本文選題：imipramine　切入點：緊密連接蛋白　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的急性肺損傷(Acute Lung Injury,ALI)/急性呼吸窘迫綜合癥(Acute Respiratory Distress Syndrome,ARDS)均是呼吸內(nèi)科常見的危重疾患,且具有較高的病死率。目前,臨床上仍缺乏有效的治療措施。在ALI發(fā)生發(fā)展中最顯著的病理變化是彌漫性肺泡上皮細胞損傷,因此,增強肺泡上皮細胞屏障功能可成為治療ALI一個靶點。Imipramine是常見的三環(huán)類抗抑郁藥,也是動物實驗中常用的酸性鞘磷脂酶(Acid Sphingomyelinase,ASMase)生物活性抑制劑,其具有抗炎、抗凋亡的作用。本文主要探討imipramine對脂多糖(LPS)誘導的小鼠ALI肺泡上皮屏障功能是否有保護作用,并探討其可能機制。方法將SPF級雄性Balb/c小鼠隨機分為4組,即正常對照組、Imipramine組、LPS組、LPS+Imipramine組。采用LPS(20mg/kg)腹腔注射建立小鼠ALI模型,并在腹腔注射LPS前30分鐘給予imipramine(25mg/kg)干預,處死前10 min,給予尾靜脈注射FITC-FD4,12 h后麻醉下放血處死小鼠,隨后取出支氣管肺泡灌洗液(BALF)及肺組織。常規(guī)HE染色觀察肺組織病理變化及病理學評分;肺組織濕/干比(W/D)和BALF/血清FD4比值的測定分別評估肺水腫和肺泡上皮細胞通透性;采用real-time PCR、Western blot和免疫組織化學方法檢測不同組緊密連接蛋白Occludin、Claudin-4和ZO-1的表達情況;免疫熒光法檢測每組ASMase的生物活性。數(shù)據(jù)分析采用SPSS 16.0軟件,多組間的均數(shù)比較采用單因素方差分析,組間兩兩比較采用LSD-t檢驗。結果腹腔注射LPS誘導小鼠ALI,模型簡單、穩(wěn)定且均勻,肺組織病理學變化符合ALI病理改變。正常對照組和Imipramine組小鼠肺組織病理學無明顯變化,LPS+Imipramine組肺組織病理學評分小于LPS組,差異具有統(tǒng)計學意義(P0.001);LPS+Imipramine組肺組織濕/干比小于LPS組,差異具有統(tǒng)計學意義(P=0.001);LPS+Imipramine組較LPS組相比,BALF/血清FD4比值下降差異具有統(tǒng)計學意義P=0.002);與LPS組相比,LPS+Imipramine組小鼠肺緊密連接蛋白Occludin、Claudin-4、ZO-1 m RNA及蛋白的表達水平均升高(P0.05);免疫組化結果顯示,緊密連接蛋白Occludin、Claudin-4主要位于肺泡上皮細胞膜,ZO-1主要位于肺泡上皮細胞胞漿內(nèi),在正常對照組和Imipramine組蛋白表達明顯,LPS組蛋白表達降低(P0.05),LPS+Imipramine組較LPS組蛋白表達有所恢復(P0.05);免疫熒光法檢測ASMase活性結果顯示,LPS+Imipramine組較LPS組相比,ASMase活性降低,差異具有統(tǒng)計學意義(P0.05),說明Imipramine能抑制應激狀態(tài)下ASMase的活性,進而減少神經(jīng)酰胺的生成。結論通過腹腔注射LPS建立小鼠ALI在ALI動物模型中值得推廣;Imipramine可改善小鼠ALI病理變化,降低肺水腫程度;Imipramine可能通過抑制應激狀態(tài)下ASMase的活性,減少神經(jīng)酰胺的生成,進而改善肺泡上皮細胞緊密連接蛋白的表達以增強肺泡上皮細胞的屏障功能,最終保護LPS誘導的小鼠ALI。
[Abstract]:Objective Acute Lung injury / acute Respiratory Distress Syndrome ARDS are common serious diseases in respiratory medicine department, and have high mortality. The most significant pathological change in the development of ALI is diffuse alveolar epithelial cell injury. Enhancing the function of alveolar epithelial cell barrier is a target of ALI. Imipramine is a common tricyclic antidepressant and a bioactive inhibitor of acid sphingolipase acid sphingomyelinase (ASMase). To investigate the protective effect of imipramine on ALI alveolar epithelial barrier induced by lipopolysaccharide (LPS) and its possible mechanism. Methods male Balb/c mice of SPF grade were randomly divided into 4 groups. The ALI model of mice was established by intraperitoneal injection of LPS-20 mg / kg. The mice were treated with imipramine 25mg / kg 30 minutes before intraperitoneal injection of LPS. The mice were sacrificed 10 min before death and anesthetized with FITC-FD412 h after intravenous injection of FITC-FD412 h. Then the bronchoalveolar lavage fluid (BALF) and lung tissue were taken out. The pathological changes and pathological scores of lung tissue were observed by routine HE staining, the lung tissue wet / dry ratio (WR / D) and the ratio of serum FD4 of BALF/ were measured to evaluate pulmonary edema and alveolar epithelial cell permeability, respectively. Real-time PCR Western blot and immunohistochemistry were used to detect the expression of cladin-4 and ZO-1 in different groups, and immunofluorescence assay was used to detect the bioactivity of ASMase in each group. The data were analyzed by SPSS 16.0 software. Single factor analysis of variance (ANOVA) and LSD-t test were used to compare the mean of multiple groups. Results the model was simple, stable and uniform in mice induced by intraperitoneal injection of LPS. The lung histopathological changes were consistent with the pathological changes of ALI in normal control group and Imipramine group. The lung histopathological score in Imipramine group was lower than that in LPS group, and the difference was statistically significant (P 0.001). The wet / dry ratio of lung tissue in LPs Imipramine group was lower than that in LPS group. Compared with LPS group, the ratio of BALF-1 / serum FD4 in LPS-LPs group was significantly lower than that in LPS group, and the expression of clondin-4ZO-1 m RNA and protein in LPS-LPs Imipramine group was significantly higher than that in LPS-#en4# group (P 0.05). Claudin-4 is mainly located in the cytoplasm of alveolar epithelial cells, and ZO-1 is mainly located in the cytoplasm of alveolar epithelial cells. In the normal control group and the Imipramine histone expression group, the expression of LPS histone protein was significantly decreased in the Imipramine group compared with that in the LPS group, and the results of immunofluorescence assay showed that the ASMase activity in the LPS-#en4# group was lower than that in the LPS group. The difference was statistically significant (P 0.05), which indicated that Imipramine could inhibit the activity of ASMase and decrease the production of ceramide. Conclusion the establishment of mouse ALI by intraperitoneal injection of LPS is worth popularizing in ALI animal model, which can improve the pathological changes of ALI in mice. Reducing the degree of pulmonary edema and imipramine may enhance the barrier function of alveolar epithelial cells by inhibiting the activity of ASMase and reducing the production of ceramide, and then improving the expression of tight junction protein in alveolar epithelial cells. The final protection of LPS induced ALI in mice.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R563.8
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本文編號：1584275