發(fā)布時間：2018-04-10 16:34

  本文選題：川芎嗪 + 急性呼吸窘迫綜合征��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的水通道蛋白是一組與液體平衡有關(guān)的細胞膜轉(zhuǎn)運蛋白,大量證據(jù)表明AQPs與ARDS肺水的代謝和轉(zhuǎn)運密切相關(guān),其中AQP1和AQP5對維持肺內(nèi)外液體平衡共同發(fā)揮著重要作用。川芎嗪的有效成分是TMP,研究業(yè)已證實其具有緩解肺水腫的藥理作用,但TMP對肺水腫的改善作用是否與水通道蛋白1、5密切相關(guān),目前尚不清楚。為了證實這一假設,我們通過細胞和動物兩個層面,構(gòu)建ARDS的體外和體內(nèi)模型,觀察川芎嗪對急性呼吸窘迫綜合征非心源性肺水腫的影響,并探討水通道蛋白1、5表達的調(diào)節(jié)是否為其作用的可能靶點之一。方法構(gòu)建ARDS的體外和體內(nèi)模型,將A549細胞和SD大鼠隨機分為正常對照組(N組)、TMP處理組(T組)、LPS刺激組(L組)、TMP治療組(LT組)。MTT法檢測細胞體外增殖,實時定量PCR技術(shù)(RT-q PCR)檢測各組細胞AQP1、AQP5m RNA水平。通過觀察大鼠的一般情況及動脈血氣分析證實造模是否成功,肺組織病理學改變和濕/干重比驗證肺水腫變化,RT-q PCR檢測各組大鼠肺組織AQP1和AQP5m RNA水平,免疫組化和免疫印跡技術(shù)(Western blot)檢測AQP1、AQP5蛋白表達。結(jié)果經(jīng)MTT法檢測LPS和TMP對A549細胞增殖的影響后,確定LPS的最佳作用濃度為5-10mg/L、TMP的安全范圍為10-50mg/L。觀察大鼠一般情況及動脈血氣分析結(jié)果證實尾靜脈注射10mg/kg LPS復制ARDS模型成功,通過肺組織病理改變和濕/干重比觀察到TMP處理組肺水腫緩解。RT-q PCR檢測各組細胞和大鼠肺組織AQP1、AQP5m RNA結(jié)果顯示TMP處理組AQP1和AQP5m RNA水平高于對照組(P0.05);而LPS刺激組兩者水平降低(P0.05);同時,與LPS刺激組相比,TMP治療組AQP1、AQP5轉(zhuǎn)錄水平上升,但低于N、T兩組,差異有統(tǒng)計學意義(P0.05)。免疫組化和Western blot檢測各組大鼠肺組織AQP1、AQP5蛋白表達情況與m RNA水平一致。結(jié)論川芎嗪可有效緩解ARDS時的非心源性肺水腫,其可能機制之一或與上調(diào)受抑制的AQP1和AQP5表達有關(guān),水通道蛋白1、5可能作為ARDS治療的其中一個潛在靶點。
[Abstract]:Objective aquaporins are a group of membrane transporters related to fluid balance. There is a great deal of evidence that AQPs is closely related to the metabolism and transport of lung water in ARDS. Among them, AQP1 and AQP5 play an important role in maintaining the fluid balance inside and outside the lung.The effective component of ligustrazine is TMP, which has been proved to have pharmacological effects on pulmonary edema. However, it is not clear whether the amelioration of TMP on pulmonary edema is closely related to aquaporin 1 / 5.To verify this hypothesis, we established in vitro and in vivo models of ARDS at both cellular and animal levels to observe the effects of ligustrazine on non-cardiogenic pulmonary edema in patients with acute respiratory distress syndrome.And to explore whether the regulation of aquaporin 1 5 expression is one of the possible targets.Methods the model of ARDS in vitro and in vivo was established. A549 cells and SD rats were randomly divided into two groups: normal control group (n group) and control group (n group).Real time quantitative PCR technique was used to detect the AQP5 RNA level in the cells of each group.By observing the general situation of rats and arterial blood gas analysis, we confirmed the success of the model. Lung histopathology and wet / dry weight ratio were used to verify the changes of pulmonary edema. RT-q PCR was used to detect the levels of AQP1 and AQP5m RNA in lung tissue of rats in each group.The expression of AQP1 and AQP5 protein was detected by immunohistochemistry and Western blotting.Results after the effects of LPS and TMP on the proliferation of A549 cells were detected by MTT assay, the optimal concentration of LPS was determined to be 5-10 mg / L ~ 10 mg / L ~ (-1) 10 ~ (-50) mg 路L ~ (-1) 路L ~ (-1).Observing the general situation of rats and the results of arterial blood gas analysis confirmed that the model of ARDS was successfully established by injecting 10mg/kg LPS into caudal vein.Lung tissue pathological changes and wet / dry weight ratio were observed in TMP treated group. RT-q PCR was used to detect the levels of AQP1 and AQP5m RNA in the cells of each group and the lung tissue of rats. The results showed that the levels of AQP1 and AQP5m RNA in the TMP treated group were higher than those in the control group (P 0.05), while in the LPS stimulated group, the levels of AQP1 and AQP5m RNA were higher than those in the control group (P 0.05).At the same time,Compared with the LPS stimulation group, the AQP1AAQP5 transcription level in the TMPtreated group was increased, but lower than that in the NT-treated group. The difference was statistically significant (P 0.05).Immunohistochemical staining and Western blot were used to detect the expression of AQP1 and AQP5 protein in lung tissue of rats. The expression of AQP5 protein was consistent with the level of m RNA.Conclusion Ligustrazine can effectively relieve non-cardiogenic pulmonary edema in patients with ARDS, which may be related to up-regulation of the expression of AQP1 and AQP5. Aquaporin 1 / 5 may be one of the potential targets for the treatment of ARDS.
【學位授予單位】：重慶醫(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：1732070