人源基因核糖体DNA区靶向载体VEGF165的构建及其在细胞中的定点表达研究

发布时间：2018-04-11 17:31

本文选题：人源基因载体 + VEGF165　；参考：《中南大学》2007年博士论文

【摘要】： 血管内皮生长因子具有特异性促进内皮细胞增殖，诱发新生血管生长的作用。利用血管内皮生长因子(VEGF)基因诱导治疗性新生血管生成，从而治疗缺血性心脏病和缺血性下肢疾病，是一种很有前景的方法。VEGF基因成功治疗肢体血管闭塞症和心脏缺血性疾病的临床试验在国外已有较多的报道。将外源基因VEGF导入到机体或细胞内是基因治疗的前提，目前，已有很多种技术和方法可将外源基因转移到真核细胞中。病毒载体因其体内、外转染效率均较高，而成为在当前VEGF基因治疗的基础和临床研究中应用得最多的载体。然而，病毒载体的安全性仍然是一个不容忽视的问题。病毒载体的安全性问题主要存在于两个方面，首先，由于它们易被机体识别为异源物质而可能具有免疫原性(immunogenicity)，另外一个更大的威胁是它可能将外源基因或者自身基因片段随机插入到宿主基因转录与／或调控区域，造成宿主细胞正常功能基因的失活与某些沉默基因的激活，，从而可能引发肿瘤或其它疾病。因此，病毒载体并不是基因治疗最理想的载体。医学遗传学国家重点实验室构建的人源基因载体—核糖体基因(rDNA)区靶向表达载体pHrneo能携带外源基因以较高的效率(10~(-4)-10~(-5))定点整合至人rDNA基因区。人每细胞有约600-800拷贝的rDNA基因，破坏一个拷贝对细胞维持正常的功能没有影响，是安全的基因治疗靶位点；而另一方面rDNA基因区编码核糖体RNA(rRNA)，是转录异常活跃的染色体区域，外源基因整合于rDNA基因区可以有效的表达。因此，以人源基因载体pHrneo将外源基因VFGE定点整合于人rDNA基因区，将能实现外源基因安全、稳定和有效的表达。本实验先成功地克隆出了VEGF基因片段，然后构建了携带VEGF基因的人源基因载体pHrneo-VEGF165，将构建的载体转入细胞内，测定定点整合及筛选定点细胞克隆，通过ELSA、RT-PCR、Western-blot等实验方法，检测出了VEGF的定点表达，为利用VEGF基因治疗缺血性心脏病和缺血性下肢疾病提供新的视角。
[Abstract]:Vascular endothelial growth factor (VEGF) can promote endothelial cell proliferation and induce neovascularization.Vascular endothelial growth factor (VEGF) gene was used to induce therapeutic neovascularization to treat ischemic heart disease and ischemic lower extremity disease.VEGF gene has been successfully used in the treatment of limb vascular occlusive disease and ischemic heart disease.The introduction of exogenous gene VEGF into organism or cell is the premise of gene therapy. At present, there are many techniques and methods to transfer exogenous gene into eukaryotic cells.Because of its high transfection efficiency in vivo and in vitro, virus vectors have become the most widely used vectors in the basic and clinical studies of VEGF gene therapy.However, the safety of virus vectors is still a problem that can not be ignored.The security problem of virus vector mainly exists in two aspects.Because they may be immunogenic because they are easily recognized as heterogenous substances, a greater threat is that they may be randomly inserted into the transcription and / or regulatory regions of host genes.Inactivation of normal functional genes and activation of some silencing genes in host cells may lead to tumor or other diseases.Therefore, viral vector is not the most ideal vector for gene therapy.The targeted expression vector pHrneo constructed by the State key Laboratory of Medical Genetics can carry foreign gene into human rDNA gene region with high efficiency.Human cells have about 600-800 copies of the rDNA gene per cell. Destroying one copy does not affect the normal function of the cell and is a safe target for gene therapy. On the other hand, the rDNA gene region encodes ribosomal RNA-rRNAs, which is a chromosomal region with abnormal transcriptional activity.The integration of exogenous genes into the rDNA gene region can be expressed effectively.Therefore, the integration of exogenous gene VFGE into human rDNA gene region by human gene vector pHrneo can realize the safe, stable and effective expression of exogenous gene.In this experiment, VEGF gene fragment was cloned successfully, and then the human gene vector pHrneo-VEGF165 was constructed. The constructed vector was transferred into the cells, and the targeted integration was determined and the targeted cell clone was screened. Western blot was used to detect the expression of pEGF165. by ELSART-PCRX Western-blot.The expression of VEGF was detected, which provided a new perspective for the treatment of ischemic heart disease and ischemic lower extremity disease with VEGF gene.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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