【作者】 李文雷；

【导师】 李守军；

【作者基本信息】 北京协和医学院 ， 外科学， 2014， 博士

【摘要】 目的婴幼儿在经历过采用深低温低流量(DHLF)体外循环(CPB)转机方法的心脏手术后,由于多种病理生理原因,其肺损伤往往较年长儿严重,已成为患儿术后并发症的主要死因。近年来,有临床及实验报道,体外循环期间经肺动脉灌注肺保护液可减轻体外循环所造成的肺损伤。深低温低流量体外循环中未成熟肺损伤机制研究还不十分完善,在肺保护液的选择上尚无定论。目前,人们发现microRNA作为一类重要的基因调控因子在多种病理生理过程和疾病中发挥着多效作用。本研究中,我们从临床出发,通过建立稳定的乳猪深低温低流量体外循环模型,向肺动脉灌注乌司他丁肺保护液,探讨其对深低温低流量体外循环下未成熟肺脏的保护作用。并检测肺组织microRNA表达谱变化及对其潜在调控的靶基因和相关功能进行研究,进而为肺保护的研究提供理论依据。方法15只乳猪(14-21天,2.4-7.Okg)随机分为深低温低流量体外循环致肺损伤组(C组)、不含乌司他丁肺保护液灌注组(P组)、乌司他丁肺保护液灌注组(U组),各5例。均模拟临床操作建立体外循环模型。P组及U组于主动脉阻断心脏灌停后经肺动脉分别灌注不含乌司他丁肺保护液和乌司他丁肺保护液,C组无保护液灌注,主动脉阻断时间2小时期间使用深低温低流量(25℃,50ml/kg/min)转机1小时,维持循环稳定2小时。分别于CPB转机前,CPB停机、停机后1小时、2小时检测血流动力学指标,通气情况,血气变化,计算相关呼吸参数以比较和评价肺功能改变,同期各时间点抽取5ml肺静脉血离心后提取血清,实验结束后取右下肺组织匀浆离心提取上清,ELISA法测定血清及肺组织中肿瘤坏死因子a (TNF-αa),白细胞介素6(IL-6),白细胞介素10(IL-10),髓过氧化物(MPO),丙二醛(MDA),超氧化物歧化酶(SOD)含量,EMSA法检测肺组织中核转录因子kappa B(NF-κB)的活性,并对肺组织病理学评分、细胞凋亡情况、肺湿重干重比值进行比较。同时使用microRNA芯片技术对C组及U组肺组织miRNA表达与同龄同体重正常乳猪肺组织进行检测比较,筛选差异表达的microRNA,选取差异较明显的microRNA实时定量聚合酶链反应(qRT-PCR)进行验证。使用miRanda预测潜在靶基因,运用DAVID数据库对差异表达miRNA的可能靶基因进行Gene Ontology分析和KEGG信号通路分析,并以qRT-PCR和ELISA分别检测肺组织中可能靶基因的表达。结果DHLF-CPB模型建立稳定成功,所有乳猪均出现肺损伤,并无明显血流动力学差异。实验终点时U组中肺功能指标、病理学评分明显优于C组及P组,同时湿干比及凋亡细胞比例较该两组明显降低。ELISA结果显示U组在实验终点时肺静脉血清中TNF-α,IL-6,MPO水平明显低于C组及P组,IL。-10,SOD水平则明显高于C组及P组,肺组织中IL-6,MPO含量明显低于C组及P组,IL-10,SOD含量明显高于C组及P组。实验终点时血清中MDA表达及组织TNF α,MDA含量U组与P组相比无明显差异,但均明显低于C组。EMSA结果显示U组NF-κ B活性要明显低于C组及P组。通过miRNA microarray技术检测,同正常乳猪肺组织miRNA表达相比,C组中16个miRNAs,U组中8个miRNAs的表达发生了显著改变(P<0.05),运用荧光定量PCR对miR-21, miR-127, miR-145, miR-204表达进行验证,C组中,miR-127, miR-145,miR-204的水平明显降低,miR-21水平明显上升,U组除miR-204表达变化无统计学差异,其余miRNAs变化趋势与C组相同,具有统计学差异。与C组相比,U组miR-127,miR-204表达均抬高,miR-21表达降低。通过对miRanda预测的miRNA靶点进行归类分析,得到在体外循环肺损伤期间相关的重要通路,包括T细胞受体信号通路,P13K-Akt-NF-K B信号通路,RAS通路。预测靶基因磷脂酰肌醇3-激酶(PIK3CG)、血管紧张素转化酶(ACE)、前列腺素内过氧化物合酶2(PTGS2) PCR验证和ELISA检测结果均提示U组ACE、PTGS2表达水平及C组PIK3CG、ACE、PTGS2均明显升高。同时,U组ACE、PTGS2、PIK3CG表达水平均较C组显著降低。结论深低温低流量体外循环术后可造成严重的肺损伤,乌司他丁肺保护液能较好地改善未成熟肺肺功能指标,减轻炎性反应和氧化应激,减少肺内细胞凋亡,减轻肺损伤,可能与其抑制NF-K B活性有关。miRNA表达变化提示miRNA参与肺损伤和肺动脉灌注肺保护的过程。C组和U组肺组织中可能具有抑炎作用的miR-127,miR-145, miR-204表达下调与促炎作用的miR-21表达上调,提示miRNA参与肺损伤的作用机制与炎性反应、氧化应激密切相关。而U组中miRNA变化程度均小于C组,说明乌司他丁肺灌注液可调节相关miRNA达到肺保护的效果。通过对miRNA及其相关分子通路的发现将有助于今后我们对体外循环肺损伤机制研究及肺保护的研究。更多还原

【Abstract】 ObjectiveAfter cardiac surgery performed under deep hypothermia with low-flow (DHLF) cardiopulmonary bypass (CPB) in infants, the lung injury often becomes more severe than elder children driven by diverse pathogenic etiologies, which has always been a major cause of infant patients’ death during the post-operation time. Previous studies reported the potential advantages of additional lung protective solution perfusion on alleviate lung injury during cardiac surgery with CPB. For now, the mechanism of DHLF-CPB induced lung injury is inconclusive, a suitable lung preservation solution is still required. Due to their pleiotropic actions, microRNAs (miRNAs) are potential candidates involved in a diverse pathophysiological processes and diseases via regulating gene expression.In this study, we mimicked clinical procedure and established a stable piglet model undergoing deep hypothermia with low-flow (DHLF) cardiopulmonary bypass (CPB) with pulmonary artery perfusion as a lung protective strategy, aimed to evaluate the protective efficacy of pulmonary artery perfusion with urinary trypsin inhibitor (UTI) on immature lung. Furthermore, we investigated the changed miRNAs and their potential target genes and relevant function in neonatal piglet lungs in response to DHLF-CPB and lung protection with pulmonary perfusion with ulinastatin, in order to provide theoretical basis for CPB related immature lung injury and lung protection solution improvement.Methods15piglets aged14-21day, weighting2.4-7.0kg were randomly divided into3groups, with5piglets in each group:DHLF-CPB related lung injury group (Group C), the pulmonary artery perfusion without ulinastatin group (Group P) and the pulmonary infusion with ulinastatin group (Group U). A systemic CPB flow was established via aortic cannula and venous cannula in the right atrial appendage via median sternotomy. In group P and U, lung protective solution without or with ulinastatin was perfused via pulmonary artery respectively after cardiac arrest was obtained by aortic cross clamp and antegrade St. Thomas’cardioplegia through the aortic root. The DHLF-CPB was performed for60minutes (25℃,50ml/kg/min) during in the2hours aortic cross-clamping. After the piglets weaned from CPB, then received another120min observation. The hemodynamics and respiratory indices, blood gas analysis were recorded at four defined time-points:before CPB, at the end of CPB, and60minutes,120minutes after CPB. Pulmonary venous blood was sampled at the same preceding time interval. Right lung lower lobe was harvested at the end of the study. The levels of cytokines (TNF-α, IL-6, MPO, MDA and SOD) in pulmonary venous serum and lung tissue and the activity of NF-kappa B in lung tissue were measured by enzyme-linked immunosorbent assay (ELISA) and elctrophoresis mobility shift assay (EMSA), respectively. Morphological comparison was assessed by light and electronic microscopy. A TUNEL technique was performed to evaluate the state of cell apoptosis in the lungs. The wet to dry ratio of lung tissue is calculated to assess the extent of tissue edema. We applied miRNA microarray and qRT-PCR analysis to compare the miRNA expression of group C and group U with normal piglets in lung tissue. Meanwhile, predicted mRNA targets were obtained from miRanda. Then the predicted mRNA target genes had a Gene Ontology analysis and KEGG analysis performed by DAVID to obtain the functional annotation and pathway mapping. qRT-PCR and ELISA are used for further validation for the chosen target genes.ResultsAll the15piglets were accomplished CPB successfully with stable hemodynamics, and demonstrated a state of lung injury. While pulmonary artery perfusion with UTI significantly ameliorated lung function and histopathological changes and we found a decrease in W/D ratio and the number of TUNEL-positive apoptotic cells in group U lungs when compared with that in the other two group lungs. There is a great decrease in the serum levels of TNF-α,IL-6, MPO and lung tissue levels of IL-6, MPO in group U compared to group C and group P. Also, we found an increase in the level of IL-10, SOD in group U lungs compared with that in lungs and serum of group C and group P which correlated with a strongly inhibition in the activity of NF-κB. We found no significant difference between group U and group P in the concentration of MDA in pulmonary venous serum and level of TNF-α and MDA in lung tissue.Using miRNA microarray we identified that when compared to normal piglets, the expression of16miRNAs in group C,8miRNAs in group U were significantly changed (P<0.05). The qRT-PCR analyses verified up-regulation of miR-21and down-regulation of miR-127, miR-145miR-204in group C. The same trend was also found in group U except miR-204. But there are up-regulation of miR-127, miR-204and down-regulation of miR-21in group U when compared to group C.Target genes were cross-referenced to the molecular pathways after obtained from miRanda, suggesting that the differentially expressed miRNAs were related to series of important biology pathway related to CPB-induced lung injury, including T Cell receptor signaling pathway, PI3K-Akt-NF-κB signaling pathway and RAS signaling pathway. We performed qRT-PCR and ELISA of3putatively target genes ACE, PTGS2and PIK3CG for further validation. The results revealed that the expression of the3target genes were up-regulated in group C. And there is an up-regulation of ACE and PTGS2in group U. And the extent of up-regulation of the3target genes in group C was higher than group U. Conclusion Deep hypothermic low-flow cardiopulmonary bypass surgery can induce severe lung damage. Pulmonary artery perfusion with UTI ameliorated immature pulmonary injury in the lungs via inhibiting the activity of NF-κB to reduce the extent of oxidative shock, inflammatory response and apoptosis of cells.Our results showed that dynamic changes of miRNA expression in piglet lungs of DHLF-CPB induced immature lung injury and pulmonary perfusion with ulinastatin lung protective solution. The level of putative anti-inflammatory miRNA:miR-127, miR-145and miR-204down-regulated and putative pro-inflammatory miRNA:miR-21up-regulated in group C and group U indicated that miRNA involved in the process of lung injury with close correlation to inflammatory response and oxidative stress. While the degree of miRNA level change was lower in group U suggested that ulinastatin alleviated DHLF-CPB induced lung injury by regulate relevant miRNA. Moreover, by understanding how the changed miRNAs regulate lung injury may provide a basis for understanding the pathogenesis of DHLF-CPB induced immature lung injury and pave a way to develop a novel therapeutic approach for lung protection.更多还原