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.

建立了大鼠离体肺灌注模型，用含4%白蛋白的Krebs-Henseleit液灌流，分离左、右肺动脉，使其优先灌注于每一肺，同时保持双肺通气量相同。我们的假设是，缺血肺组织中的炎症细胞因子基因可能被诱导而导致缺血再灌注损伤。左肺缺血60-100 min再灌注0-20 min后，在灌注肺和缺血肺中分别获得数条重约100 mg的条带。采用Chomczynski和Sacchi的方法提取肺组织总RNA，采用MPCR引物、最适温度和最适循环次数进行RT-PCR，获得TNF-α、IL-1β、IL-10和IFN-γ的DNA。将获得的DNA在添加有溴化乙锭的琼脂糖凝胶中电泳。TNF-α、IL-1β和IL-6基因在对照右肺叶的表达高于左肺叶。缺血肺组织中IL-10基因表达明显高于对照右叶。干扰素-γ在两个脑叶间无显著性差异。对照组右肺组织中炎性细胞因子的含量高于缺血组左肺组织的原因可能与标准牛血清白蛋白中的内毒素含量有关，而标准牛血清白蛋白在左肺缺血时优先经右肺灌注。虽然左右肺均出现了炎性细胞因子，但左肺仅出现缺血再灌注损伤，提示炎性细胞因子所诱导的介质与氧自由基相互作用，攻击细胞膜，导致肺水肿。