去铁胺预处理对大鼠肺缺血再灌注损伤的保护作用研究

发布时间：2018-08-22 07:06

【摘要】：目的：肺缺血再灌注损伤是指中断血供肺脏发生的缺血、缺氧性损伤随血流灌注恢复进一步加重的病理现象，广泛存在于心胸外科领域。研究表明，缺血再灌注时期游离铁增多，后者可以催化氧自由基生成，介导炎症反应。提自于多绒链霉菌中的去铁敏是一种高选择性的铁离子螯合剂，最初用于治疗血液疾病。其特定的羟胺基团具有同机体内游离铁离子相结合的能力，减少由其参与产生的活性氧基团对生物膜的攻击；同时诱导低氧预适应，调节机体经受缺血、缺氧、炎症攻击的能力，减少组织凋亡。目前去铁胺(Deferoxamine,DFO)逐渐成为组织保护领域焦点，国内外实验均证明在心肌保护，缺血缺氧性脑损伤治疗、肝脏保存液改良等的有较可靠的效果，但尚缺乏对肺缺血再灌注损伤保护作用研究的报道。本实验旨在观察DFO预处理对大鼠原位肺缺血再灌注损伤的作用，并对其可能机制做初步探讨。方法：取健康清洁SD大鼠90只,体重300g士25g,采用随机数字表法分为3组(n=30)：假手术组(NC组)，生理盐水预处理组(NS组),去铁胺预处理组(DFO组)。NS组开胸前连续3d经腹腔注射生理盐水，阻断左肺门45min再灌注120min；DFO组术前连续3d经腹腔注射DFO预处理，再建立鼠原位肺缺血再灌注模型；NC组左前外侧胸壁第3-5肋间切口开胸显露肺门，不进行肺缺血再灌注处理。各组均于再灌注30min、60min、120min共3个时间点分别留取左心尖血、肺组织标本后处死动物10只，各时间点血液标本检测动脉血氧分压(Pa02)、TNF-α浓度，评估肺氧合功能、炎症水平。各时间点肺组织标本计算湿／干重比值(W／D)评价肺水肿程度；制作肺组织匀浆并检测其中丙二醛含量观察脂质氧化反应；末端转移酶标记法测定肺脏组织内细胞凋亡水平；光镜观察组织和细胞病理学改变。结果：1.肺氧合功能：所测得的动脉血氧分压（Pa02）中，NS组和DFO组Pa02值出现下降，与NC组比较，在再灌注R60min、R120min时间点均存在统计学差异（P0.05）；与NS组相比，DFO组Pa02值在R60min、R120min时间点增高(P0.05)。2.肺干湿重比值：所测得的肺干湿重比（W/D）中，NS组和DFO组表现为W/D值增高，与NC组比较，在再灌注R30min、R60min、 R120min时间点均存在统计学差异（P0.05）；与NS组相比，W/D值在R30min、R60min、R120min时间点降低(P0.05)。3.脂质氧化反应： DFO组和NS组肺组织MDA含量随时间逐步上升，与NC组比较在再灌注R30min、R60min、 R120min时间点均存在统计学差异（P0.05）；与NS组相比，DFO组中R60min、R120min时间点肺组织MDA含量降低(P0.05)。4.炎症反应强度： DFO组和NS组肺组织血清TNF-α浓度随时间逐步上升，与NC组比较在再灌注R30min、R60min、 R120min时间点均存在统计学差异（P0.05）；与NS组相比，DFO组中R30min、R60min、R120min时间点血清TNF-α浓度浓度降低(P0.05)。5.病理评价：各时间点NC组大鼠肺组织结构清晰肺泡完整，肺泡无充血、水肿、渗出等改变。NS组各时间点大鼠肺组织均见肺泡萎陷或不张，大量炎性细胞浸润表现，泡腔内红细胞渗出，随再灌注时间延长加重，肺泡损伤指数较NC组增高(P0.05)。DFO组与NS组相比各时间点大鼠肺组织亦有炎性浸润、肺泡结构破坏伴少量出血，DFO组各时间点肺损伤指数低于NS组(P0.05)。6.TUNEL染色：各时间点上NS组大鼠肺组织均有大量棕褐色颗粒，呈明显凋亡表现，随再灌注时间延长加重，凋亡指数较NC组显著增高（P0.05)。DFO组与NS组相比各时间点大鼠肺组织亦有上述改变，但R30min、R120min凋亡指数低于NS组(P0.05)，，NC组存在凋亡现象不明显。结论：1.DFO对大鼠原位肺缺血再灌注损伤具有改善通气功能、减轻水肿，具备一定的保护作用。2.在大鼠LIRI模型中，DFO干预后可有效抑制脂质氧化反应，减轻炎症反应的作用。3.在大鼠LIRI模型中, DFO预处理有抑制肺组织因LIRI导致的凋亡。
[Abstract]:AIM: Pulmonary ischemia-reperfusion injury (PIRI) refers to the pathological phenomenon of ischemia and hypoxic injury aggravated with the recovery of blood perfusion. It is widely found in cardiothoracic surgery. Deferrin in mold is a highly selective iron chelating agent originally used in the treatment of blood diseases. Its specific hydroxylamine groups have the ability to combine with free iron ions in the body, reducing the aggression of reactive oxygen species produced by them to biofilms, and inducing hypoxic preconditioning to regulate the body's ability to withstand ischemia and hypoxia. Deferoxamine (DFO) has gradually become the focus in the field of tissue protection. Domestic and foreign experiments have proved that it has a reliable effect on myocardial protection, treatment of ischemic and hypoxic brain injury, and improvement of liver preservation fluid. However, there is no report on the protective effect of DFO on lung ischemia-reperfusion injury. Methods: Ninety healthy and clean SD rats weighing 300 g and 25g were randomly divided into three groups (n=30), sham operation group (NC group), normal saline group (NS group) and desferriamine pretreatment group (DFO group). NS group received intraperitoneal injection of normal saline for 3 days before thoracotomy to block the left hilum for 45 minutes and reperfusion for 120 minutes; DFO group received intraperitoneal injection of DFO preconditioning for 3 days before operation to establish in situ pulmonary ischemia-reperfusion model in rats; NC group received thoracotomy to expose the hilum through the third-fifth intercostal incision on the left anterolateral chest wall, without ischemia-reperfusion treatment. Blood samples of left ventricular apex were collected at 3 time points of in, 60 min and 120 min, and then 10 animals were sacrificed. The arterial oxygen partial pressure (Pa02), TNF-a concentration, pulmonary oxygenation function and inflammation level were measured at each time point. Results: 1. Pulmonary oxygenation function: Pa02 values in NS and DFO groups decreased, compared with NC group, in reperfusion R60. Compared with NS group, the Pa02 value in DFO group increased at R60 min and R120 min (P 0.05). 2. The ratio of dry to wet lung weight: The W/D value in NS group and DFO group increased significantly at R30 min, R60 min and R120 min of reperfusion compared with NC group. Compared with NS group, W/D value decreased at R30 min, R60 min, R120 min time point (P 0.05). 3. Lipid oxidation reaction: MDA content in lung tissue of DFO group and NS group increased gradually with time, and there were statistical differences between DFO group and NC group at R30 min, R60 min, R120 min time point of reperfusion (P 0.05); compared with NS group, there were statistical differences between DFO group at R60 min, R120 min time point of lung. Tissue MDA content decreased (P 0.05). 4. Inflammation intensity: The serum TNF-alpha concentration in DFO group and NS group increased gradually with time, and there were significant differences between the two groups at the time points of reperfusion R30 min, R60 min, R120 min (P 0.05); compared with NS group, the serum TNF-alpha concentration in DFO group decreased at the time points of R30 min, R60 min, R120 min (P 0.05). 5. Pathological evaluation: The pulmonary tissue of NC group was clear, alveolar intact, alveolar no congestion, edema, exudation and other changes. In NS group, alveolar atrophy or atelectasis, a large number of inflammatory cells infiltration, alveolar erythrocyte exudation, with the extension of reperfusion time, alveolar injury index was higher than NC group (P 0.05). Compared with NS group, the lung tissue of DFO group also had inflammatory infiltration and alveolar structure damage with a little bleeding. The lung injury index of DFO group was lower than NS group (P 0.05). 6. TUNEL staining showed that there were a lot of brown granules in lung tissue of NS group at each time point, showing obvious apoptosis. With the prolongation of reperfusion time, the apoptosis index increased. Compared with NC group, DFO group also had the above changes in lung tissue at each time point, but the apoptosis index at R30 min and R120 min was lower than NS group (P 0.05). There was no obvious apoptosis in NC group. Conclusion: 1. DFO can improve ventilation function and reduce edema in rats with lung ischemia-reperfusion injury in situ. 2. In rat LIRI model, DFO intervention can effectively inhibit lipid oxidation and alleviate inflammation. 3. In rat LIRI model, DFO preconditioning can inhibit lung tissue apoptosis induced by LIRI.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R655.3

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