【摘要】：目的 研究補(bǔ)腎組分淫羊藿素對(duì)香煙煙霧提取物(CSE)誘導(dǎo)的人肺泡II型上皮細(xì)胞(A549細(xì)胞)氧化應(yīng)激的影響,并從固有的抗氧化通路(PI3K-Akt和Nrf2通路)探討其機(jī)制。 方.法 (1)以2.5%,5%,10%CSE刺激A549細(xì)胞,24小時(shí)后于倒置顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化并用CCK-8法檢測(cè)細(xì)胞活力,選出合適濃度的CSE進(jìn)行試驗(yàn)； (2)以luM,10μM,100μM ICT干預(yù)A549細(xì)胞,24小時(shí)后于倒置顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化并用CCK-8法檢測(cè)細(xì)胞活力,選出適合濃度的ICT進(jìn)行試驗(yàn)。 (3)以10μM ICT預(yù)培養(yǎng)A549細(xì)胞1小時(shí)后加入5%、10%CSE刺激24小時(shí)后于倒置顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化并用CCK-8法檢測(cè)細(xì)胞活力,與單純CSE刺激組相對(duì)比。 (4)培養(yǎng)A549細(xì)胞,并分組。 A組：正常對(duì)照組； B組：5%CSE刺激模型組； C組：ICT10μM干預(yù)組； D組：ICT10μM干預(yù)1小時(shí)后予5%CSE刺激組； E組：AKT抑制劑LY294002干預(yù)1小時(shí)后加ICT10μM干預(yù)1小時(shí)后予5%CSE刺激組； F組：Nrf2siRNA轉(zhuǎn)染48小時(shí)后加入ICT10μM干預(yù)1小時(shí)后予5%CSE刺激組； (5)檢測(cè)指標(biāo)如下： 1)倒置顯微鏡下觀察各組細(xì)胞形態(tài)學(xué)變化； 2)流式細(xì)胞儀檢測(cè)各組細(xì)胞內(nèi)ROS變化； 3)GSH檢測(cè)試劑盒檢測(cè)細(xì)胞內(nèi)GSH變化； 4) Western blot檢測(cè)細(xì)胞內(nèi)Nrf2、P-Akt蛋白的變化； 5) Real-time PCR檢測(cè)細(xì)胞內(nèi)GCLM mRNA變化。 結(jié)果 (1)不同濃度CSE均可抑制細(xì)胞增殖,且該作用呈濃度依賴性。 (2)中、低濃度(1μM,10μM) ICT對(duì)細(xì)胞增殖具有促進(jìn)作用；高濃度(100μ M)ICT對(duì)細(xì)胞增殖無明顯影響。 (3)10μM ICT對(duì)CSE誘導(dǎo)的細(xì)胞損傷具有保護(hù)作用。 (4)CSE可顯著引起細(xì)胞內(nèi)ROS釋放,而ICT提前干預(yù)則可降低ROS產(chǎn)生。 (5)與單獨(dú)CSE刺激組相比,ICT提前干預(yù)1小時(shí)可增加細(xì)胞內(nèi)GSH含量。 (6)與單獨(dú)CSE刺激組相比,ICT促進(jìn)GCLM基因轉(zhuǎn)錄,與此同時(shí),Nrf2蛋白核轉(zhuǎn)位和細(xì)胞內(nèi)磷酸化Akt蛋白明顯增加。 (7)采用siRNA技術(shù)沉默Nrf2后,ICT對(duì)Nrf2下游的抗氧化基因GCLM的轉(zhuǎn)錄無促進(jìn)作用。 (8)采用Akt抑制劑LY294002干預(yù)后,ICT對(duì)Nrf2的核轉(zhuǎn)位及其下游抗氧化基因GCLM的轉(zhuǎn)錄無促進(jìn)作用,同時(shí)對(duì)ROS的釋放無明顯抑制作用。 結(jié)論 1.CSE可誘導(dǎo)A549細(xì)胞氧化應(yīng)激,從而引起細(xì)胞損傷和死亡。 2-補(bǔ)腎組分淫羊藿素是一種新型抗氧化劑,對(duì)于CSE誘導(dǎo)的肺泡II型上皮細(xì)胞損傷具有保護(hù)作用,可為COPD的抗氧化治療提供新選擇； 3.補(bǔ)腎組分淫羊藿素可通過激活細(xì)胞固有的抗氧化通路,發(fā)揮抗氧化應(yīng)激作用。具體而言,淫羊藿素可激活PI3K-Akt通路,增加Nrf2核轉(zhuǎn)位,繼而啟動(dòng)GCL基因轉(zhuǎn)錄并最終增加GSH水平從而減少ROS釋放。
[Abstract]:Objective to study the effects of epimedium on oxidative stress induced by cigarette smoke extract (CSE) in human alveolar II epithelial cells (A549 cells) and to explore its mechanism from the intrinsic antioxidant pathways (PI3K-Akt and Nrf2 pathways). Fang. Methods (1) A549 cells were stimulated with 2.5 and 10 CSE respectively. After 24 hours, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 method. The appropriate concentration of CSE was selected for the experiment. (2) A549 cells were treated with 100 渭 M luM,10 渭 M ICT. After 24 hours, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 assay. The suitable concentration of ICT was selected for the experiment. (3) A549 cells were precultured with 10 渭 M ICT for 1 hour. After 24 hours of stimulation with 10 渭 M ICT, the morphological changes of A549 cells were observed under inverted microscope and the viability of A549 cells was detected by CCK-8 assay. (4) A549 cells were cultured and grouped. Group A: normal control group, group B: 5%CSE stimulation model group, group C: ICT10 渭 M intervention group, group D: 5%CSE stimulation group after 1 hour of ICT10 渭 M intervention; Group E was treated with AKT inhibitor LY294002 for 1 hour and then treated with ICT10 渭 M for 1 hour. Group F was treated with 5%CSE after 48 hours of Nrf2siRNA transfection and ICT10 渭 M for 1 hour. (5) the indexes were as follows: 1) observe the changes of cell morphology under inverted microscope, 2) detect the changes of intracellular ROS by flow cytometry, 3) detect the changes of intracellular GSH by GSH assay kit. ) Western blot was used to detect the changes of Nrf2,P-Akt protein and 5) Real-time PCR was used to detect the changes of intracellular GCLM mRNA. Results (1) CSE could inhibit cell proliferation in a concentration dependent manner. (2) low concentration (1 渭 m ~ (10) 渭 M) ICT) could promote cell proliferation, while high concentration (100 渭 M) ICT) had no effect on cell proliferation. (3) 10 渭 M ICT has protective effect on CSE induced cell damage. (4) CSE could significantly induce the release of intracellular ROS, while early intervention of ICT could decrease the production of ROS. (5) compared with CSE alone, ICT increased intracellular GSH content 1 hour in advance. (6) compared with CSE alone, ICT promoted the transcription of GCLM gene, at the same time, Nrf2 protein nuclear translocation and intracellular phosphorylated Akt protein increased significantly. (7) after silencing Nrf2 with siRNA technique, ICT did not promote the transcription of antioxidant gene GCLM downstream of Nrf2. (8) after the intervention of Akt inhibitor LY294002, ICT did not promote the transcription of Nrf2 nuclear translocation and its downstream antioxidant gene GCLM, but did not inhibit the release of ROS. Conclusion 1.CSE can induce oxidative stress in A549 cells, resulting in cell injury and death. Epimedium, a new component of Epimedium, is a new antioxidant, which can protect alveolar II type epithelial cells from injury induced by CSE, and provide a new choice for the antioxidation therapy of COPD. 3. Epimedium, a component of tonifying kidney, can exert antioxidant stress by activating the intrinsic antioxidant pathway of cells. Specifically, Epimediin activates the PI3K-Akt pathway, increases the Nrf2 nuclear translocation, then initiates the transcription of the GCL gene and ultimately increases the GSH level, thus reducing the release of ROS.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563.9

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