探讨靶向调控蛋白激酶D1的微小RNA及其对大鼠急性胰腺炎的影响  

The identification and roles of microRNA targeting protein kinase D1 in rat with acute pancreatitis

作　　者：徐佳佳 程旸 耿岚岚[1] 许万福 杨敏[1] 陈佩瑜[1] 许朝晖 王洪丽[1] 陈涣 叶丽萍 何李英 龚四堂[1] Xu Jiajia;Cheng Yang;Geng Lanlan;Xu Wangfu;Yang Min;Chen Peiyu;Xu Chaohui;Wang Hongli;Chen Huan;Ye Liping;He Liying;Gong Sitang(Department of Gastroenterology,Guangzhou Women and Children′s Medical Center,Guangzhou Medical University,Guangzhou 510623,China)

机构地区：[1]广州医科大学附属广州市妇女儿童医疗中心消化科,510623

出　　处：《中华实用儿科临床杂志》2018年第19期1473-1477,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81770552）

摘　　要：目的预测和印证调控蛋白激酶D1（PKD1）的微小RNA（miRNA），探讨其在蛙皮素诱导的大鼠急性胰腺炎（AP）中发挥的作用。方法生物信息学软件预测PKD1的上游调控miRNA，并通过双荧光素酶报告基因和Western blot验证目标miRNA对PKD1表达的调控。采用蛙皮素（20 μg/kg）腹腔注射（每隔1 h注射1次，连续6次）的方法构建AP模型。大鼠分为正常组（10只）、AP组（20只）和治疗组（20只），其中，正常组不做处理，AP组和治疗组分别在造模前腹腔注射对照miRNA和带CY5荧光基因的miRNA模拟体。AP组和治疗组各取10只在首次注射蛙皮素后6 h处死，其余在首次注射后24 h处死。所有动物下腔静脉采血检测血清淀粉酶、脂肪酶活力；采集胰腺组织包埋切片行免疫组织化学染色检测PKD1的表达，HE染色进行AP病理评分。结果TargetSacn7.1软件预测miR-128-3p为PKD1潜在的调控miRNA，双荧光素酶报告基因和Western blot证实miR-128-3p与PRKD1 mRNA 3′UTR结合，抑制PKD1的蛋白表达。造模后6 h，AP组与治疗组血清淀粉酶和脂肪酶活力较正常组增高[13 313.00（9 424.00～15 995.00） U/L、13 552.00（10 399.50～18 408.25） U/L比1 430.50（1 214.25～1 543.25） U/L；547.00（515.00～627.00） U/L、857.50（522.00～1 222.25） U/L比34.00（32.50～34.75） U/L]，差异均有统计学意义（χ^2＝8.715，P〈0.05；χ^2＝9.115，P〈0.05），提示造模成功。造模后24 h，治疗组PKD1免疫组织化学评分[0.50（0～2.75）分]较正常组[4.00（4.00～8.00）分]和AP组[4.00（3.75～8.00）分]均下降，差异有统计学意义（χ^2＝18.302，P〈0.05）。炎性细胞浸润和组织坏死明显好转，病理评分总分显著低于AP组（3.80±0.85比6.90±1.15，t＝4.481，P〈0.01）。结论miR-128-3p是PKD1的上游调控miRNA，可抑制PKD1介导的AP大鼠的组织坏死和炎性细胞浸润。ObjectiveTo predict and verify the upstream regulatory microRNA （miRNA） of protein kinase D1 （PKD1）, and to investigate its role in cerulein induced acute pancreatitis （AP） in rats.MethodsPotential upstream regulatory miRNA of PKD1 was predicted by using bioinformatics software.Dual luciferase reporter gene system and Western blot were applied to verify the regulation of PKD1 by the selected miRNA.Experimental AP was induced by 6 intraperitoneal injection of cerulein （20 μg/kg） at hourly intervals after administration of the CY5-labeled notarget control （AP group, n=20） or selected miRNA （treatment group, n=20）, respectively by intraperitoneal injection into rats.Other rats were divided randomly into a normal control group （n=10） without any treatment.Besides 10 rats in either AP or treatment group were sacrificed 6 hours after the first injection of cerulein, and the rats were all sacrificed 24 hours after the first injection.The blood samples and pancreatic tissues of each rat were collected to test serum amylase and lipase activities, or to make hematoxylin-eosin stain for AP pathological scores as well as PKD1 immunohistochemical staining, respectively.ResultsTargetScan 7.1 software analysis showed that miR-128-3p was the potential upstream regulatory miRNA of PKD1, which was verified by dual luciferase reporter gene system and Western blot detection.Compared to the normal control group, serum amylase and lipase activities after 6 h exposure to cerulein increased in both AP group and the treatment group[13 313.00（9 424.00-15 995.00） U/L, 13 552.00（10 399.50-18 408.25） U/L vs.1 430.50（1 214.25-1 543.25） U/L; 547.00 （515.00-627.00） U/L, 857.50（522.00-1 222.25） U/L vs.34.00（32.50-34.75） U/L], and the differences were significant（χ^2=8.715, P〈0.05; χ^2=9.115, P〈0.05）, which indicated that the rat models of AP were successfully established.The immunohistochemical scores of PKD1 after 24 h exposure to cerulein decreased in the treatment group[0.50（0-2

关 键 词：急性胰腺炎 miR-128-3p 蛋白激酶D1 坏死 

分 类 号：R576[医药卫生—消化系统]