表皮生长因子受体在体外循环急性肺损伤中的调控作用
发布时间：2018-09-03 10:10
【摘要】：体外循环(CPB)已经成为心脏外科手术中一项非常重要的关键技术。体外循环(CPB)对心脏手术的发展带来了革命性的变化,同时也很大程度上改善患片的预后。但是,CPB术后本身也会给病人造成一些不可避免的急性肺损伤,导致一系列并发症的发生。为此,我们设计以下实验,探究体外循环术后急性肺损伤的机制以及可能的治疗方案。材料与方法1.体外循环模型的建立:沿兔胸骨正中开胸,分别经主动脉插入6F动脉导管,导管末端沿降主动脉方向;经右心房插入静脉引流管。转流开始,阻断钳完全夹闭主动脉、肺动脉,灌注Thomas液,维持肛温33℃。心脏停搏30min后,开放主、肺动脉,终止体外转流,中和肝素,监测生命体征,观察4小时。2.实验分组:新西兰兔随机进行分为4组:CTRL组(术前3小时口服15%Captisol 6.25ml/Kg,仅开胸不进行体外循环手术)、Erlotinib组(术前3小时口服溶于15%Captisol的Erlotinib 100mg/Kg,仅开胸不进行体外循环手术)、CPB组(术前3小时口服15%Captisol 6.25ml/Kg,开胸进行体外循环手术)、CPB+Erlotinib 组(术前 3 小时口 服溶于 15%Captisol 的 Erlotinib 100mg/Kg,开胸进行体外循环手术)。3.ELISA法检测血清TNF-a的表达:使用酶联免疫吸附法(ELISA)检测血清TNF-a表达水平。4.免疫荧光检测兔肺组织PARP-1的表达水平:收集样本,使用免疫荧光法检测术后肺组织PARP-1的表达水平。5.Western blot检测各组肺组织ERK1/2、P38蛋白磷酸化水平:收集术后样本,使用Western blot法检测组织ERK1/2、P38蛋白磷酸化水平。6.统计学分析:采用SPSS20.0统计学软件进行统计分析,计量资料均数±标准差(mean±SD)表示。不同时间点采用重复测量;两组比较采用两独立样本t检验;多组比较采用单因素方差分析(one wayANVOA),比较组间差异时,方差齐用LSD检验,方差不齐用Dunnett'sT3检验。P0.05差异有统计学意义。作图使用GraphPad Prism 5.0作图软件。实验结果1.体外循环术对肺泡上皮细胞死亡方式的影响与CTRL相比,CPB肺泡上皮细胞PARP-1的表达水平明显升高,有统计学意义(p0.001)。2.EGFR抑制剂对体外循环诱导的TNF-α生成的影响收集各组术后4h动脉血,使用酶联免疫吸附测定ELISA检测血清中TNF-α的表达水平。与CTRL组相比,CPB组、CPB+Erlotinib组动脉血液中TNF-α的表达水平明显升高(p0.001),有统计学意义;CTRL组与Erlotinib组相比,TNF-α的表达水平无明显差异(p =0.974)。与CPB组相比,CPB+Erlotinib组TNF-α的表达水平显著降低,有统计学意义(p0.001)。3.EGFR抑制剂对ERK1/2和P38蛋白磷酸化的影响与CTRL组相比,CPB组与CPB+Erlotinib组ERK1/2的磷酸化水平显著升高(p0.001);而与Erlotinib组,ERK1/2蛋白的磷酸化水平无明显差异(p=0.656)。与CPB组相比,CPB+Erlotinib组ERK1/2蛋白的磷酸化水平明显被降低,差异有统计学意义(p0.001)。与CTRL组相比,CPB组(p0.001)与CPB+Erlotinib组(p0.05)P38的磷酸化水平显著升高;而Erlotinib组,P38蛋白的磷酸化水平无明显差异(p=0.277)。与CPB组相比,CPB+Erlotinib组P38蛋白的磷酸化水平明显降低,差异有统计学意义(p0.05)。结论1、体外循环术后TNF-α水平明显升高,大量肺泡上皮细胞发生Parthanatos。2、表皮生长因子受体EGFR参与体外循环诱导的TNF-α生成。3、体外循环导致体内炎症反应水平升高,其机制可能是通过某种途径激活EGFR,诱导MAPK信号途径的ERK1/2和P38激活,进而ERK1/2和P38的磷酸化,引起TNF-α生成和释放,促进急性肺损伤发生。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R654.1
本文编号：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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R654.1
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