人骨髓内皮祖细胞的实验研究

发布时间：2018-07-07 16:31

本文选题：人低氧诱导因子1-α(HIF1-α) + 基因转染　；参考：《吉林大学》2008年硕士论文

【摘要】： 近来研究发现,在人的外周血中存在血管内皮祖细胞,它来源于骨髓,在组织缺血的情况下,内皮祖细胞可以从骨髓中动员,迁移并结合到活跃的血管再生部位,增殖、分化为成熟内皮细胞,参与局部新生血管的形成这一过程称为血管发生。目前,在成人的外周血、骨髓、胎儿脐血中都证实含有内皮祖细胞。HIF-1是低氧条件下广泛存在于哺乳动物和人体内的一种转录因子,可调节多种靶基因如VEGF、红细胞生长因子(EPO)等的表达。本实转染HIF基因到内皮祖细胞验,通过将基因与EPCs治疗结合起来,一方面可以提高EPCs的“利用率”,另一方面,为基因进入体内找到合适“载体”,达到更强的治疗效果。本实验采用密度梯度法分离人骨髓中的内皮祖细胞,RT-PCR法检测内皮细胞特异性成分ecNOS,flk-1(KDR)的表达;采用硝酸酶还原法测定培养基中NOS活性的变化,间接证实内皮细胞的功能;用acLDL-Dil和FITC-UEA-1对细胞染色后,通过激光共聚焦显微镜鉴定,证实从人骨髓中成功分离出并诱导培养出内皮祖细胞。采用LipofectamineTm2000介导PCDNA3.0-HIF1α质粒转染内皮祖细胞,反向聚合酶链式反应(RT-PCR)检测HIF1α的转录;酶联免疫吸附试验(ELISA)检测VEGF表达,证实转染HIF1α的内皮祖细胞上清中VEGF含量比未转染HIF1α的内皮祖细胞上清中VEGF含量增加约5倍;免疫印迹法(Westem blot)检测HIF1蛋白表达,最终确认PCDNA3.0-HIF1α质粒转染内皮祖细胞成功。下一步的研究中,我们将探讨转染HIF基因的内皮祖细胞对心肌梗塞心肌血管修复能力及血管再生能力修复,进而为修复病损心脏组织提供另一条方案,为在细胞水平促进缺血心肌组织血管新生寻找新的途径,为心肌梗塞细胞生物治疗开创一条新的方法。
[Abstract]:Recent studies have found that vascular endothelial progenitor cells exist in human peripheral blood, which are derived from bone marrow. In the case of tissue ischemia, endothelial progenitor cells can mobilize from bone marrow, migrate and combine with active vascular regeneration sites to proliferate. The process of differentiation into mature endothelial cells involved in the formation of local neovascularization is called angiogenesis. At present, in adult peripheral blood, bone marrow, fetal umbilical cord blood have confirmed that the endothelial progenitor cell. HIF-1 is a transcription factor widely existed in mammals and human body under hypoxia. It can regulate the expression of many target genes such as VEGF, erythrocyte growth factor (EPO), etc. HIF gene was transfected into endothelial progenitor cells (EPCs). The combination of HIF gene and EPCs therapy can improve the "utilization ratio" of EPCs on the one hand, on the other hand, find the appropriate "vector" for gene entry into the body to achieve a stronger therapeutic effect. In this experiment, endothelial progenitor cells from human bone marrow were isolated by density gradient method to detect the expression of ecNOSflk-1 (KDR), the activity of NOS in culture medium was determined by nitrate reduction method, and the function of endothelial cells was confirmed indirectly. After staining with acLDL-Dil and FITC-UEA-1, it was confirmed by laser confocal microscopy that endothelial progenitor cells were successfully isolated from human bone marrow and induced to culture endothelial progenitor cells. PCDNA3.0-HIF1 伪 plasmid was transfected into endothelial progenitor cells by Lipofectamine Tm2000, reverse polymerase chain reaction (RT-PCR) was used to detect HIF1 伪 transcription, and VEGF expression was detected by enzyme-linked immunosorbent assay (Elisa). It was confirmed that the VEGF content in the supernatant of endothelial progenitor cells transfected with HIF1 伪 was increased by about 5 times than that in the supernatant of endothelial progenitor cells without HIF1 伪 transfection, and the expression of HIF1 protein was detected by Western blotting, and it was confirmed that PCDNA3.0-HIF1 伪 plasmid was successfully transfected into endothelial progenitor cells. In our next study, we will explore the repair of vascular repair and vascular regeneration by endothelial progenitor cells transfected with HIF gene in myocardial infarction. To find a new way to promote angiogenesis in ischemic myocardial tissue at cell level and to open up a new method for biotherapy of myocardial infarction cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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