非转移性黑色素瘤糖蛋白B在急性肾缺血再灌注损伤中的表达及与巨噬细胞表型的关系  

Expression of Gpnmb in the acute ischemia-reperfusion injury and the relationship betweenGpnmb and macrophage phenotypes

作　　者：周乐天[1] 刘伏友[1] 

机构地区：[1]中南大学湘雅二医院肾脏病研究所,长沙410011

出　　处：《中华肾脏病杂志》2013年第7期509-514,共6页Chinese Journal of Nephrology

基　　金：中南大学青年教师助推专项资助项目（2011QNZTl82）

摘　　要：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（CDl63、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GpnmbmRNA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。GpnmbmRNA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（CDl63、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。Objective To observe the expression of glycoprotein non- metastatic melanoma protein B （Gpnmb） in the kidney and urine after ischemie- reperfusion injury （IRI）, and explore the relationship between Gpnmb and maerophage 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 microvaseular 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 cytometryand 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 inc

关 键 词：缺血再灌注 急性肾损伤 巨噬细胞 非转移性黑色素瘤糖蛋白B 

分 类 号：R692[医药卫生—泌尿科学]