表皮生長因子受體在體外循環(huán)急性肺損傷中的調(diào)控作用
發(fā)布時間：2018-09-03 10:10
【摘要】：體外循環(huán)(CPB)已經(jīng)成為心臟外科手術(shù)中一項非常重要的關(guān)鍵技術(shù)。體外循環(huán)(CPB)對心臟手術(shù)的發(fā)展帶來了革命性的變化,同時也很大程度上改善患片的預(yù)后。但是,CPB術(shù)后本身也會給病人造成一些不可避免的急性肺損傷,導(dǎo)致一系列并發(fā)癥的發(fā)生。為此,我們設(shè)計以下實驗,探究體外循環(huán)術(shù)后急性肺損傷的機(jī)制以及可能的治療方案。材料與方法1.體外循環(huán)模型的建立:沿兔胸骨正中開胸,分別經(jīng)主動脈插入6F動脈導(dǎo)管,導(dǎo)管末端沿降主動脈方向;經(jīng)右心房插入靜脈引流管。轉(zhuǎn)流開始,阻斷鉗完全夾閉主動脈、肺動脈,灌注Thomas液,維持肛溫33℃。心臟停搏30min后,開放主、肺動脈,終止體外轉(zhuǎn)流,中和肝素,監(jiān)測生命體征,觀察4小時。2.實驗分組:新西蘭兔隨機(jī)進(jìn)行分為4組:CTRL組(術(shù)前3小時口服15%Captisol 6.25ml/Kg,僅開胸不進(jìn)行體外循環(huán)手術(shù))、Erlotinib組(術(shù)前3小時口服溶于15%Captisol的Erlotinib 100mg/Kg,僅開胸不進(jìn)行體外循環(huán)手術(shù))、CPB組(術(shù)前3小時口服15%Captisol 6.25ml/Kg,開胸進(jìn)行體外循環(huán)手術(shù))、CPB+Erlotinib 組(術(shù)前 3 小時口 服溶于 15%Captisol 的 Erlotinib 100mg/Kg,開胸進(jìn)行體外循環(huán)手術(shù))。3.ELISA法檢測血清TNF-a的表達(dá):使用酶聯(lián)免疫吸附法(ELISA)檢測血清TNF-a表達(dá)水平。4.免疫熒光檢測兔肺組織PARP-1的表達(dá)水平:收集樣本,使用免疫熒光法檢測術(shù)后肺組織PARP-1的表達(dá)水平。5.Western blot檢測各組肺組織ERK1/2、P38蛋白磷酸化水平:收集術(shù)后樣本,使用Western blot法檢測組織ERK1/2、P38蛋白磷酸化水平。6.統(tǒng)計學(xué)分析:采用SPSS20.0統(tǒng)計學(xué)軟件進(jìn)行統(tǒng)計分析,計量資料均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。不同時間點(diǎn)采用重復(fù)測量;兩組比較采用兩獨(dú)立樣本t檢驗;多組比較采用單因素方差分析(one wayANVOA),比較組間差異時,方差齊用LSD檢驗,方差不齊用Dunnett'sT3檢驗。P0.05差異有統(tǒng)計學(xué)意義。作圖使用GraphPad Prism 5.0作圖軟件。實驗結(jié)果1.體外循環(huán)術(shù)對肺泡上皮細(xì)胞死亡方式的影響與CTRL相比,CPB肺泡上皮細(xì)胞PARP-1的表達(dá)水平明顯升高,有統(tǒng)計學(xué)意義(p0.001)。2.EGFR抑制劑對體外循環(huán)誘導(dǎo)的TNF-α生成的影響收集各組術(shù)后4h動脈血,使用酶聯(lián)免疫吸附測定ELISA檢測血清中TNF-α的表達(dá)水平。與CTRL組相比,CPB組、CPB+Erlotinib組動脈血液中TNF-α的表達(dá)水平明顯升高(p0.001),有統(tǒng)計學(xué)意義;CTRL組與Erlotinib組相比,TNF-α的表達(dá)水平無明顯差異(p =0.974)。與CPB組相比,CPB+Erlotinib組TNF-α的表達(dá)水平顯著降低,有統(tǒng)計學(xué)意義(p0.001)。3.EGFR抑制劑對ERK1/2和P38蛋白磷酸化的影響與CTRL組相比,CPB組與CPB+Erlotinib組ERK1/2的磷酸化水平顯著升高(p0.001);而與Erlotinib組,ERK1/2蛋白的磷酸化水平無明顯差異(p=0.656)。與CPB組相比,CPB+Erlotinib組ERK1/2蛋白的磷酸化水平明顯被降低,差異有統(tǒng)計學(xué)意義(p0.001)。與CTRL組相比,CPB組(p0.001)與CPB+Erlotinib組(p0.05)P38的磷酸化水平顯著升高;而Erlotinib組,P38蛋白的磷酸化水平無明顯差異(p=0.277)。與CPB組相比,CPB+Erlotinib組P38蛋白的磷酸化水平明顯降低,差異有統(tǒng)計學(xué)意義(p0.05)。結(jié)論1、體外循環(huán)術(shù)后TNF-α水平明顯升高,大量肺泡上皮細(xì)胞發(fā)生Parthanatos。2、表皮生長因子受體EGFR參與體外循環(huán)誘導(dǎo)的TNF-α生成。3、體外循環(huán)導(dǎo)致體內(nèi)炎癥反應(yīng)水平升高,其機(jī)制可能是通過某種途徑激活EGFR,誘導(dǎo)MAPK信號途徑的ERK1/2和P38激活,進(jìn)而ERK1/2和P38的磷酸化,引起TNF-α生成和釋放,促進(jìn)急性肺損傷發(fā)生。
[Abstract]:Cardiopulmonary bypass (CPB) has become a very important key technique in cardiac surgery. CPB has brought revolutionary changes to the development of cardiac surgery and greatly improved the prognosis of the patients. However, CPB itself can also cause some unavoidable acute lung injury, resulting in a series of complications. Material and Methods 1. Establishment of a cardiopulmonary bypass model: thoracotomy along the median sternum of rabbits was performed, 6F catheter was inserted through the aorta, the end of the catheter was inserted along the descending aorta, and venous drainage was performed through the right atrium. After 30 minutes of cardiac arrest, the aorta and pulmonary arteries were opened, the extracorporeal circulation was terminated, heparin was neutralized, and vital signs were monitored for 4 hours. 2. New Zealand rabbits were randomly divided into four groups: CTRL group (15% Captisol 6.25 ml/Kg orally 3 hours before operation). Only thoracotomy without cardiopulmonary bypass was performed, Erlotinib group (Erlotinib 100 mg/kg dissolved in 15% Captisol 3 hours before operation, only thoracotomy without cardiopulmonary bypass), CPB group (15% Captisol 6.25 ml/kg 3 hours before operation, open thoracotomy with cardiopulmonary bypass), CPB + Erlotinib group (Erlotinib dissolved in 15% Captisol 3 hours before operation) Detection of serum TNF-a expression by ELISA: Detection of serum TNF-a expression by enzyme-linked immunosorbent assay (ELISA). 4. Immunofluorescence detection of rabbit lung tissue PARP-1 expression level: Collection of samples, using immunofluorescence detection of postoperative lung tissue PARP-1 expression level. 5. Western blot detection of each The phosphorylation levels of ERK1/2 and P38 proteins in lung tissues of group B were detected by Western blot after operation. 6. Statistical analysis: SPSS20.0 software was used for statistical analysis, and the mean (+ SD) of measurement data was expressed. Sample t test; one way ANVOA was used to compare the differences between groups. LSD was used to test the variance, and Dunnett's T3 was used to test the variance. P 0.05 was statistically significant. The expression level of PARP-1 in alveolar epithelial cells of CPB was significantly higher than that of control group (p0.001). 2. The effect of EGFR inhibitor on TNF-a production induced by cardiopulmonary bypass was observed by enzyme-linked immunosorbent assay (ELISA). The expression level of TNF-alpha in blood was significantly increased (p0.001), with statistical significance; there was no significant difference between CTRL group and Erlotinib group (p = 0.974). Compared with CPB group, the expression level of TNF-alpha in CPB + Erlotinib group was significantly decreased, with statistical significance (p0.001). 3. The effect of EGFR inhibitor on the phosphorylation of ERK1/2 and P38 proteins was statistically significant (p0.001). The phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly higher than that in CPB + Erlotinib group (p0.001), but there was no significant difference between CPB and Erlotinib group (p = 0.656). Compared with CPB group, the phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly lower (p0.001). Compared with CRB group, the phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly lower (p0.001). The phosphorylation level of P38 protein in CPB + Erlotinib group (p0.05) was significantly higher than that in Erlotinib group (p = 0.277). Compared with CPB group, the phosphorylation level of P38 protein in CPB + Erlotinib group was significantly lower than that in CPB group (p0.05). Conclusion 1. After CPB, the level of TNF - alpha was significantly higher and there was a large number of alveoli. Epidermal growth factor receptor EGFR participates in the production of TNF-a induced by cardiopulmonary bypass. 3. Cardiopulmonary bypass leads to elevated levels of inflammatory response in vivo. The mechanism may be that EGFR is activated by some pathway, ERK1/2 and P38 of MAPK signaling pathway are activated, ERK1/2 and P38 are phosphorylated, and TNF-a is produced and released. Promote the occurrence of acute lung injury.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R654.1
本文編號：2219667
[Abstract]:Cardiopulmonary bypass (CPB) has become a very important key technique in cardiac surgery. CPB has brought revolutionary changes to the development of cardiac surgery and greatly improved the prognosis of the patients. However, CPB itself can also cause some unavoidable acute lung injury, resulting in a series of complications. Material and Methods 1. Establishment of a cardiopulmonary bypass model: thoracotomy along the median sternum of rabbits was performed, 6F catheter was inserted through the aorta, the end of the catheter was inserted along the descending aorta, and venous drainage was performed through the right atrium. After 30 minutes of cardiac arrest, the aorta and pulmonary arteries were opened, the extracorporeal circulation was terminated, heparin was neutralized, and vital signs were monitored for 4 hours. 2. New Zealand rabbits were randomly divided into four groups: CTRL group (15% Captisol 6.25 ml/Kg orally 3 hours before operation). Only thoracotomy without cardiopulmonary bypass was performed, Erlotinib group (Erlotinib 100 mg/kg dissolved in 15% Captisol 3 hours before operation, only thoracotomy without cardiopulmonary bypass), CPB group (15% Captisol 6.25 ml/kg 3 hours before operation, open thoracotomy with cardiopulmonary bypass), CPB + Erlotinib group (Erlotinib dissolved in 15% Captisol 3 hours before operation) Detection of serum TNF-a expression by ELISA: Detection of serum TNF-a expression by enzyme-linked immunosorbent assay (ELISA). 4. Immunofluorescence detection of rabbit lung tissue PARP-1 expression level: Collection of samples, using immunofluorescence detection of postoperative lung tissue PARP-1 expression level. 5. Western blot detection of each The phosphorylation levels of ERK1/2 and P38 proteins in lung tissues of group B were detected by Western blot after operation. 6. Statistical analysis: SPSS20.0 software was used for statistical analysis, and the mean (+ SD) of measurement data was expressed. Sample t test; one way ANVOA was used to compare the differences between groups. LSD was used to test the variance, and Dunnett's T3 was used to test the variance. P 0.05 was statistically significant. The expression level of PARP-1 in alveolar epithelial cells of CPB was significantly higher than that of control group (p0.001). 2. The effect of EGFR inhibitor on TNF-a production induced by cardiopulmonary bypass was observed by enzyme-linked immunosorbent assay (ELISA). The expression level of TNF-alpha in blood was significantly increased (p0.001), with statistical significance; there was no significant difference between CTRL group and Erlotinib group (p = 0.974). Compared with CPB group, the expression level of TNF-alpha in CPB + Erlotinib group was significantly decreased, with statistical significance (p0.001). 3. The effect of EGFR inhibitor on the phosphorylation of ERK1/2 and P38 proteins was statistically significant (p0.001). The phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly higher than that in CPB + Erlotinib group (p0.001), but there was no significant difference between CPB and Erlotinib group (p = 0.656). Compared with CPB group, the phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly lower (p0.001). Compared with CRB group, the phosphorylation level of ERK1/2 protein in CPB + Erlotinib group was significantly lower (p0.001). The phosphorylation level of P38 protein in CPB + Erlotinib group (p0.05) was significantly higher than that in Erlotinib group (p = 0.277). Compared with CPB group, the phosphorylation level of P38 protein in CPB + Erlotinib group was significantly lower than that in CPB group (p0.05). Conclusion 1. After CPB, the level of TNF - alpha was significantly higher and there was a large number of alveoli. Epidermal growth factor receptor EGFR participates in the production of TNF-a induced by cardiopulmonary bypass. 3. Cardiopulmonary bypass leads to elevated levels of inflammatory response in vivo. The mechanism may be that EGFR is activated by some pathway, ERK1/2 and P38 of MAPK signaling pathway are activated, ERK1/2 and P38 are phosphorylated, and TNF-a is produced and released. Promote the occurrence of acute lung injury.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R654.1
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