發(fā)布時(shí)間：2018-02-21 17:51

  本文關(guān)鍵詞： PR39 低氧誘導(dǎo) 基因治療 血管生成　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:為了探討PR39對(duì)急性缺血心肌的保護(hù)作用,本研究構(gòu)建腺相關(guān)病毒(AAV)介導(dǎo)的融和基因NT4-TAT-His-PR39,驗(yàn)證其在人臍靜脈內(nèi)皮細(xì)胞(ECV304細(xì)胞株)表達(dá);在缺氧ECV304細(xì)胞內(nèi)HIF-1α表達(dá)及抗缺氧應(yīng)激凋亡作用及其對(duì)缺氧環(huán)境雞胚促血管生成作用。 方法:1.應(yīng)用PCR技術(shù)和T載體克隆法克隆PR39基因,酶切鑒定,并進(jìn)行DNA測(cè)序及分析。2.將編碼PR39、穿膜肽TAT、6×His標(biāo)簽肽及NT4信號(hào)肽的四個(gè)基因片段用DNA連接酶相連,構(gòu)建pBV220/NT4-TAT-His-PR39融合基因載體,酶切獲取NT4-TAT-His-PR39融合基因片段,將其裝入質(zhì)粒pSSHG/NT4-TAT-His-PR39。3.采用磷酸鈣沉淀法三質(zhì)粒共轉(zhuǎn)染293細(xì)胞,獲得重組攜帶NT4-TAT-His-PR39的AAV載體,斑點(diǎn)雜交方法測(cè)定重組病毒滴度。4.AAV-PR39和空病毒載體(EV)分別感染ECV304,免疫細(xì)胞化學(xué)檢測(cè)6×His表達(dá)情況,以此驗(yàn)證融合基因的表達(dá)。5.將ECV304分為AAV-PR39組和PBS組,在1%O2條件培養(yǎng),免疫細(xì)胞化學(xué)測(cè)定ECV304中缺氧誘導(dǎo)因子1α(HIF-1α)表達(dá)。6.流式細(xì)胞儀檢測(cè)(FCM)分析低氧環(huán)境下ECV304細(xì)胞凋亡情況。7.30只雞胚隨機(jī)分為AAV-PR39組、EV組和PBS組,將各組細(xì)胞分別在低氧(5%O_2)和常氧(21%O_2)條件培養(yǎng),圖象軟件Image Pro Plus (IPP)分析雞胚尿膜囊(CAM)血管密度。 結(jié)果: 1 . PR39基因經(jīng)DNA測(cè)序與Genebank序列一致。2.NT4-TAT-His-PR39融合基因經(jīng)瓊脂糖凝膠電泳鑒定正確,測(cè)序結(jié)果與實(shí)驗(yàn)設(shè)計(jì)的序列完全一致。3.經(jīng)酶切鑒定將融合基因成功插入至pSSHG載體中;斑點(diǎn)雜交測(cè)定病毒的滴度為3.4×10~9 PFU/ml。4.免疫細(xì)胞化學(xué)檢測(cè)到實(shí)驗(yàn)組ECV304中6×His表達(dá)。5.免疫細(xì)胞化學(xué)檢測(cè)到缺氧ECV304細(xì)胞內(nèi)HIF-1α蛋白表達(dá)高于PBS組(t=11.23, P0.001)。6.流式細(xì)胞儀分析低氧環(huán)境下ECV304細(xì)胞周期圖,實(shí)驗(yàn)組ECV304中凋亡率明顯小于PBS組。7.低氧環(huán)境下,實(shí)驗(yàn)組的雞胚尿膜囊血管密度明顯大于PBS組及EV組。在常氧環(huán)境下,三組之間血管密度并無明顯差別。 結(jié)論:1.成功克隆PR39基因。2.成功構(gòu)建具有穿膜功能的“NT4-TAT-His-PR39”融合基因。3.成功構(gòu)建pSSHG/ NT4-TAT-His-PR39重組腺相關(guān)病毒載體,并成功包裝較高濃度的重組病毒。4.重組攜帶NT4-TAT-His-PR39的AAV載體能在ECV304中進(jìn)行分泌表達(dá),并提高缺氧ECV304中HIF-1α蛋白的表達(dá)水平。5.PR39具有抗缺氧應(yīng)激細(xì)胞凋亡作用。6.重組AAV-PR39載體對(duì)缺氧雞胚血管生成具有顯著的促進(jìn)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 偽 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 偽 (HIF-1 偽) 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 偽 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 偽 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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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