大鼠肠缺血再灌注时肠组织微小RNA表达的变化

Changes in expression of intestinal microRNAs during intestinal ischemia-reperfusion in rats

目的 探讨大鼠肠缺血再灌注时肠组织微小RNA（miRNA）表达的变化.方法 成年雄性SD大鼠12只,体重230～250 g,采用随机数字表法分为2组（n=6）：假手术组（S组）和缺血再灌注组（Ⅰ/R组）.采用阻断肠系膜上动脉60 mim再灌注120 min的方法制备肠缺血再灌注损伤模型.再灌注结束后观察小肠组织病理学结果,行Chiu评分,测定小肠含水率和肠黏膜组织乳酸含量.对肠黏膜组织进行miRNA表达谱芯片分析,采用qRT-PCR法验证差异表达的miRNA.另取成年雄性SD大鼠24只,体重230～ 250 g,采用随机数字表法分为4组（n=6）：miR-378抑制剂组（An组）、miR-378激动剂组（Ag组）、抑制剂对照组（AnC组）和激动剂对照组（AgC组）分别经尾静脉注射miR-378抑制剂antagomir、激动剂agomir、antagomir阴性对照、agomir阴性对照溶液100μl（10nmol/ml）,24 h后制备肠缺血再灌注损伤模型.再灌注结束后观察小肠组织病理学结果,并行Chiu评分,测定肠含水率和小肠黏膜乳酸含量.结果 与S组比较,其余5组Chiu评分、肠含水率和肠黏膜乳酸含量升高（P＜0.01）.与Ⅰ/R组比较,An组Chiu评分、肠含水率和肠黏膜乳酸含量升高,Ag组上述指标降低（P＜0.05）,AnC组及AgC组上述指标差异无统计学意义（P＞0.05）.An组肠组织病理学损伤较Ⅰ/R组加重,Ag组肠组织病理学损伤较Ⅰ/R组减轻.miRNA芯片分析共筛选出19个差异表达的miRNA,其中miR-292-3p表达上调,包括miR-378在内的18个miRNA表达下调（P＜0.05）.qRT-PCR验证9个miRNA表达下调（P＜0.05或0.01）,与芯片分析结果一致.结论 大鼠肠缺血再灌注时肠组织有19个miRNA表达发生变化,且明确miR-378表达下凋与大鼠肠缺血再灌注损伤有关.

Objective To investigate the changes in the expression of intestinal microRNAs （miRNAs） during intestinal ischemia-reperfusion （Ⅰ/R） in rats.Methods Twelve adult male SpragueDawley rats,weighing 230-250 g,were randomly divided into 2 groups （n =6 each） using a random number table：sham operation group （group S） and Ⅰ/R group.Intestinal Ⅰ/R was induced by 60 min occlusion of superior mesenteric artery followed by 120 min reperfusion in group Ⅰ/R.At the end of reperfusion,the intestinal specimens were obtained for microscopic examination of pathological changes which were graded using Chiu＇s scoring system and for determination of small intestinal water content and intestinal mucosal lactic acid content,miRNA microarray chip analysis was performed in the intestinal mucosal tissues,and quantitative real-time PCR was used to verify the differentially expressed miRNAs.Another 24 adult male Sprague-Dawley rats,weighing 230-250 g,were randomly divided into 4 groups （n =6 each） using a random number table：miR-378 inhibitor antagomir group （group An）,miR-378 agonist agomir group （group Ag）,inhibitor control group （group AnC） and agonist control group （group AgC）.Antagomir,agomir,antagomir negative control and agomir negative control solutions l00 μl （10 nmol/ml） were injected via the tail vein in An,Ag,AnC and AgC groups,respectively.Intestinal Ⅰ/R was then produced 24 h later.The intestinal specimens were obtained for microscopic examination of pathological changes which were graded using Chiu＇s scoring system and for determination of small intestinal water content and intestinal mucosal lactic acid content.Results Compared with group S,Chiu＇s score,intestinal water content and intestinal mucosal lactic acid content were significantly increased in the other 5 groups.Compared with group Ⅰ/R,Chiu＇s score,intestinal water content and intestinal mucosal lactic acid content were were significantly increased in group An,the parameters mentioned above were decreased in group Ag,and no significant change was found in the parameters mentioned above in AnC and AgC groups.The pathological damage was aggravated in group An and was attenuated in group Ag as compared with group Ⅰ/R.miRNA microarray chip analysis revealed that a total of 19 miRNAs were found to be differentially expressed.Among the 19 differentially expressed miRNAs,the expression of miR-292-3p was significantly up-regulated,and the expression of 18 miRNAs including miR-378 was down-regulated.The expression of 9 miRNAs verified by quantitative real-time PCR was down-regulated,which was consistent with the results of miRNA microarray chip analysis.Conclusion Nineteen miRNA expression changes after Ⅰ/R,and it is confirmed that down-regulation of miR-378 expression is related to the intestinal Ⅰ/R injury in rats.