高糖通过ERK_(1/2)途径调节足细胞产生明胶酶B  

High glucose regulates the production of MMP-9 in podocyte through ERK_(1/2) signal pathway

作　　者：白亚玲[1] 黄海长[1] 李惊子[1] 赵玉庸[1] 王海燕[1] 

机构地区：[1]北京大学第一医院肾内科北京大学肾脏病研究所,现工作于河北医科大学第四医100034

出　　处：《中华医学杂志》2005年第21期1451-1455,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30270612);北京大学"十五211工程"基金资助项目;人类功能基因与疾病基因学研究学科群基金资助项目

摘　　要：目的 探讨高糖持续刺激对足细胞培养上清液明胶酶活性、Ⅳ型胶原的影响及可能的信号途径。方法 小鼠足细胞系分为正常糖组、高糖组和甘露醇高渗对照组。应用明胶酶谱法观察三组细胞培养液上清明胶酶活性的动态变化,Western印迹分析法检测细胞培养液上清中Ⅳ型胶原α5链蛋白水平及足细胞ERK信号途径活化的变化。RT-PCR方法检测MMP-9 mRNA的表达。结果正常糖组和甘露醇高渗对照组培养液上清MMP-2和MMP-9活性在10天实验时间内无明显变化。高糖组细胞培养液上清MMP-2的活性没有明显变化;MMP-9活性于第2天开始升高,第3天达高峰为正常糖组的144.2±18.7%(P=0.006),第5天下降,第7天回到基础水平为正常糖组的76.6%±16.4%(P=0.218),直至第10天。足细胞培养上清中有较高基础水平的Ⅳ型胶原α5链蛋白;高糖组细胞培养上清Ⅳ型胶原α5链蛋白表达第2天开始下降,第3天达最低水平41.9%±25.5%(P=0.047),第5天回升到基础水平持续至第7天。上清MMP-9的活性与Ⅳ型胶原α5链蛋白的表达量呈负相关(r=-0.577,P=0.006)。正常糖组和甘露醇高渗对照组细胞培养上清Ⅳ型胶原α5链蛋白无变化。高糖刺激足细胞2天MMP-9 mRNA水平增加为正常糖组的199.8%±40.2%(P=0.003),刺激5天时为正常糖组的90.9±8.8%(P=0.411)。高糖刺激足细胞30 min可以活化ERK1/2,6 h达高峰,持续至24 h,48 h降至基础水平;在足细胞上未能检测到p38和JNK活化。ERK活化特异抑制剂PD98059预处理细胞后,能阻断高糖引起的MMP-9的活性和MMP-9 mRNA增加及Ⅳ型胶原α5链蛋白的下降。结论 ERK信号传导途径介导了高糖刺激足细胞MMP-9生成及相应的Ⅳ型胶原α5链蛋白动态反向改变。Objective To assess the effect of high glucose on the production of gelatinase and collagen α5 (Ⅳ) protein in podocytes and its possible signal pathway. Methods Mouse podocytes of an immortalized cell line were cultured and divided into 3 groups: NG group, treated with normal concentration of D-glucose (100 mg/dl) , HG group, treated with high concentration of D-glucose (450 mg/dl) , and MN group, treated with mannitol (350 mg/dl) plus D-glucose (100 mg/dl). The culture medium supernatants were collected every day. The activity of MMP-9 and MMP-2 was detected by gelatin zymography, the level of collagen α5 (Ⅳ) protein and the activation of MAPKs ( Erk, p38, and JNK) signaling pathway in podocytes were detected by Western blot analysis, and the level of MMP-9 mRNA was detected by RT-PCR. Another podocytes were pretreated by PD9805 , a specific inhibitor of MEK1 activation, and then divided into 3 groups as mentioned above so as to detect the effects of high glucose on the MMP-9 activation, and expression of MMP-9 mRNA and collagen α5 (Ⅳ) protein. Results The MMP-2 and MMP-9 activity in the medium supernatants of the NG and MN groups remained constant during the 10 days' incubation. High glucose incubation also did not affect the activity of MMP-2. The MMP-9 activity in the supernatant of the HG group began to increase in the 2nd day, reached the maximum in the 3rd day (144. 2±18. 7% that of the NG group, P =0. 006) , then began to decline since the 5th day, back to the basal level in the 7th day (76. 6±16. 4% that of the NG group,P =0. 218) , and remained at the basal level until the 10 th day. The basal level of collagen α5 (Ⅳ) protein in the supernatant of the NG group was quite high. The collagen α5 (Ⅳ) protein level in the supernatant of the HG group began to decrease since the 2nd day, reached the minimum in the 3rd day (41. 9±25. 5% that of the NG group,P =0. 047) , then backed to the basal levels in the 5th day, and retained at that level to the 7th days. The MMP9 activity in the supernata

关 键 词：ERK1/2 足细胞 明胶酶B MMP-9MRNA MMP-9活性 Western印迹 RT-PCR方法 细胞培养液 Ⅳ型胶原 细胞培养上清液 ERK信号途径 PD98059 MMP-2 基础水平 调节 mRNA水平 高糖刺激 信号传导途径 明胶酶谱法 链蛋白 甘露醇 动态变化 

分 类 号：R587.2[医药卫生—内分泌]