本文選題：熱休克蛋白70　切入點(diǎn)：腺病毒　出處：《新疆醫(yī)科大學(xué)》2015年博士論文

【摘要】：目的:1、探討腺病毒介導(dǎo)熱休克蛋白70(HSP70)基因轉(zhuǎn)染液的制備。2、探討腺病毒介導(dǎo)HSP70基因轉(zhuǎn)染到新生大鼠肺組織內(nèi)最佳轉(zhuǎn)染途徑、轉(zhuǎn)染量和轉(zhuǎn)染時(shí)間。3、探討通過基因轉(zhuǎn)染技術(shù),觀察經(jīng)HSP70基因轉(zhuǎn)染后缺氧性肺動(dòng)脈高壓(HPH)新生大鼠的肺動(dòng)脈壓力、肺血管結(jié)構(gòu)及重塑指標(biāo)改變的情況,以及肺組織中HSP70、缺氧誘導(dǎo)因子-1α(HIF-1α)及其下游靶基因內(nèi)皮素-1(ET-1)、誘導(dǎo)型一氧化氮合酶(iNOS)的表達(dá)情況,了解HSP70對(duì)HPH新生大鼠肺動(dòng)脈壓力、肺血管重塑的作用,進(jìn)一步推斷HSP70能否通過下調(diào)HIF-1α及其下游靶基因的表達(dá),進(jìn)而延緩新生大鼠HPH的發(fā)生發(fā)展,為臨床防治該病提供理論依據(jù)。方法:1、通過pHBAd-MCMV-GFP-HSP70質(zhì)粒的構(gòu)建,利用雙質(zhì)粒共轉(zhuǎn)染293細(xì)胞,同源重組產(chǎn)生重組腺病毒,進(jìn)行毒種的鑒定、生產(chǎn)、純化及滴度測(cè)定。2、(1)將48只新生大鼠隨機(jī)分為A:尾靜脈注射組,B:腹腔注射組,將5ul×1010 PFU/ml腺病毒HSP70(Ad-HSP70)轉(zhuǎn)染液注入新生大鼠體內(nèi),分別在觀察時(shí)間點(diǎn)將各組新生大鼠處死,留取肺組織標(biāo)本,用熒光顯微鏡判斷HSP70轉(zhuǎn)染到新生大鼠肺組織的途徑。(2)將54只新生大鼠隨機(jī)分為:空病毒組,病毒HSP70組,對(duì)照組,分別給予2.5ul×1010PFU/ml、5ul×1010 PFU/ml、10ul×1010 PFU/ml的轉(zhuǎn)染液劑量,經(jīng)過尾靜脈注射轉(zhuǎn)染后72h將各組新生大鼠處死,留取肺組織標(biāo)本,用RT-PCR及Western blot檢測(cè)HSP70的mRNA及蛋白質(zhì)水平,確定腺病毒介導(dǎo)的HSP70轉(zhuǎn)染的最佳轉(zhuǎn)染量。(3)確定腺病毒介導(dǎo)HSP70轉(zhuǎn)染的最佳途徑、最佳轉(zhuǎn)染量后,按照不同轉(zhuǎn)染時(shí)間將48只新生大鼠隨機(jī)分為24h組,48h組,72h組,96h組,同時(shí)設(shè)立鹽水空白對(duì)照組,按照時(shí)間點(diǎn)處死新生大鼠,留取肺組織標(biāo)本,采用RT-PCR及Western blot檢測(cè)HSP70的mRNA及蛋白質(zhì)水平,明確HSP70在新生大鼠肺組織內(nèi)表達(dá)的最佳轉(zhuǎn)染時(shí)間。3、將128只新生大鼠隨機(jī)分為2組:HPH組(H)和空白對(duì)照組(C)。H組:根據(jù)轉(zhuǎn)染液不同再分為H1:鹽水組、H2:空病毒組(pHBAd-MCMV-GFP)、H3:病毒HSP70組(pHBAd-MCMV-GFP-HSP70)。H組新生大鼠注射病毒轉(zhuǎn)染液或無(wú)菌鹽水后立即建立HPH模型,并分別于缺氧3d、7d、10d、14d和對(duì)照組同步進(jìn)行觀察。(1)在不同觀察時(shí)間點(diǎn)測(cè)定肺動(dòng)脈壓力(mPAP),比較各組mPAP的變化情況,了解腺病毒介導(dǎo)的hsp70是否有降低mpap的作用。(2)用光學(xué)顯微鏡、電子顯微鏡分別觀察肺小動(dòng)脈組織結(jié)構(gòu)及肺血管超微結(jié)構(gòu)變化,測(cè)定肺小血管重塑指標(biāo)(mt%、ma%)。(3)用免疫組化、rt-pcr和westernblot法分別檢測(cè)不同時(shí)間點(diǎn)各組新生大鼠肺組織中hsp70、hif-1α、et-1、inos的mrna及蛋白質(zhì)的表達(dá)情況,比較四種因子在各組之間的差異,從而進(jìn)一步推斷腺病毒介導(dǎo)的hsp70阻斷hif-1α及其靶基因在新生大鼠hph中的作用。結(jié)果:1、重組質(zhì)粒phbad-mcmv-gfp-hsp70序列測(cè)定與genebank的登記序列一致,確定為hsp70基因。2、(1)尾靜脈注射組新生大鼠48小時(shí)熒光顯微鏡下見少量綠色熒光信號(hào)標(biāo)記蛋白,72及96小時(shí)見明顯的綠色熒光信號(hào)標(biāo)記蛋白。(2)5ul×1010pfu/mlad-hsp70轉(zhuǎn)染劑量的新生大鼠肺組織內(nèi)hsp70mrna及蛋白質(zhì)表達(dá)量增高。(3)轉(zhuǎn)染新生大鼠72h后,新生大鼠肺組織內(nèi)hsp70mrna及蛋白質(zhì)表達(dá)量較24h、48h增高(p0.05),與96h比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。3、(1)h1組與h2組缺氧3d、7d、10d、14d的mpap水平與對(duì)照組比較顯著增高,差異有統(tǒng)計(jì)學(xué)意義(p0.05);h3組缺氧3d、7d、10d的mpap水平與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05),缺氧14d的mpap水平與同日齡h1及h2組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。(2)he、vg染色光鏡下觀察h3組缺氧3d、7d、10d無(wú)肺血管重塑,14d肺血管重塑較輕;電鏡下肺超微結(jié)構(gòu)顯示h3組缺氧7d、10d、14d肺血管重塑減輕;缺氧7d、10d各組間肺血管ma%、mt%比較差異有統(tǒng)計(jì)學(xué)意義(p0.05),c組及h3組與h1、h2組比較差異有統(tǒng)計(jì)學(xué)意義(p0.05);缺氧14d各組間肺血管ma%、mt%比較差異有統(tǒng)計(jì)學(xué)意義(p0.01),c組與hph各組間比較差異有統(tǒng)計(jì)學(xué)意義(p0.01)。(3)免疫組化顯示對(duì)照組肺組織中hsp70、hif-1α、et-1及inos免疫反應(yīng)強(qiáng)度呈陰性,hph各組hsp70、hif-1α、et-1及inos免疫反應(yīng)強(qiáng)度呈不同程度的陽(yáng)性反應(yīng),其表達(dá)多見于肺血管壁內(nèi)皮細(xì)胞質(zhì)與細(xì)胞上;缺氧3d、7d、10d各組間hsp70免疫組化表達(dá)強(qiáng)度比較差異有統(tǒng)計(jì)學(xué)意義(p0.05),hph各組與c組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.01),h3組與h1、h2組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.01);缺氧3d、7d、10d各組間hif-1α、et-1及inos的免疫組化表達(dá)強(qiáng)度比較差異有統(tǒng)計(jì)學(xué)意義(p0.01),hph各組均表達(dá)增強(qiáng),h3與h1、h2組比較表達(dá)降低,差異有統(tǒng)計(jì)學(xué)意義(p0.01)。(4)缺氧3d、7d、10dhsp70的mrna表達(dá)各組間比較差異有統(tǒng)計(jì)學(xué)意義(p0.01),hph各組與c組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.05),h3組與h1、h2組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.05),14dhph組與c組比較差異有統(tǒng)計(jì)學(xué)意義(p0.05);缺氧3d、7d、10d各組間hsp70蛋白質(zhì)表達(dá)比較差異有統(tǒng)計(jì)學(xué)意義(p0.05),h1、h2組與c組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.01),h3組與h1、h2組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(5)缺氧3d、7d、10dhif-1αmrna、et-1mrna、iNOSmRNA表達(dá)各組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.01),H1、H2組與C組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(P0.05),H3組與H1、H2組比較表達(dá)降低差異有統(tǒng)計(jì)學(xué)意義(P0.05);各組間HIF-1α、ET-1、iNOS的蛋白質(zhì)比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),H3組與H1、H2組比較表達(dá)降低差異有統(tǒng)計(jì)學(xué)意義(P0.05)。缺氧14d各組間iNOSmRNA表達(dá)比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),HPH各組與C組比較表達(dá)增強(qiáng)差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:1、成功構(gòu)建了HSP70基因重組腺病毒載體及制備高滴度的重組腺病毒。2、確定腺病毒介導(dǎo)的HSP70在新生大鼠體內(nèi)轉(zhuǎn)染的最佳途徑:尾靜脈,轉(zhuǎn)染量:5ul×1010PFU/ml及轉(zhuǎn)染后最早觀察時(shí)間點(diǎn):72 h。3、缺氧應(yīng)激可以引起內(nèi)源性HSP70表達(dá),腺病毒介導(dǎo)的HSP70可以提高HPH新生大鼠肺組織外源性HSP70的表達(dá),下調(diào)HIF-1α、ET-1、iNOS的表達(dá),降低肺動(dòng)脈壓力,減輕肺血管重塑,可以成為治療新生兒HPH的一種新策略。
[Abstract]:Objective: To investigate 1 adenoviral mediated heat shock protein 70 (HSP70).2 gene transfection liquid preparation, study of adenovirus mediated HSP70 gene transfection to the best transfection pathways in the lung tissue of neonatal rats, and transfection transfection time.3, explore the gene transfection technique, observe the transfection of HSP70 gene after hypoxic pulmonary arterial hypertension (HPH) and pulmonary artery pressure in neonatal rats, the changes of pulmonary vascular structure and remodeling index, and lung tissue HSP70, hypoxia inducible factor -1 alpha (HIF-1 alpha) and its downstream target gene of endothelin -1 (ET-1), inducible nitric oxide synthase (iNOS) expression and understanding HSP70 on pulmonary artery pressure in neonatal HPH rats, pulmonary vascular remodeling, further infer that HSP70 can down regulate expression of HIF-1 alpha and its downstream target genes, and thus delay the occurrence and development of neonatal rat HPH, and provide a theoretical basis for the prevention and treatment of the disease. Methods: 1 through pHBA The construction of d-MCMV-GFP-HSP70 plasmid, the two plasmids were co transfected into 293 cells to produce recombinant adenovirus by homologous recombination, identification, virus production, purification and titer of.2, (1) 48 newborn A: rats were randomly divided into intravenous injection group, intraperitoneal injection of B: group, HSP70 5ul * 1010 PFU/ml gland virus (Ad-HSP70) was injected into the body fluid of neonatal rats were sacrificed in the observation time points, each newborn rat lung samples were judged by fluorescence microscopy, HSP70 was transfected into pathway in lung tissue of newborn rats. (2) 54 neonatal rats were randomly divided into empty virus group, virus group HSP70 and the control group were treated with 2.5ul * 1010PFU/ml, 5ul * 1010 PFU/ml * 1010 PFU/ml 10ul transfection liquid dose, after intravenous injection of 72h after transfection to all neonatal rats were sacrificed and lung samples were used, the mRNA and protein levels of RT-PCR and Western blot detection HSP70, To determine the optimal amount of HSP70 transfected with adenovirus mediated human. (3) the best way to determine the adenovirus mediated HSP70 transfection, the best transfection amount, according to the different transfection time 48 neonatal rats were randomly divided into 24h group, 48h group, 72h group, 96h group, while the establishment of saline blank control group. According to the time after neonatal rat lung samples were used, the mRNA and protein levels of RT-PCR Western and blot HSP70 detection, the best transfection time.3 clear expression of HSP70 in lung tissue of neonatal rats, 128 neonatal rats were randomly divided into 2 groups: HPH group (H) and blank control group (C).H group: according to the different transfection solution divided into H1: group, H2: group, empty virus (pHBAd-MCMV-GFP), H3: HSP70 (pHBAd-MCMV-GFP-HSP70) virus group immediately HPH model group.H newborn rats injected with sterile saline solution or after transfection, and were lack of oxygen 3D, 7d, 10d, 14d and control group step Line. (1) observed in the pulmonary artery pressure was measured at different observation time points (mPAP), mPAP changes were compared, understand the adenovirus mediated Hsp70 is to reduce the role of mPAP. (2) using optical microscopy and electron microscope were used to observe the pulmonary tissue structure and ultrastructure of pulmonary vascular changes, determination small pulmonary vascular remodeling index (mt%, ma%). (3) were detected by immunohistochemical, HSP70, lung tissue of each group neonatal rats at different time points in RT-PCR and Westernblot HIF-1 alpha, ET-1, expression of mRNA and protein of iNOS, the difference between the four kinds of factors among the groups, to further infer adenovirus mediated HSP70 inhibition of HIF-1 alpha and its target gene in neonatal rat HPH. Results: 1, determination and registration sequence of genebank recombinant plasmid phbad-mcmv-gfp-hsp70 was identified as HSP70 gene sequence,.2, (1) intravenous injection of newborn rats in group 48 When you see a little green fluorescence under the fluorescence microscope signal marker protein, 72 and 96 hours to see the green fluorescent protein marker signal obviously. (2) in lung tissue of neonatal rats with 5ul * 1010pfu/mlad-hsp70 transfection dose and protein expression of HSP70mRNA increased. (3) 72h rats after transfected newborn, the expression of HSP70mRNA and protein content was 24h the lung tissue of neonatal rats, increased 48h (P0.05), no statistically significant difference compared with 96h (P0.05).3, (1) H1 group and H2 group, 3D 7d, 10d, hypoxia, the level of mPAP 14d increased significantly compared with the control group, the difference was statistically significant (P0.05); group H3 hypoxia 3D 7d, there was no significant difference between mPAP levels of 10d and the control group (P0.05), no statistically significant difference between the levels of mPAP and 14d in hypoxia of the same age in H1 and H2 group (P0.05). (2) he, VG staining was observed in the H3 group and hypoxia 3D, under light microscope, 7d, 10d and pulmonary vascular remodeling, 14d pulmonary vascular remodeling with light microscope; The lung ultrastructure showed group H3 hypoxia 7d, 10d, 14d, pulmonary vascular remodeling reduces; hypoxia 7d 10d group. Pulmonary vascular ma%, mt% had significant difference (P0.05), C group and H3 group and H1 group H2 had statistically significant difference (P0.05); hypoxic pulmonary vascular ma% 14d among the groups. Mt% was statistically significant difference (P0.01), there was significant difference between C group and HPH group (P0.01). (3) immunohistochemistry showed that the control group in the lung tissue HSP70, HIF-1 alpha, ET-1 and iNOS immunoreactive intensity were negative, HPH groups HSP70, HIF-1 alpha, ET-1 and iNOS immunoreactive intensity positive reaction in different degree, its expression in the cytoplasm and cell wall of pulmonary vascular endothelium; hypoxia 3D, 7d, 10d groups were HSP70 immunohistochemical expression difference was statistically significant (P0.05), HPH group compared with the C group expression difference was statistically significant (P0.01), H3 group and H1. Table H2 group 杈懼寮哄樊寮傛湁緇熻瀛︽剰涔，

本文編號(hào)：1669973