敲除Vanin-1促进肾缺血再灌注损伤后功能恢复的作用及机制  

Effect and mechanism of vanin-1 knockout in promoting recovery of kidney function after renal ischemia reperfusion injury in mice

作　　者：王玲[1] 汪晓月 封蕾[1] 王梨名 罗佳[1] 陈佳[1] 陈客宏[1] WANG Ling;WANG Xiaoyue;FENG Lei;WANG Liming;LUO Jia;CHEN Jia;CHEN Kehong(Department of Nephrology,Daping Hospital,Army Medical University(Third Military Medical University),Chongqing,400042,China)

机构地区：[1]陆军军医大学(第三军医大学)大坪医院肾内科,重庆400042

出　　处：《第三军医大学学报》2021年第7期593-598,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81770731);重庆市技术创新与应用发展专项面上项目(CSTC2019jscx-msxmX0258)。

摘　　要：目的探讨血管非炎症分子1(vascular noninflammatory molecule-1,Vanin-1)在缺血再灌注损伤后肾组织的表达水平及其与肾脏功能恢复的关系。方法选取8周龄BALB/c野生型、Vanin-1基因敲除雄鼠,采用完全随机法分为假手术组、缺血再灌注组、敲除组、敲除+缺血再灌注组,每组6只。缺血再灌注组、敲除+缺血再灌注组分离双侧肾蒂后夹闭35 min,后松开夹闭,恢复血流;假手术组、敲除组仅暴露肾蒂,不夹闭。术后0、3、14 d,留取血清、尿液、肾组织标本,检测血清肌酐、尿素氮水平,检测尿液TGF-β水平,PAS染色明确肾组织损伤严重程度,免疫组化检测肾组织Vanin-1表达水平。提取野生型和Vanin-1基因敲除鼠原代肾小管上皮细胞,传代至第2代,设置对照组、对照+损伤组、敲除组、敲除+损伤组,损伤条件为缺氧(1%O2,94%N2,5%CO2,48 h)、复氧(5%CO2,95%空气,24 h),模拟体内缺血再灌注损伤模型,收取肾小管上皮细胞和上清,检测Vanin-1蛋白和上清中TGF-β表达水平。结果缺血再灌注损伤后第3天,与假手术组相比,缺血再灌注组、敲除+缺血再灌注组血清肌酐、尿素氮水平明显升高(P<0.05),PAS染色提示肾组织损伤明显;但缺血再灌注组与敲除+缺血再灌注组小鼠比较,血清肌酐、尿素氮水平无明显差异,PAS染色提示两组肾组织损伤无明显差异。缺血再灌注后第14天,该两组血清肌酐、尿素氮、PAS肾组织损伤均比缺血再灌注后第3天显著恢复,敲除+缺血再灌注组的恢复速度均明显快于缺血再灌注组,PAS肾组织慢性化改变明显轻于缺血再灌注组(P<0.05),且敲除+缺血再灌注组尿中TGF-β水平明显低于缺血再灌注组(P<0.05)。免疫组化结果提示:损伤后第14天,肾组织Vanin-1表达明显升高(P<0.01),且主要表达于受损肾小管上皮细胞。细胞实验结果显示:予以缺氧-复氧损伤后,对照+损伤组肾小管上皮细胞Vanin-1蛋白表达明显升高(P<0.01)Objective To explore the expression level of vascular noninflammatory molecule-1(Vanin-1) in renal tissue after renal ischemia-reperfusion(I/R) injury and its relationship with the recovery of renal function. Methods Eight-week-old male BALB/c wild-type and Vanin-1 knockout mice were subjected in this study. They were randomly divided into sham operation group, I/R group, knockout group, knockout+ I/R group, with 6 mice in each group. In the I/R model groups, the bilateral renal pedicles were separated and clamped for 35 min, and then the clamp was released to restore blood flow. For the sham operation and knockout groups, only the renal pedicles were exposed without clipping. Serum, urine, and kidney tissue specimens were collected at 0, 3, and 14 d after surgery to determine blood creatinine and urea nitrogen levels and urine TGF-β level. PAS staining was used to determine the severity of renal tissue damage, and immunohistochemical assay was employed to test the expression of Vanin-1 in renal tissue. After renal tubular epithelial cells were isolated from the kidneys of the wild-type and Vanin-1 knockout mice, the cells were primarily cultured and then passed down to the second generation. Then the cells were divided into control group, injury group, knockout group, and knockout+injury group. The cell injury was inflicted with culture condition of hypoxia(1%O2, 94%N2, 5%CO2) for 48 h followed by reoxygenation(5%CO2, 95% air) for 24 h to simulate in vitro I/R injury model. The expression of Vanin-1 and supernatant content of TGF-β were measured. Results In 3 d after I/R injury, serum creatinine and urea nitrogen levels were significantly higher(P<0.05), and PAS staining indicated obvious renal tissue damage in the I/R and knockout+I/R groups when compared the sham operation group. But there were no significant differences in the levels and the tissue damages between the I/R and knockout+I/R groups. However, on the 14 th day after I/R injury, the 2 groups got the serum creatinine and urea nitrogen levels and r

关 键 词：血管非炎症分子1 再灌注损伤 肾小管细胞 转化生长因子Β 肾脏功能恢复 小鼠 基因敲除 

分 类 号：R363.21[医药卫生—病理学] R364.12[医药卫生—基础医学]