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

【摘要】：背景:ARDS是嚴重感染、創(chuàng)傷、休克等多種病因下引起肺泡上皮細胞和毛細血管內皮細胞損傷,從而導致急性呼吸衰竭綜合征。近年來針對ARDS的發(fā)病機制和診斷標準進行了深入了研究和不斷更新,其發(fā)病率較前下降,但臨床調查顯示其病死率較高,仍然缺乏有效的治療手段。因此深入探討ARDS的發(fā)病機制并尋求干預是目前研究的重點。ARDS發(fā)病機制復雜,炎性反應是其本質。核因子κB(Nuclear factorκB,NF-κB)是細胞內最重要的核轉錄因子,在多種炎性刺激介導的細胞信號轉錄調控中起著至關重要的作用,NF-κB通道的激活與ARDS的發(fā)生發(fā)展密切相關。前B細胞克隆增強因子(pre-B cell colony-enhancing factor,PBEF)是一種具有多重生理功能的細胞因子,參與了細胞的代謝、炎性反應和免疫調節(jié)功能�，F(xiàn)研究發(fā)現(xiàn)PBEF可通過促進肺泡上皮細胞損傷、肺微血管內皮損傷、肺泡通透性增加參與ARDS的病理生理過程,而抑制PBEF的活性可減輕ARDS炎性反應。研究表明PBEF和NF-κB可作為研究ARDS發(fā)病機制的靶點,為明確二者之間的聯(lián)系,有學者在臍靜脈內皮細胞和肺微血管內皮細胞炎性反應中做了探索性研究,目前尚不清楚PBEF和NF-κB二者在肺泡上皮細胞炎性反應中的具體聯(lián)系。川芎嗪(Tetramethylpyrazine,TMP)是從中藥川芎根中提取的活性成分,具有鈣離子通道拮抗劑作用,臨床上廣泛應用于心腦血管疾病。有研究發(fā)現(xiàn)TMP能通過抑制肺泡巨噬細胞NF-κB活化減輕家兔失血性休克合并內毒素誘發(fā)的急性肺損傷;該課題前期實驗發(fā)現(xiàn)在油酸誘導的大鼠ARDS模型中,TMP能下調PBEF等炎性因子的表達,發(fā)揮肺保護作用。但目前TMP在ARDS炎性反應中的具體作用機制并不清晰。目的:(1)體外培養(yǎng)人II型肺泡上皮細胞(A549細胞,來源于人肺腺癌),觀察LPS和TMP對A549細胞增殖活性的影響,篩選下一步實驗選用的LPS和TMP濃度。(2)體外探討LPS作用于A549細胞后PBEF和其他炎癥因子的變化。(3)探討PBEF與其他炎癥因子的聯(lián)系以及和NF-κB的關聯(lián)。(4)探討TMP對LPS誘導的A549炎性反應的保護作用及機制。方法:(1)體外培養(yǎng)人II型肺泡上皮細胞(A549細胞,來源于人肺腺癌),MTT法檢測LPS和TMP藥物對A549細胞的增殖作用的影響,并選擇LPS造模的最佳濃度和TMP作用于A549細胞的安全濃度范圍。(2)設正常對照組(Control)、LPS組、LPS+TMP組和LPS+FK866組。LPS(終濃度5mg/L)刺激A549細胞6h、12h和24h后建立炎癥模型,分別加入TMP(終濃度10mg/L)和PBEF抑制劑FK866(終濃度10nmol/L)進行干預,q-PCR和Western Blot分別檢測炎癥因子腫瘤壞死因子α(tumor necrosis factor,TNF-a)、白細胞介素-1β(interleukin-1,IL-1β)、白細胞介素-8(interleukin-8,IL-8)、PBEF的m RNA和蛋白表達水平。(3)通過Western Blot檢測3個時間段的細胞核和細胞質內磷酸化P65蛋白水平動態(tài)變化來反映NF-κB的激活情況。結果:(1)LPS對A549細胞增殖影響在同一時間點,6h的5mg/L和10mg/L、20mg/L和50mg/L實驗組間細胞抑制率(Inhibition rate,IR)比較差異無統(tǒng)計學意義(P0.05),其余實驗組在同一時間段間兩兩比較差異有統(tǒng)計學意義(P0.05);1mg/L LPS刺激6h、12h和24h后IR差異無統(tǒng)計學意義(P0.05),其余實驗組在任何時間點組間兩兩比較差異有統(tǒng)計學意義(P0.05)。(2)TMP對A549細胞增殖影響在同一時間點,10mg/L和50mg/L的TMP對A549細胞(survival rate,SR)比較差異不具有統(tǒng)計意義(P0.05),6h的200mg/L和300mg/L實驗組比較差異不具有統(tǒng)計學意義(P0.05),其余實驗組在同一時間組間亮亮比較差異具有統(tǒng)計學意義(P0.05);在不同時間點,10mg/L和50mg/L實驗組差異不具有統(tǒng)計學意義(P0.05),200mg/L和300mg/L實驗組在12h和24h組間比較差異不具有統(tǒng)計學意義(P0.05),其余實驗組在任何時間點組間比較差異有統(tǒng)計學意義(P0.05)。(3)藥物干預對TNF-a、IL-1β、IL-8和PBEFm RNA表達的影響LPS刺激A549細胞后TNF-a、IL-1β、IL-8和PBEF炎癥因子m RNA的表達均較對照組明顯增高(P0.001),并隨著作用時間延長LPS組間比較明顯升高(P0.001)。FK866干預后TNF-a、IL-1β和IL-8m RNA的表達較同一時間段內LPS組降低(P0.05)。TMP干預后TNF-a、IL-1β、IL-8和PBEFm RNA的表達較同一時間段內LPS組降低(P0.05)。(4)藥物干預對TNF-a、IL-1β、IL-8和PBEF蛋白表達的影響LPS作用于A549細胞后TNF-a、IL-1β、IL-8和PBEF蛋白較對照組表達升高(P0.05),隨著作用時間延長,上述炎癥因子蛋白表達升高,LPS組在12h和24h與6h比較差異有統(tǒng)計學意義(P0.05)。在加入FK866干預后TNF-a、IL-1β和IL-8蛋白表達較同一時間段內LPS組下降(P0.05),TMP干預后TNF-a、IL-1β、IL-8和PBEF蛋白的表達較同一時間段內LPS組降低(P0.05)。(5)藥物干預對NF-κB活性的影響LPS刺激A549細胞后后細胞核磷酸化P65蛋白表達較對照組顯著增高(3個時間段均P0.001),并隨著作用時間延長逐漸升高,LPS組間兩兩比較差異具有統(tǒng)計學意義(P0.05)。在FK866干預后細胞核磷酸化P65蛋白較LPS組明顯下降(3個時間段均P0.001),上述指標在TMP干預后也較LPS組降低(P0.01)。LPS刺激A549細胞后后細胞質磷酸化P65蛋白表達較對照組顯著增高(3個時間段均P0.001),隨著作用時間延長磷酸化P65蛋白逐漸開始下降,LPS組間兩兩比較差異具有統(tǒng)計學意義(P0.05)。在FK866干預后細胞質磷酸化P65蛋白較LPS組下降(P0.01),不同時間段FK866干預組間兩兩比較差異具有統(tǒng)計學意義(P0.05)。TMP干預后同一時間段細胞質磷酸化P65蛋白也較LPS組降低(P0.05),在6h和24h時間段TMP組間比較差異具有統(tǒng)計學意義(P0.05)。結論:(1)LPS對A549細胞增殖抑制呈劑量-時間依賴效應。(2)低濃度時TMP對A549細胞增殖無影響,100~300 mg/L時則抑制生長。(3)PBEF可通過NF-κB參與調控LPS誘導的A549細胞炎性因子TNF-a、IL-1β和IL-8的表達和釋放。(4)TMP能降低LPS誘導A549細胞的PBEF的表達和NF-κB的活性,并降低炎癥因子TNF-a、IL-1β和IL-8的表達和釋放。因此,TMP可能通過降低PBEF的表達,抑制NF-κB的活性,從而減輕II型肺泡上皮細胞炎性反應,發(fā)揮ARDS保護作用。
[Abstract]:Background: ARDS is a serious infection, trauma, shock and other causes that cause damage to alveolar epithelial cells and capillary endothelial cells, resulting in acute respiratory failure syndrome. In recent years, the pathogenesis and diagnostic criteria of ARDS have been deeply studied and updated, the incidence of which is lower than before, but the clinical investigation shows its disease. The death rate is high and still lacks effective treatment. Therefore, it is very complex to explore the pathogenesis of ARDS and to seek intervention. The inflammatory response is its essence. Nuclear factor kappa B (Nuclear factor kappa B, NF- kappa B) is the most important nuclear transcription factor in the cell, and it is mediated by a variety of inflammatory stimuli. Regulation plays a vital role. The activation of NF- kappa B channel is closely related to the development of ARDS. The pre B cell clone enhancement factor (pre-B cell colony-enhancing factor, PBEF) is a cytokine with multiple physiological functions and participates in cell metabolism, inflammatory response and immunoregulation. The present study found PBEF can be passed. Promoting alveolar epithelial cell injury, pulmonary microvascular endothelial damage and increased alveolar permeability are involved in the pathophysiological process of ARDS, and the inhibition of PBEF activity can reduce the ARDS inflammatory response. The study shows that PBEF and NF- kappa B can be used as a target for the study of the pathogenesis of ARDS, and to clarify the association between the two and some scholars in the umbilical vein endothelial cells and pulmonary Microblood. PBEF and NF- kappa B two are not clearly understood in the inflammatory response of endothelium cells. Ligustrazine (Tetramethylpyrazine, TMP) is an active ingredient extracted from Ligusticum chuanxiong root, which has calcium ion channel antagonist, and is widely used in cardiovascular and cerebrovascular diseases. Some studies have found that TMP can reduce the activation of NF- kappa B in the alveolar macrophages to alleviate acute lung injury induced by hemorrhagic shock and endotoxin in rabbits. In the earlier experiment, it was found that in the ARDS model of oleic acid induced rat, TMP could reduce the expression of PBEF and other inflammatory factors and play the role of lung protection. But at present, TMP is in the inflammatory response of ARDS. The specific mechanism is not clear. Objective: (1) in vitro culture of human type II alveolar epithelial cells (A549 cells, derived from human lung adenocarcinoma), observe the effects of LPS and TMP on the proliferation of A549 cells, select the LPS and TMP concentrations in the next experiment. (2) to explore the changes in PBEF and other inflammatory factors after the action of LPS in A549 cells in vitro. (3) to explore PBEF Association with other inflammatory factors and the association with NF- kappa B. (4) to explore the protective effect and mechanism of TMP on LPS induced A549 inflammatory response. Methods: (1) human II alveolar epithelial cells (A549 cells, derived from human lung adenocarcinoma) were cultured in vitro. MTT assay was used to detect the effect of LPS and TMP drugs on the proliferation of A549 cells, and selected the best LPS model. The concentration and TMP effect on the safe concentration range of A549 cells. (2) the normal control group (Control), the LPS group, the LPS+TMP group and the LPS+FK866 group.LPS (final concentration 5mg/L) stimulated A549 cell 6h, 12h and 24h to establish the inflammatory model. Detection of inflammatory factor tumor necrosis factor (TNF-a), interleukin -1 beta (interleukin-1, IL-1 beta), interleukin -8 (interleukin-8, IL-8), PBEF m and protein expression levels. (3) the dynamic changes of phosphorylated protein levels in the nucleus and cytoplasm of the 3 time periods were detected to reflect the nuclear kappa Results: (1) the effects of LPS on the proliferation of A549 cells at the same time point, 5mg/L and 10mg/L of 6h, and the cell inhibition rate (Inhibition rate, IR) between the 20mg/L and 50mg/L experimental groups were not statistically significant (P0.05), and the other experimental groups were statistically significant in the same time period. There was no statistically significant difference in IR after 4H (P0.05), and in the rest of the experimental groups, there was a significant difference between 22 groups in any time point group (P0.05). (2) the effect of TMP on A549 cell proliferation at the same time point, 10mg/L and 50mg/L TMP to A549 cells (survival rate) was not statistically significant. The difference was not statistically significant (P0.05), and the difference between the other experimental groups was statistically significant (P0.05) at the same time. The difference between 10mg/L and 50mg/L was not statistically significant (P0.05) at different time points (P0.05), and there was no statistical difference between the 200mg/L and 300mg/L groups in the 12h and 24h groups (P0.05), but the rest were not statistically significant (P0.05). There were significant differences between the experimental groups at any time point (P0.05). (3) the effects of drug intervention on the expression of TNF-a, IL-1 beta, IL-8 and PBEFm RNA were significantly higher than those of the control group after LPS stimulation of A549 cells, and the expression of IL-1 beta, IL-8 and PBEF inflammatory factors increased significantly. After.FK866, the expression of IL-1 beta and IL-8m RNA was lower than that of LPS group in the same time period (P0.05).TMP intervention. The expression of TNF-a, IL-1 beta, IL-8 and PBEFm decreased in the same time period. (4) the effect of drug intervention on the expression of beta, beta, and egg white in the same time period. The expression of white was higher than that in the control group (P0.05). The expression of the above inflammatory factor protein increased with the prolongation of the action time. There was a significant difference between the 12h and 24h in the LPS group and 6h (P0.05). The expression of IL-1 beta and IL-8 protein was lower than that in the LPS group (P0.05). The expression of LPS group was lower than that of the same time period (P0.05). (5) the effect of drug intervention on the activity of NF- kappa B was significantly higher than that of the control group (P0.001) after LPS stimulated A549 cells (all 3 time periods were P0.001), and with the prolongation of action time, the difference between 22 of LPS groups was statistically significant (P0.05). After intervention, the nuclear phosphorylation P65 protein was significantly lower than that in the LPS group (3 time periods P0.001), and the above indexes were also lower than those in the LPS group after TMP intervention (P0.01).LPS stimulated A549 cells after A549 cells, and the expression of P65 protein in the cytoplasm was significantly higher than that in the control group (3 periods of P0.001). As the time extended the phosphorylation P65 protein gradually began to begin. The difference between the 22 groups in the LPS group was statistically significant (P0.05). After FK866 intervention, the cytoplasmic phosphorylation P65 protein was lower than that in the LPS group (P0.01), and the difference between the FK866 intervention groups at different time periods was statistically significant (P0.05), and the cytoplasmic phosphorylation P65 protein was also lower than the LPS group (P0.05) at the same time after the intervention of.TMP (P0.05). The difference between the TMP groups in the time period was statistically significant (P0.05). Conclusion: (1) LPS has a dose time dependence effect on the proliferation inhibition of A549 cells. (2) TMP has no effect on the proliferation of A549 cells at low concentration, while 100~300 mg/L inhibits the growth. (3) PBEF can be involved in regulating LPS induced inflammatory cytokines through NF- kappa B. (4) (4) TMP can reduce the expression of PBEF and the activity of NF- kappa B in A549 cells, and reduce the expression and release of inflammatory factors TNF-a, IL-1 beta and IL-8. Therefore, TMP may reduce the activity of NF- kappa by reducing PBEF expression, thus alleviated the inflammatory response of alveolar epithelial cells and played a protective role.
【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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