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  本文關鍵詞：纈沙坦通過抑制periostin的表達對脂多糖誘導的急性肺損傷大鼠的肺保護作用　出處：《大連醫(yī)科大學》2016年碩士論文　論文類型：學位論文

  更多相關文章： periostin 急性肺損傷 纈沙坦 AngⅡ受體拮抗劑

【摘要】：目的:Priostin(POSTN)是動物體內(nèi)成骨細胞和成纖維細胞分泌的一種細胞外分泌蛋白,在多種生理及病理狀態(tài)下均會表達,病理狀態(tài)下尤為顯著。急性肺損傷(Acute Lung Injury.ALI)是一種肺組織的系統(tǒng)性炎癥,常見原因有膿毒癥、重癥肺炎及創(chuàng)傷等。治療不當或不及時可增加患者急性呼吸窘迫綜合癥(Acute Respiratory Distress Syndrome.ARDS)的發(fā)生率,且此類患者的死亡率較高,目前尚無顯著高效的總體治療策略,因此對于急性肺損傷的研究仍然有很強的必要性。有研究表明血管緊張素Ⅱ(AngiotensinⅡ.AngⅡ)受體拮抗劑能降低大鼠體內(nèi)POSTN表達水平。本研究通過在大鼠體內(nèi)應用AngⅡ受體拮抗劑纈沙坦來降低ALI大鼠肺組織POSTN的表達,來探討纈沙坦是否能夠保護膿毒癥導致的急性肺損傷大鼠的肺臟。方法:1.ALI模型建立及動物分組:體重分布200-320g的45只SPF級S-D大鼠按照隨機數(shù)表法分為空白對照組,脂多糖實驗組(LPS組),纈沙坦低量干預組(10只)、纈沙坦中劑量干預組(10只)和纈沙坦高劑量干預組(10只)。動物稱重后記錄數(shù)據(jù),對照組給予1ml無菌生理鹽水經(jīng)大鼠尾靜脈注入,其余4組按照每公斤體重5mg LPS溶入1ml生理鹽水中經(jīng)大鼠尾靜脈注入,纈沙坦干預組分別按照每公斤體重40mg、80mg、120mg腹腔注射纈沙坦混懸液。2.標本留取:各組大鼠經(jīng)上述處置后正常喂食喂水,模型建立后6h麻醉處死大鼠,取大鼠右肺上葉放在-80℃冰箱中保存,用于實時定量PCR(RT-PCR)來檢測肺組織periostin的表達程度。右肺下葉用于測肺組織干濕重比,用止血鉗夾住主氣管,用磷酸鹽溶液反復沖洗支氣管及肺泡多次,留取肺泡灌洗液(Bronchoalveolar Lavage Fluid.BALF),BALF離心后留取上清于-20℃冰箱中保存,留作炎癥因子檢測。將左葉肺組織放入中性甲醛溶液中,中性甲醛溶液起到組織固定作用。用HE染色制作組織病理標本。從腹主靜脈抽取血液,經(jīng)離心機離心后取上清液置于-80℃冰箱中。用ELISA法檢測炎癥因子IL-6、IL-13、TNF-α在BALF中的含量。3.濕干重比的測量:將取出的大鼠右肺下葉放入烤箱中,烤箱設置溫度為80℃,持續(xù)烘烤24h后,應用微量電子稱稱重并記錄。4.組織病理學檢測:取出固定完全的肺組織,常規(guī)石蠟包埋,采用HE染色,應用電子顯微鏡觀察肺部組織炎癥反應的程度。5.實時熒光定量PCR(Real-Rime PCR)檢測periostin mRNA在肺組織中的表達Periostin mRNA的表達:首先提取肺組織中的總mRNA,然后根據(jù)RT-PCR試劑盒提供的產(chǎn)品說明書在反應體系內(nèi)加入實驗所需的各種試劑。設定PCR的反應條件,進行逆轉(zhuǎn)錄循環(huán),循環(huán)45次后,反應體系在72℃繼續(xù)延伸10分鐘。根據(jù)Gen Bank中大鼠的periostin(基因登錄號NM001108550)及β-actin(基因登錄號NM031144)序列設計引物。Periostin上游引物為5’-AGGAGCCGTGTTTGAGACCAT-3’下游引物為5’-CGGTGAAAGTGGTTTGCTGTTT-3’β-actin上游引物為5’-TCCTCCCTGGAGAAGAGCTA-3’下游引物為5’-TCAGGAGGAGCAATGATCTTG-3’。將反應結(jié)果采用相對定量的方法在熒光定量儀進行分析。以β-actin基因mRNA的表達為內(nèi)參基因,根據(jù)公式計算相對表達量的差值,即為2-△△Ct,2-△△Ct的意義為實驗組的目的基因的表達相對于對照組的表達倍數(shù)。6.Western Blot方法測量肺組織中periostin蛋白含量:將肺組織取出后在冰面上用組織研磨器進行研磨,研磨完成后,應用試劑盒提供的說明書測量肺組織中總蛋白得濃度,統(tǒng)一濃度后,進行電泳,轉(zhuǎn)膜,封閉,抗體雜交,發(fā)光后凝膠成像,采取圖樣。7.用ELISA法檢測炎癥因子IL-4、IL-13及TNF-α在BALF中的濃度。8.數(shù)據(jù)統(tǒng)計分析:本研究中實驗數(shù)據(jù)均采用SPSS18軟件進行統(tǒng)計學分析及處理。規(guī)定P0.05時,可認為比較各組間的差異具有統(tǒng)計學意義。結(jié)果:(一)各組大鼠右下肺葉的濕/干重比值與正常對照組(4.01±1.14)相比,LPS組大鼠肺組織濕/干重比(5.36±0.30)明顯升高,差異顯著,有統(tǒng)計學意義(p0.05),說明模型的制作是成功的。纈沙坦低(4.91±0.15)、中(4.81±0.10)、高(4.85±0.11)干預組之間差異無統(tǒng)計學意義(p0.05)。纈沙坦干預組與LPS組差異具有統(tǒng)計學意義(p0.05)。(二)組織病理學檢測用光學顯微鏡在高倍鏡下觀察病理切片,可見正常組大鼠肺組織結(jié)構(gòu)清晰,細胞大小正常,肺泡間隔正常,肺泡腔內(nèi)炎癥細胞的浸潤較少,無肺組織明顯水腫。急性肺損傷組大鼠肺組織可見肺泡間隔明顯消失并呈明顯增厚表現(xiàn),組織可見大量出血點及水腫,肺泡腔內(nèi)可見異常大量炎癥細胞的浸潤。纈沙坦干預組上述病理改變比LPS組大鼠表現(xiàn)有輕度減少。(三)RT-PCR檢測肺組織PeriostinmRNA的表達水平應用相對定量的計算辦法,以β-action為內(nèi)參基因,結(jié)果以LPS組目的基因periostin mRNA的表達相對于內(nèi)參基因的變化倍數(shù)表示。結(jié)果分析,LPS實驗組相對于空白對照組比較,LPS組結(jié)果升高明顯,差異顯著,差異具有統(tǒng)計學意義;纈沙坦干預組(低、中、高)與LPS組相對表達量差異具有統(tǒng)計學意義。(四)肺組織Periostin蛋白Western Blot檢測肺組織Western Blot檢測,根據(jù)凝膠成像分析系統(tǒng)檢測,其相應條帶對應蛋白含量=(樣品條帶的光密度×面積)/(GAPDH的光密度×面積)。肺組織periostin蛋白含量:相對于對照組,LPS組大鼠肺組織periostin蛋白含量明顯增高(2.48±0.07vs0.74±0.05,p0.05);相對于對照組,纈沙坦干預組大鼠肺組織periostin蛋白含量差異具有統(tǒng)計學意義(1.43±0.03vs0.74±0.05、1.65±0.07vs0.74±0.05、1.48±0.04vs0.74±0.05,p0.05),纈沙坦低、中、高劑量干預組組間比較,periostin蛋白含量差異無統(tǒng)計學意義(1.43±0.03vs1.65±0.07vs1.48±0.04,p0.05)。(五)BALF中炎癥因子IL-6、IL-13、TNF-α的含量與對照組相比(170±19)pg/ml,LPS組大鼠的BALF中IL-6的含量(310±37)pg/ml升高明顯,差異有統(tǒng)計學意義(p0.05),干預組與LPS組相比差異有統(tǒng)計學意義(p0.05)。與對照組相比(83±8)pg/ml,LPS組大鼠的BALF中IL-13的含量(53±9)pg/ml降低明顯,差異有統(tǒng)計學意義(p0.05),干預組與LPS組相比差異有統(tǒng)計學意義(p0.05)。與對照組相比(149±12)pg/ml,LPS組大鼠的BALF中TNF-α含量(280±40)pg/ml呈明顯升高,差異有統(tǒng)計學意義(p0.05),干預組與LPS組相比差異有統(tǒng)計學意義(p0.05)。結(jié)論:1.急性肺損傷大鼠肺組織中的POSTN表達水平明顯升高。2.纈沙坦可以通過拮抗AngⅡ受體來降低POSTN的表達,從而降低肺組織的炎癥反應及水腫程度,也就是降低了ALI大鼠肺組織的損傷程度,起到肺保護作用。說明纈沙坦對膿毒癥導致的ALI有一定的保護作用。
[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.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R563

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