CAJ | 학술논문

目的观察非转移性黑色素瘤糖蛋白B(glycoprotein non-metastatic melanoma protein B,Gpnmb)在急性肾缺血再灌注损伤(IRI)肾脏和尿液中的表达,分析其与M1、M2表型巨噬细胞的相关性,探讨其在IRI免疫炎性反应损伤中的作用.方法 C57BL/6J小鼠被随机分为3组:正常对照组(n=4)、假手术组(n=4)、IRI组(n=12).以双侧肾蒂血管阻断30min建立IRI模型.PAS染色观察肾脏病理改变；免疫荧光双染色及流式细胞术检测Gpnmb和巨噬细胞标志物F4/80蛋白的表达及分布；实时荧光定量PCR检测Gpnmb及M1型巨噬细胞表型(CD40、CCR7)及M2型巨噬细胞表型(CD163、MMR)在肾脏中的mRNA表达；Western印迹和ELISA法检测小鼠尿液中Gpnmb的表达.结果 IRI小鼠肾脏肾小管上皮细胞脱落,肾间质内可见大量炎性细胞浸润.实时荧光定量PCR结果显示,与正常对照组、假手术组比较,IRI组小鼠肾脏Gpnmb mRNA于造模后第1天和第2天升高,差异有统计学意义(P＜ 0.01),第3天下降至接近正常水平.免疫荧光双染色及流式细胞术检测结果均显示Gpnmb蛋白主要表达在F4/80阳性的巨噬细胞内.Gpnmb mRNA表达与M1型巨噬细胞表型(CD40、CCR7)的mRNA表达无相关性,与M2型巨噬细胞表型(CD163、MMR)的mRNA表达呈正相关.Western印迹和ELISA结果示IRI组小鼠在造模后第1天尿液中的Gpnmb表达显著升高,与正常对照组和假手术组比较差异有统计学意义(P＜0.01),第3天降至接近正常水平.结论 Gpnmb在IRI肾脏的巨噬细胞和尿液中呈高表达,并与M2型巨噬细胞相关,提示Gpnmb可能参与了巨噬细胞的分型和急性肾损伤的炎性反应过程,临床上也可用作AKI早期诊断的生物学标志物.
목적 관찰비전이성흑색소류당단백B(glycoprotein non-metastatic melanoma protein B,Gpnmb)재급성신결혈재관주손상(IRI)신장화뇨액중적표체,분석기여M1、M2표형거서세포적상관성,탐토기재IRI면역염성반응손상중적작용.방법 C57BL/6J소서피수궤분위3조:정상대조조(n=4)、가수술조(n=4)、IRI조(n=12).이쌍측신체혈관조단30min건립IRI모형.PAS염색관찰신장병리개변；면역형광쌍염색급류식세포술검측Gpnmb화거서세포표지물F4/80단백적표체급분포；실시형광정량PCR검측Gpnmb급M1형거서세포표형(CD40、CCR7)급M2형거서세포표형(CD163、MMR)재신장중적mRNA표체；Western인적화ELISA법검측소서뇨액중Gpnmb적표체.결과 IRI소서신장신소관상피세포탈락,신간질내가견대량염성세포침윤.실시형광정량PCR결과현시,여정상대조조、가수술조비교,IRI조소서신장Gpnmb mRNA우조모후제1천화제2천승고,차이유통계학의의(P＜ 0.01),제3천하강지접근정상수평.면역형광쌍염색급류식세포술검측결과균현시Gpnmb단백주요표체재F4/80양성적거서세포내.Gpnmb mRNA표체여M1형거서세포표형(CD40、CCR7)적mRNA표체무상관성,여M2형거서세포표형(CD163、MMR)적mRNA표체정정상관.Western인적화ELISA결과시IRI조소서재조모후제1천뇨액중적Gpnmb표체현저승고,여정상대조조화가수술조비교차이유통계학의의(P＜0.01),제3천강지접근정상수평.결론 Gpnmb재IRI신장적거서세포화뇨액중정고표체,병여M2형거서세포상관,제시Gpnmb가능삼여료거서세포적분형화급성신손상적염성반응과정,림상상야가용작AKI조기진단적생물학표지물.
Objective To observe the expression of glycoprotein non-metastatic melanoma protein B (Gpnmb) in the kidney and urine after ischemic-reperfusion injury (IRI),and explore the relationship between Gpnmb and macrophage phenotypes in the IRI kidney.Methods Male C57BL/6J mice were randomly divided into control group (n =4),sham group (n =4) and IRI group (n =12).Both renal pedieles of mice in IRI group were identified and occluded with microvascular clamps for 30 min.Renal pathological injury was observed by PAS staining.The expression of Gpnmb was examined by real-time PCR and immunofluoresence staining.The location of Gpnmb was observed by flow cytometry and double immunofluoresence staining with F4/80.The mRNA expressions of Gpnmb,CD40,CRR7,CD163 and MMR were examined by real-time PCR.The expression of Gpnmb in the urine was examined by Western blotting and ELISA.Results PAS-stained IRI kidney section showed desquamative epithelia,necrosis debris and a large number of inflammatory cell infiltration.Real-time PCR results showed that there was little expression of Gpnmb in the kidney of control group and sham group.However,the Gpnmb mRNA level in IRI kidneys was highly up-regulated at day 1 and day 2 (both P ＜ 0.01) and followed by a decrease that was similar to the control level at day 3.Double immunofluoresence staining of kidney sections from IRI mice revealed that Gpnmb was predominantly detected in F4/80 positive macrophages.The mRNA expression of Gpnmb was not correlated with M1 macrophage phenotypes CD40 and CCR7,but positively correlated with M2 macrophages phenotypes CD163 and MMR.Western blotting and ELISA result showed that there was significant increase of Gpnmb expression in the urine from IRI mice compared to those of the control group and the sham group (P ＜ 0.01).Conclusions Gpnmb expression is up-regulated in IRI kidney and is associated to M2 macrophages.It may play a role in the process of acute kidney injury.Gpnmb expression is also increased in urine after IR injury and it may be a new biomarker to diagnose AKI.