川芎嗪对脂多糖诱导急性呼吸窘迫综合征水通道蛋白1、5表达影响的研究

发布时间：2018-04-10 16:34

本文选题：川芎嗪 + 急性呼吸窘迫综合征　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的水通道蛋白是一组与液体平衡有关的细胞膜转运蛋白,大量证据表明AQPs与ARDS肺水的代谢和转运密切相关,其中AQP1和AQP5对维持肺内外液体平衡共同发挥着重要作用。川芎嗪的有效成分是TMP,研究业已证实其具有缓解肺水肿的药理作用,但TMP对肺水肿的改善作用是否与水通道蛋白1、5密切相关,目前尚不清楚。为了证实这一假设,我们通过细胞和动物两个层面,构建ARDS的体外和体内模型,观察川芎嗪对急性呼吸窘迫综合征非心源性肺水肿的影响,并探讨水通道蛋白1、5表达的调节是否为其作用的可能靶点之一。方法构建ARDS的体外和体内模型,将A549细胞和SD大鼠随机分为正常对照组(N组)、TMP处理组(T组)、LPS刺激组(L组)、TMP治疗组(LT组)。MTT法检测细胞体外增殖,实时定量PCR技术(RT-q PCR)检测各组细胞AQP1、AQP5m RNA水平。通过观察大鼠的一般情况及动脉血气分析证实造模是否成功,肺组织病理学改变和湿/干重比验证肺水肿变化,RT-q PCR检测各组大鼠肺组织AQP1和AQP5m RNA水平,免疫组化和免疫印迹技术(Western blot)检测AQP1、AQP5蛋白表达。结果经MTT法检测LPS和TMP对A549细胞增殖的影响后,确定LPS的最佳作用浓度为5-10mg/L、TMP的安全范围为10-50mg/L。观察大鼠一般情况及动脉血气分析结果证实尾静脉注射10mg/kg LPS复制ARDS模型成功,通过肺组织病理改变和湿/干重比观察到TMP处理组肺水肿缓解。RT-q PCR检测各组细胞和大鼠肺组织AQP1、AQP5m RNA结果显示TMP处理组AQP1和AQP5m RNA水平高于对照组(P0.05);而LPS刺激组两者水平降低(P0.05);同时,与LPS刺激组相比,TMP治疗组AQP1、AQP5转录水平上升,但低于N、T两组,差异有统计学意义(P0.05)。免疫组化和Western blot检测各组大鼠肺组织AQP1、AQP5蛋白表达情况与m RNA水平一致。结论川芎嗪可有效缓解ARDS时的非心源性肺水肿,其可能机制之一或与上调受抑制的AQP1和AQP5表达有关,水通道蛋白1、5可能作为ARDS治疗的其中一个潜在靶点。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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