Elucidation of pathophysiology of ischemia reperfusion injury jn isolated perfused rat lungs

离体灌注大鼠肺缺血再灌注损伤病理生理学的阐明

• 批准号: 11470317
• 负责人: IMAI Takasuke
• 金额: $ 8万
• 依托单位: Tokyo Medical & Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470317/
• 关键词: ischemia-reperfusion; isolated rat lung; cyotokine; unilateral ischemia; inflammatory cytokines; RT-PCR; Inteferon-γ; Interferon-γ; 肺遊離環流標本; 虚血・再環流障害; 低酸素症; IL-1 beta; IL-6; 虚血・再潅流障害; 肺循環; 遊離潅流肺標本; mRNA

Isolated rat lung model perfused with Krebs-Henseleit solution added with 4% albumin was developed, in which right and left pulmonary arteries were isolated to enable preferential perfusion to each one of lungs with the same ventilation to the both lung maitained. Our hypothesis is that the genes of inflammatory cytokines would be induced in ischemic lung to cause the ischemia-reperfusion injury. After the left lung were exposed to ischemia for 60-100 min followed by reperfusion for 0-20 min, a few strips weighing about 100 mg were obtained separately in the perfused and ischemic lungs. Total RNA was extracted from these lung specimens by the method described by Chomczynski and Sacchi and DNAs of TNF-α, IL-1β, IL-10 and Interferon-γ were obtained by RT-PCR using MPCR primer, optimal temperatures and cycles. The obtained DNAs were electrophoresed in the agarose gel added with ethidium bromide. The genes of TNF-α, IL-1β and IL-6 in the control right lung lobe were detected more than in the left ischemic lung. Contrarily, the gene of IL-10 in the ischemic lung was detected more than in the control right lobe. Interfeorn-γ did not show significent difference between the both lobes. The reason why the inflammatory cyotokines appeared greater in the control right lung lobe than in the ischemic left lobe was supposed to the amount of endotoxin contained in the standard bovine serum albumin which were preferentially perfused through right lung during ischemia of left lung. Although the inflammatory cytokines were induced in the right and left lungs, left lung only showed ischemia reperfusion injury which suggested that interactions of any mediators induced by inflammatory cytokines and oxygen radicals would attack the cell membrane and cause the pulmonary edema.

开发了用添加了 4% 白蛋白的 Krebs-Henseleit 溶液灌注的离体大鼠肺模型，其中分离右肺动脉和左肺动脉，以便能够优先灌注到每个肺，同时维持双肺相同的通气量。左肺缺血60-100分钟后，会在缺血肺中诱导炎症细胞因子基因引起缺血再灌注损伤。再灌注0-20分钟，按照Chomczynski和Sacchi描述的方法从这些肺标本中分别提取几条重约100 mg的条带，提取TNF-α、IL-1β的DNA。使用MPCR引物、最佳温度和循环，通过RT-PCR获得IL-10和干扰素-γ，将获得的DNA在添加有琼脂糖凝胶中进行电泳。对照右肺叶中检测到的TNF-α、IL-1β和IL-6基因多于左缺血肺，相反，缺血肺中检测到的IL-10基因多于左肺叶。对照右肺叶中的干扰素-γ在两个肺叶之间没有表现出显着差异，这就是对照右肺叶中炎症细胞因子比缺血左肺中出现更多的原因。推测标准牛血清白蛋白中内毒素的含量，在左肺缺血时优先通过右肺灌注，尽管在右肺和左肺中诱导了炎性细胞因子，但左肺仅表现出缺血再灌注损伤。炎症细胞因子和氧自由基诱导的任何介质的相互作用都会攻击细胞膜并引起肺水肿。