缬沙坦通过抑制periostin的表达对脂多糖诱导的急性肺损伤大鼠的肺保护作用

发布时间：2018-01-10 11:03

本文关键词：缬沙坦通过抑制periostin的表达对脂多糖诱导的急性肺损伤大鼠的肺保护作用　出处：《大连医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:Priostin(POSTN)是动物体内成骨细胞和成纤维细胞分泌的一种细胞外分泌蛋白,在多种生理及病理状态下均会表达,病理状态下尤为显著。急性肺损伤(Acute Lung Injury.ALI)是一种肺组织的系统性炎症,常见原因有脓毒症、重症肺炎及创伤等。治疗不当或不及时可增加患者急性呼吸窘迫综合症(Acute Respiratory Distress Syndrome.ARDS)的发生率,且此类患者的死亡率较高,目前尚无显著高效的总体治疗策略,因此对于急性肺损伤的研究仍然有很强的必要性。有研究表明血管紧张素Ⅱ(AngiotensinⅡ.AngⅡ)受体拮抗剂能降低大鼠体内POSTN表达水平。本研究通过在大鼠体内应用AngⅡ受体拮抗剂缬沙坦来降低ALI大鼠肺组织POSTN的表达,来探讨缬沙坦是否能够保护脓毒症导致的急性肺损伤大鼠的肺脏。方法:1.ALI模型建立及动物分组:体重分布200-320g的45只SPF级S-D大鼠按照随机数表法分为空白对照组,脂多糖实验组(LPS组),缬沙坦低量干预组(10只)、缬沙坦中剂量干预组(10只)和缬沙坦高剂量干预组(10只)。动物称重后记录数据,对照组给予1ml无菌生理盐水经大鼠尾静脉注入,其余4组按照每公斤体重5mg LPS溶入1ml生理盐水中经大鼠尾静脉注入,缬沙坦干预组分别按照每公斤体重40mg、80mg、120mg腹腔注射缬沙坦混悬液。2.标本留取:各组大鼠经上述处置后正常喂食喂水,模型建立后6h麻醉处死大鼠,取大鼠右肺上叶放在-80℃冰箱中保存,用于实时定量PCR(RT-PCR)来检测肺组织periostin的表达程度。右肺下叶用于测肺组织干湿重比,用止血钳夹住主气管,用磷酸盐溶液反复冲洗支气管及肺泡多次,留取肺泡灌洗液(Bronchoalveolar Lavage Fluid.BALF),BALF离心后留取上清于-20℃冰箱中保存,留作炎症因子检测。将左叶肺组织放入中性甲醛溶液中,中性甲醛溶液起到组织固定作用。用HE染色制作组织病理标本。从腹主静脉抽取血液,经离心机离心后取上清液置于-80℃冰箱中。用ELISA法检测炎症因子IL-6、IL-13、TNF-α在BALF中的含量。3.湿干重比的测量:将取出的大鼠右肺下叶放入烤箱中,烤箱设置温度为80℃,持续烘烤24h后,应用微量电子称称重并记录。4.组织病理学检测:取出固定完全的肺组织,常规石蜡包埋,采用HE染色,应用电子显微镜观察肺部组织炎症反应的程度。5.实时荧光定量PCR(Real-Rime PCR)检测periostin mRNA在肺组织中的表达Periostin mRNA的表达:首先提取肺组织中的总mRNA,然后根据RT-PCR试剂盒提供的产品说明书在反应体系内加入实验所需的各种试剂。设定PCR的反应条件,进行逆转录循环,循环45次后,反应体系在72℃继续延伸10分钟。根据Gen Bank中大鼠的periostin(基因登录号NM001108550)及β-actin(基因登录号NM031144)序列设计引物。Periostin上游引物为5’-AGGAGCCGTGTTTGAGACCAT-3’下游引物为5’-CGGTGAAAGTGGTTTGCTGTTT-3’β-actin上游引物为5’-TCCTCCCTGGAGAAGAGCTA-3’下游引物为5’-TCAGGAGGAGCAATGATCTTG-3’。将反应结果采用相对定量的方法在荧光定量仪进行分析。以β-actin基因mRNA的表达为内参基因,根据公式计算相对表达量的差值,即为2-△△Ct,2-△△Ct的意义为实验组的目的基因的表达相对于对照组的表达倍数。6.Western Blot方法测量肺组织中periostin蛋白含量:将肺组织取出后在冰面上用组织研磨器进行研磨,研磨完成后,应用试剂盒提供的说明书测量肺组织中总蛋白得浓度,统一浓度后,进行电泳,转膜,封闭,抗体杂交,发光后凝胶成像,采取图样。7.用ELISA法检测炎症因子IL-4、IL-13及TNF-α在BALF中的浓度。8.数据统计分析:本研究中实验数据均采用SPSS18软件进行统计学分析及处理。规定P0.05时,可认为比较各组间的差异具有统计学意义。结果:(一)各组大鼠右下肺叶的湿/干重比值与正常对照组(4.01±1.14)相比,LPS组大鼠肺组织湿/干重比(5.36±0.30)明显升高,差异显著,有统计学意义(p0.05),说明模型的制作是成功的。缬沙坦低(4.91±0.15)、中(4.81±0.10)、高(4.85±0.11)干预组之间差异无统计学意义(p0.05)。缬沙坦干预组与LPS组差异具有统计学意义(p0.05)。(二)组织病理学检测用光学显微镜在高倍镜下观察病理切片,可见正常组大鼠肺组织结构清晰,细胞大小正常,肺泡间隔正常,肺泡腔内炎症细胞的浸润较少,无肺组织明显水肿。急性肺损伤组大鼠肺组织可见肺泡间隔明显消失并呈明显增厚表现,组织可见大量出血点及水肿,肺泡腔内可见异常大量炎症细胞的浸润。缬沙坦干预组上述病理改变比LPS组大鼠表现有轻度减少。(三)RT-PCR检测肺组织PeriostinmRNA的表达水平应用相对定量的计算办法,以β-action为内参基因,结果以LPS组目的基因periostin mRNA的表达相对于内参基因的变化倍数表示。结果分析,LPS实验组相对于空白对照组比较,LPS组结果升高明显,差异显著,差异具有统计学意义;缬沙坦干预组(低、中、高)与LPS组相对表达量差异具有统计学意义。(四)肺组织Periostin蛋白Western Blot检测肺组织Western Blot检测,根据凝胶成像分析系统检测,其相应条带对应蛋白含量=(样品条带的光密度×面积)/(GAPDH的光密度×面积)。肺组织periostin蛋白含量:相对于对照组,LPS组大鼠肺组织periostin蛋白含量明显增高(2.48±0.07vs0.74±0.05,p0.05);相对于对照组,缬沙坦干预组大鼠肺组织periostin蛋白含量差异具有统计学意义(1.43±0.03vs0.74±0.05、1.65±0.07vs0.74±0.05、1.48±0.04vs0.74±0.05,p0.05),缬沙坦低、中、高剂量干预组组间比较,periostin蛋白含量差异无统计学意义(1.43±0.03vs1.65±0.07vs1.48±0.04,p0.05)。(五)BALF中炎症因子IL-6、IL-13、TNF-α的含量与对照组相比(170±19)pg/ml,LPS组大鼠的BALF中IL-6的含量(310±37)pg/ml升高明显,差异有统计学意义(p0.05),干预组与LPS组相比差异有统计学意义(p0.05)。与对照组相比(83±8)pg/ml,LPS组大鼠的BALF中IL-13的含量(53±9)pg/ml降低明显,差异有统计学意义(p0.05),干预组与LPS组相比差异有统计学意义(p0.05)。与对照组相比(149±12)pg/ml,LPS组大鼠的BALF中TNF-α含量(280±40)pg/ml呈明显升高,差异有统计学意义(p0.05),干预组与LPS组相比差异有统计学意义(p0.05)。结论:1.急性肺损伤大鼠肺组织中的POSTN表达水平明显升高。2.缬沙坦可以通过拮抗AngⅡ受体来降低POSTN的表达,从而降低肺组织的炎症反应及水肿程度,也就是降低了ALI大鼠肺组织的损伤程度,起到肺保护作用。说明缬沙坦对脓毒症导致的ALI有一定的保护作用。
[Abstract]:Objective: Priostin (POSTN) is the animal in vivo osteogenic cells and a cell exocrine protein fiber cells, in a variety of physiological and pathological conditions are expression of pathological conditions is particularly significant. Acute lung injury (Acute Lung Injury.ALI) is a systemic inflammation of lung tissue, a common cause of sepsis sepsis, severe pneumonia and trauma. Improper treatment or not timely increase in patients with acute respiratory distress syndrome (Acute Respiratory Distress Syndrome.ARDS) the incidence and mortality in such patients is higher, the overall strategy of treatment there was no significant efficiency, so the research of acute lung injury is still very necessary. Research that angiotensin II (Angiotensin II.Ang II) receptor antagonist can decrease the expression level of POSTN rats. The study in vivo by using Ang II receptor antagonist valsartan to anti rat Reduced expression of POSTN in lung tissue of ALI rats, to investigate whether valsartan in rats with acute lung can protect septic lung injury. Methods: to establish animal model and group 1.ALI: 45 SPF S-D rats weight distribution of 200-320g randomly divided into blank control group, lipopolysaccharide experimental group (LPS group), low dose valsartan intervention group (10 rats), valsartan dose intervention group (10 rats) and high dose valsartan intervention group (10 rats). The animal weighed record data, the control group was given 1ml sterile saline injected through the tail vein, the other 4 groups per kilogram of weight of LPS dissolved in 1ml 5mg saline through the tail vein injection and valsartan intervention group respectively according to 40mg per kilogram of body weight, 80mg, intraperitoneal injection of 120mg valsartan suspension.2. specimens: rats by the treatment after the normal feeding and watering, model rats were sacrificed 6h after anesthesia, The rat right lung upper lobe on -80 C stored in the refrigerator, for real-time quantitative PCR (RT-PCR) to detect the expression level of Periostin in lung tissue. The lower lobe of the right lung to lung tissue wet weight ratio, the use of hemostatic clamp master tracheal, bronchial and alveolar wash several times repeatedly with phosphate solution, take alveolar lavage (Bronchoalveolar Lavage Fluid.BALF), BALF after centrifugation, the supernatant remained at -20 deg.c for refrigerator, for detection of inflammatory factors. The left lobe of lung tissue in Formaldehyde Solution, Formaldehyde Solution to neutral fixed tissue. HE staining was used to make pathological specimens. From the abdominal vein blood samples were collected and centrifuged after the supernatant was placed in the -80 C refrigerator. IL-13 ELISA was used to detect the inflammatory factor IL-6, TNF-, alpha content in BALF.3. in the wet to dry weight ratio measurement: the removal of the rat right lung lower lobe in the oven, the oven set temperature of 80 DEG C 24h, continuous baking after application of micro electronic balance weight and record.4. histopathological examination: remove the lung tissue completely fixed, embedded in paraffin, stained by HE, the extent of.5. real-time fluorescence quantitative PCR using electron microscopy to observe the inflammatory reaction of lung tissue. The expression of mRNA (Real-Rime PCR) to detect the expression of Periostin Periostin mRNA in the lung tissue in: total mRNA extracted from lung tissues, and then RT-PCR kit provides the product description in the reaction system with various reagents required for the experiment. Setting the reaction conditions of PCR, by reverse transcription loop, after 45 cycles, the reaction system continues for 10 minutes at 72 degrees. According to Gen in rats Bank Periostin (GenBank accession No. NM001108550) and beta -actin (GenBank accession No. NM031144) primers and.Periostin primer is 5 '-AGGAGCCGTGTTTGAGACCAT-3' downstream primers for the 5 '-CG GTGAAAGTGGTTTGCTGTTT-3 'beta -actin primer is 5' -TCCTCCCTGGAGAAGAGCTA-3 'primer is 5' -TCAGGAGGAGCAATGATCTTG-3 '. The reaction results using the method for the relative quantitative analysis in quantitative fluorescence instrument. In order to express beta -actin gene mRNA as reference gene, calculate the relative expression difference according to the formula, namely 2- Delta Ct, Periostin protein the content of the expression of 2- Delta Ct as experimental group compared with the control group the gene expression of multiple.6.Western Blot measurement method in lung tissue: the lung tissue removed in the ice with a tissue grinder for grinding, grinding after the completion of the total protein measurement of lung tissue by manual kit in concentration, unified after concentration, electrophoresis, transmembrane, closed, antibody hybridization, light gel imaging, take the pattern of inflammatory factors IL-4.7. detection by ELISA method, IL-13 and TNF- in BA Analysis of the concentration of.8. in the LF data statistics: experimental data in this study were analyzed by SPSS18 software and statistics. The provisions of P0.05, can be considered statistically significant differences between groups were compared. Results: (a) the right lobe of rats under wet / dry weight ratio and the normal control group (4.01. 1.14) compared to the lungs of rats in LPS group wet / dry weight ratio (5.36 + 0.30) was significantly increased, significant difference was statistically significant (P0.05), the model is made successfully. Valsartan low (4.91 + 0.15), in (4.81 + 0.10), high (4.85 + 0.11) intervention group the difference was not statistically significant (P0.05). Statistical significance of valsartan group and LPS group (P0.05) (two). Histopathological examination of optical microscope was used to observe the pathological sections at high magnification showed normal lung tissue of rats with clear structure, small normal cell, normal alveolar septum, alveolar inflammation Less infiltration of cells, no lung tissue edema. Lung tissue of rats showed alveolar septum thickening obviously disappeared and acute lung injury, hemorrhage and edema tissue, alveolar infiltration of abnormal inflammatory cell. The pathological changes of valsartan group was slightly lower than the LPS group rats. (three) the expression level of the application of PeriostinmRNA method for detection of RT-PCR in lung tissue relative quantitative, with beta -action as a reference gene results in expression of LPS gene in Periostin mRNA group compared to changes in multiple reference genes. Results analysis of LPS experimental group compared with the blank control group, LPS group were increased significantly, significantly. The difference was statistically significant; valsartan group (low, high) and the LPS group relative expression was statistically significant difference. (four) Periostin protein in lung tissue of Western B Detection of Western Blot in lung tissue of lot detection, according to the analysis of gel imaging system detection, the corresponding bands corresponding to the protein content (= samples of the optical density of the bands x area (GAPDH) / light density x area). The content of Periostin protein in lung tissue: compared with the control group, LPS group of rat lung tissue Periostin protein significantly increase (2.48 + 0.07vs0.74 + 0.05, P0.05); compared with the control group, valsartan difference in protein content of Periostin in lung tissue of rats with statistical significance (1.43 + 0.03vs0.74 + 0.05,1.65 + 0.07vs0.74 + 0.05,1.48 + 0.04vs0.74 + 0.05, P0.05), valsartan low, high dose intervention group, the difference was not statistically significant the protein content of Periostin (1.43 + 0.03vs1.65 + 0.07vs1.48 + 0.04, P0.05). (five) IL-13 IL-6 BALF, inflammatory cytokines, TNF- alpha content compared with the control group (170 + 19) pg/ml, IL-6 in rats of LPS group in BALF (310 + 37) pg/ Ml was significantly increased, the difference was statistically significant (P0.05), the intervention group had statistically significant difference compared to the LPS group (P0.05). Compared with the control group (83 + 8) pg/ml, IL-13 in rats of LPS group in BALF (53 + 9) pg/ml decreased significantly, the difference was statistically significant (P0.05). The intervention group was statistically significant difference compared to the LPS group (P0.05). Compared with the control group (149 + 12) pg/ml, TNF- alpha in rats of LPS group BALF (280 + 40) pg/ml increased significantly, the difference was statistically significant (P0.05), the intervention group was statistically significant difference compared to the LPS group (P0.05). Conclusion: the lung tissue of rats with POSTN expression levels increased significantly.2. to valsartan can reduce the expression of POSTN by antagonizing Ang II receptor 1. in acute lung injury, thereby reducing inflammation and edema of lung tissue, is also reduced in lung tissue of rats with ALI damage extent, to lung protection effect of valsartan. It has a certain protective effect on the ALI caused by sepsis.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

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