缺氧性肺动脉高压新生大鼠体内热休克蛋白70对HIF-1α的阻断作用研究

发布时间：2018-03-26 23:03

  本文选题：热休克蛋白70　切入点：腺病毒　出处：《新疆医科大学》2015年博士论文

【摘要】：目的:1、探讨腺病毒介导热休克蛋白70(HSP70)基因转染液的制备。2、探讨腺病毒介导HSP70基因转染到新生大鼠肺组织内最佳转染途径、转染量和转染时间。3、探讨通过基因转染技术,观察经HSP70基因转染后缺氧性肺动脉高压(HPH)新生大鼠的肺动脉压力、肺血管结构及重塑指标改变的情况,以及肺组织中HSP70、缺氧诱导因子-1α(HIF-1α)及其下游靶基因内皮素-1(ET-1)、诱导型一氧化氮合酶(iNOS)的表达情况,了解HSP70对HPH新生大鼠肺动脉压力、肺血管重塑的作用,进一步推断HSP70能否通过下调HIF-1α及其下游靶基因的表达,进而延缓新生大鼠HPH的发生发展,为临床防治该病提供理论依据。方法:1、通过pHBAd-MCMV-GFP-HSP70质粒的构建,利用双质粒共转染293细胞,同源重组产生重组腺病毒,进行毒种的鉴定、生产、纯化及滴度测定。2、(1)将48只新生大鼠随机分为A:尾静脉注射组,B:腹腔注射组,将5ul×1010 PFU/ml腺病毒HSP70(Ad-HSP70)转染液注入新生大鼠体内,分别在观察时间点将各组新生大鼠处死,留取肺组织标本,用荧光显微镜判断HSP70转染到新生大鼠肺组织的途径。(2)将54只新生大鼠随机分为:空病毒组,病毒HSP70组,对照组,分别给予2.5ul×1010PFU/ml、5ul×1010 PFU/ml、10ul×1010 PFU/ml的转染液剂量,经过尾静脉注射转染后72h将各组新生大鼠处死,留取肺组织标本,用RT-PCR及Western blot检测HSP70的mRNA及蛋白质水平,确定腺病毒介导的HSP70转染的最佳转染量。(3)确定腺病毒介导HSP70转染的最佳途径、最佳转染量后,按照不同转染时间将48只新生大鼠随机分为24h组,48h组,72h组,96h组,同时设立盐水空白对照组,按照时间点处死新生大鼠,留取肺组织标本,采用RT-PCR及Western blot检测HSP70的mRNA及蛋白质水平,明确HSP70在新生大鼠肺组织内表达的最佳转染时间。3、将128只新生大鼠随机分为2组:HPH组(H)和空白对照组(C)。H组:根据转染液不同再分为H1:盐水组、H2:空病毒组(pHBAd-MCMV-GFP)、H3:病毒HSP70组(pHBAd-MCMV-GFP-HSP70)。H组新生大鼠注射病毒转染液或无菌盐水后立即建立HPH模型,并分别于缺氧3d、7d、10d、14d和对照组同步进行观察。(1)在不同观察时间点测定肺动脉压力(mPAP),比较各组mPAP的变化情况,了解腺病毒介导的hsp70是否有降低mpap的作用。(2)用光学显微镜、电子显微镜分别观察肺小动脉组织结构及肺血管超微结构变化,测定肺小血管重塑指标(mt%、ma%)。(3)用免疫组化、rt-pcr和westernblot法分别检测不同时间点各组新生大鼠肺组织中hsp70、hif-1α、et-1、inos的mrna及蛋白质的表达情况,比较四种因子在各组之间的差异,从而进一步推断腺病毒介导的hsp70阻断hif-1α及其靶基因在新生大鼠hph中的作用。结果:1、重组质粒phbad-mcmv-gfp-hsp70序列测定与genebank的登记序列一致,确定为hsp70基因。2、(1)尾静脉注射组新生大鼠48小时荧光显微镜下见少量绿色荧光信号标记蛋白,72及96小时见明显的绿色荧光信号标记蛋白。(2)5ul×1010pfu/mlad-hsp70转染剂量的新生大鼠肺组织内hsp70mrna及蛋白质表达量增高。(3)转染新生大鼠72h后,新生大鼠肺组织内hsp70mrna及蛋白质表达量较24h、48h增高(p0.05),与96h比较差异无统计学意义(p0.05)。3、(1)h1组与h2组缺氧3d、7d、10d、14d的mpap水平与对照组比较显著增高,差异有统计学意义(p0.05);h3组缺氧3d、7d、10d的mpap水平与对照组比较差异无统计学意义(p0.05),缺氧14d的mpap水平与同日龄h1及h2组比较差异无统计学意义(p0.05)。(2)he、vg染色光镜下观察h3组缺氧3d、7d、10d无肺血管重塑,14d肺血管重塑较轻;电镜下肺超微结构显示h3组缺氧7d、10d、14d肺血管重塑减轻;缺氧7d、10d各组间肺血管ma%、mt%比较差异有统计学意义(p0.05),c组及h3组与h1、h2组比较差异有统计学意义(p0.05);缺氧14d各组间肺血管ma%、mt%比较差异有统计学意义(p0.01),c组与hph各组间比较差异有统计学意义(p0.01)。(3)免疫组化显示对照组肺组织中hsp70、hif-1α、et-1及inos免疫反应强度呈阴性,hph各组hsp70、hif-1α、et-1及inos免疫反应强度呈不同程度的阳性反应,其表达多见于肺血管壁内皮细胞质与细胞上;缺氧3d、7d、10d各组间hsp70免疫组化表达强度比较差异有统计学意义(p0.05),hph各组与c组比较表达增强差异有统计学意义(p0.01),h3组与h1、h2组比较表达增强差异有统计学意义(p0.01);缺氧3d、7d、10d各组间hif-1α、et-1及inos的免疫组化表达强度比较差异有统计学意义(p0.01),hph各组均表达增强,h3与h1、h2组比较表达降低,差异有统计学意义(p0.01)。(4)缺氧3d、7d、10dhsp70的mrna表达各组间比较差异有统计学意义(p0.01),hph各组与c组比较表达增强差异有统计学意义(p0.05),h3组与h1、h2组比较表达增强差异有统计学意义(p0.05),14dhph组与c组比较差异有统计学意义(p0.05);缺氧3d、7d、10d各组间hsp70蛋白质表达比较差异有统计学意义(p0.05),h1、h2组与c组比较表达增强差异有统计学意义(p0.01),h3组与h1、h2组比较表达增强差异有统计学意义(p0.05)。(5)缺氧3d、7d、10dhif-1αmrna、et-1mrna、iNOSmRNA表达各组间比较差异有统计学意义(P0.01),H1、H2组与C组比较表达增强差异有统计学意义(P0.05),H3组与H1、H2组比较表达降低差异有统计学意义(P0.05);各组间HIF-1α、ET-1、iNOS的蛋白质比较差异有统计学意义(P0.05),H3组与H1、H2组比较表达降低差异有统计学意义(P0.05)。缺氧14d各组间iNOSmRNA表达比较差异有统计学意义(P0.05),HPH各组与C组比较表达增强差异有统计学意义(P0.01)。结论:1、成功构建了HSP70基因重组腺病毒载体及制备高滴度的重组腺病毒。2、确定腺病毒介导的HSP70在新生大鼠体内转染的最佳途径:尾静脉,转染量:5ul×1010PFU/ml及转染后最早观察时间点:72 h。3、缺氧应激可以引起内源性HSP70表达,腺病毒介导的HSP70可以提高HPH新生大鼠肺组织外源性HSP70的表达,下调HIF-1α、ET-1、iNOS的表达,降低肺动脉压力,减轻肺血管重塑,可以成为治疗新生儿HPH的一种新策略。
[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 杈惧�炲己宸�寮傛湁缁熻�″�︽剰涔，

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