分泌表达PR39的重组腺相关病毒的构建和促进低氧环境鸡胚血管新生作用的研究

发布时间：2018-02-21 17:51

本文关键词： PR39 低氧诱导基因治疗血管生成　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:为了探讨PR39对急性缺血心肌的保护作用,本研究构建腺相关病毒(AAV)介导的融和基因NT4-TAT-His-PR39,验证其在人脐静脉内皮细胞(ECV304细胞株)表达;在缺氧ECV304细胞内HIF-1α表达及抗缺氧应激凋亡作用及其对缺氧环境鸡胚促血管生成作用。方法:1.应用PCR技术和T载体克隆法克隆PR39基因,酶切鉴定,并进行DNA测序及分析。2.将编码PR39、穿膜肽TAT、6×His标签肽及NT4信号肽的四个基因片段用DNA连接酶相连,构建pBV220/NT4-TAT-His-PR39融合基因载体,酶切获取NT4-TAT-His-PR39融合基因片段,将其装入质粒pSSHG/NT4-TAT-His-PR39。3.采用磷酸钙沉淀法三质粒共转染293细胞,获得重组携带NT4-TAT-His-PR39的AAV载体,斑点杂交方法测定重组病毒滴度。4.AAV-PR39和空病毒载体(EV)分别感染ECV304,免疫细胞化学检测6×His表达情况,以此验证融合基因的表达。5.将ECV304分为AAV-PR39组和PBS组,在1%O2条件培养,免疫细胞化学测定ECV304中缺氧诱导因子1α(HIF-1α)表达。6.流式细胞仪检测(FCM)分析低氧环境下ECV304细胞凋亡情况。7.30只鸡胚随机分为AAV-PR39组、EV组和PBS组,将各组细胞分别在低氧(5%O_2)和常氧(21%O_2)条件培养,图象软件Image Pro Plus (IPP)分析鸡胚尿膜囊(CAM)血管密度。结果: 1 . PR39基因经DNA测序与Genebank序列一致。2.NT4-TAT-His-PR39融合基因经琼脂糖凝胶电泳鉴定正确,测序结果与实验设计的序列完全一致。3.经酶切鉴定将融合基因成功插入至pSSHG载体中;斑点杂交测定病毒的滴度为3.4×10~9 PFU/ml。4.免疫细胞化学检测到实验组ECV304中6×His表达。5.免疫细胞化学检测到缺氧ECV304细胞内HIF-1α蛋白表达高于PBS组(t=11.23, P0.001)。6.流式细胞仪分析低氧环境下ECV304细胞周期图,实验组ECV304中凋亡率明显小于PBS组。7.低氧环境下,实验组的鸡胚尿膜囊血管密度明显大于PBS组及EV组。在常氧环境下,三组之间血管密度并无明显差别。结论:1.成功克隆PR39基因。2.成功构建具有穿膜功能的“NT4-TAT-His-PR39”融合基因。3.成功构建pSSHG/ NT4-TAT-His-PR39重组腺相关病毒载体,并成功包装较高浓度的重组病毒。4.重组携带NT4-TAT-His-PR39的AAV载体能在ECV304中进行分泌表达,并提高缺氧ECV304中HIF-1α蛋白的表达水平。5.PR39具有抗缺氧应激细胞凋亡作用。6.重组AAV-PR39载体对缺氧鸡胚血管生成具有显著的促进作用。
[Abstract]:Objective: to investigate the protective effect of PR39 on acute ischemic myocardium, we constructed the fusion gene NT4-TAT-His-PR39 mediated by adeno-associated virus (AAVV) and verified its expression in human umbilical vein endothelial cell line (ECV304). The expression of HIF-1 伪 in hypoxic ECV304 cells and its antiapoptotic effect on hypoxia stress and its effect on promoting angiogenesis of chicken embryo in hypoxic environment. Methods: 1. PR39 gene was cloned by PCR and T vector cloning, and identified by restriction endonuclease digestion. DNA sequencing and analysis were performed. The four gene fragments encoding PR39, transmembrane peptide TAT6 脳 His tagged peptide and NT4 signal peptide were linked with DNA ligase. The pBV220/NT4-TAT-His-PR39 fusion gene vector was constructed, the NT4-TAT-His-PR39 fusion gene fragment was digested and inserted into the plasmid pSSHG / NT4-TAT-His-PR39.3. The recombinant AAV vector carrying NT4-TAT-His-PR39 was obtained by co-transfection of three plasmids using calcium phosphate precipitation method. The recombinant virus titer. 4. AAV-PR39 and empty virus vector EV304 were detected by dot blot. The expression of the fusion gene was verified by immunocytochemistry. The ECV304 was divided into AAV-PR39 group and PBS group, and cultured in O2 condition. The expression of hypoxia-inducible factor-1 伪 (HIF-1 伪) in ECV304 was determined by immunocytochemistry. 6. Flow cytometry was used to analyze the apoptosis of ECV304 cells in hypoxic environment. 7.30 chicken embryos were randomly divided into AAV-PR39 group and PBS group. Cells in each group were cultured in hypoxia-5 cells and normal oxygen group respectively. The vascular density of chicken embryo vesicle vesicle (CAM) was analyzed by image software Image Pro Plus (IPP). Results: 1. The PR39 gene was confirmed by DNA sequencing and Genebank sequence. 2.NT4-TAT-His-PR39 fusion gene was identified by agarose gel electrophoresis, and the sequencing result was completely consistent with the designed sequence .3.The fusion gene was successfully inserted into the pSSHG vector by restriction endonuclease digestion. The titer of the virus detected by dot blot was 3.4 脳 109PFU / ml 路4.The expression of 6 脳 His was detected by immunocytochemistry in ECV304 of experimental group. The expression of HIF-1 伪 protein in hypoxic ECV304 cells was higher than that in PBS group (11.23, P0.001n.6. flow cytometry was used to analyze the cell cycle diagram of ECV304 cells in hypoxic environment. The apoptotic rate in ECV304 in experimental group was significantly lower than that in PBS group. Under hypoxia, the vascular density of chick embryo vesicle in experimental group was significantly higher than that in PBS group and EV group. In normoxic environment, there was no significant difference in vascular density among the three groups. Conclusion: 1.The PR39 gene was cloned successfully. The "NT4-TAT-His-PR39" fusion gene with transmembrane function was successfully constructed. The recombinant adeno-associated virus vector pSSHG / NT4-TAT-His-PR39 was successfully constructed. The recombinant AAV vector carrying NT4-TAT-His-PR39 can secrete and express in ECV304. The expression level of HIF-1 伪 protein in hypoxic ECV304 was increased. 5. PR39 could inhibit apoptosis of hypoxia stress cells. 6. Recombinant AAV-PR39 vector could significantly promote angiogenesis of anoxic chicken embryo.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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