网站首页杂志简介编委会成员过刊目录投稿指南在线订阅联系我们读者论坛Mil Med Res
消息通知:

 

位置:首页 >> 在线订阅
Slit2对高糖诱导的肾小管上皮细胞上皮间质转分化的作用及机制
作者:胡成芳 李艺 刘军辉 王小龙 侯世会 官涛 黄云剑 
单位:400037 重庆 陆军军医大学附属新桥医院肾内科(胡成芳、李艺、刘军辉、王小龙、侯世会、官涛、黄云剑) 
关键词:Slit2 ROBO1 上皮间质转分化 高糖 HK-2细胞 
分类号:R692.6
出版年,卷(期):页码:2019,44(8):652-658
摘要:

 [摘要]  目的  探讨Slit2/ROBO1信号通路在高糖诱导肾小管上皮细胞上皮间质细胞转分化(EMT)中的作用及机制。方法  体外培养人肾小管上皮细胞HK-2,进行高糖浓度梯度实验,随机分为正常组、对照组1、对照组2和高糖组1、高糖组2;同时进行高糖时间梯度实验,随机分为正常组、对照组、高糖24 h组、高糖36 h组和高糖48 h组,采用Western blotting检测HK-2细胞中Slit2ROBO1α-平滑肌肌动蛋白(α-SMA)和纤维连接蛋白的表达变化,筛选出最佳高糖刺激浓度和时间。采用Slit2过表达质粒及阴性对照质粒转染HK-2细胞,验证转染成功后,随机分为正常组、对照组、高糖组、高糖+空载组及高糖+Slit2组,经最佳高糖浓度及时间刺激后提取总蛋白,Western blotting检测纤维连接蛋白及α-SMA的表达变化。结果  在高糖浓度梯度实验中,与正常组相比,高糖组1、高糖组2Slit2表达明显下降(分别为0.647±0.0480.210±0.023 vs. 1.000±0.050)ROBO1表达明显下降(分别为0.703±0.0410.303±0.022 vs. 1.000±0.057),纤维连接蛋白表达明显增加(分别为1.953±0.0422.997±0.078 vs. 0.990±0.059)α-SMA表达明显增加(分别为1.767±0.0122.427±0.059 vs. 1.033±0.067),差异均有统计学意义(P<0.05)。与高糖组1相比,高糖组2 Slit2ROBO1表达下降,纤维连接蛋白和α-SMA表达增加,差异有统计学意义(P<0.05)。在高糖时间梯度实验中,与正常组相比,高糖36 h组、高糖48 h组的Slit2表达明显下降(分别为0.557±0.0200.450±0.055 vs. 0.943±0.032)ROBO1表达下降(分别为0.600±0.0230.227±0.028 vs. 1.000±0.058),高糖24 h组、高糖36 h组、高糖48 h组纤维连接蛋白表达增加(分别为1.247±0.0521.733±0.0842.780±0.090 vs. 0.970±0.040)α-SMA表达增加(分别为1.277±0.0411.767±0.1202.537±0.078 vs. 1.033±0.067),差异均有统计学意义(P<0.05)。与高糖24 h组相比,高糖36 h组、高糖48 h组的Slit2表达下降(分别为0.557±0.0200.450±0.055 vs. 0.893±0.034)ROBO1表达下降(分别为0.600±0.0230.227±0.028 vs. 0.930±0.025),纤维连接蛋白及α-SMA表达增加,差异均有统计学意义(P<0.05)。与高糖36 h组相比,高糖48 h组的Slit2ROBO1表达下降,纤维连接蛋白和α-SMA表达增加,差异均有统计学意义(P<0.05)。在高糖环境下,Slit2过表达及阴性对照质粒转染成功后,观察Slit2过表达对肾小管EMT的影响发现,与正常组相比,高糖组、高糖+空载组、高糖+Slit2组的纤维连接蛋白表达增加(分别为2.760±0.0122.667±0.0271.460±0.034 vs. 1.000±0.058)α-SMA表达水平增加(分别为2.487±0.0482.557±0.0371.270±0.017 vs. 1.000±0.050),差异有统计学意义(P<0.05)。与高糖+空载组相比,高糖+Slit2组的纤维连接蛋白和α-SMA表达下降,差异均有统计学意义(P<0.05)结论  Slit2ROBO1的表达下降参与了高糖诱导的肾小管上皮细胞EMT过程,过表达Slit2可显著抑制高糖诱导的EMT

 [Abstract]  Objective  To explore the effect of Slit2/ROBO1 protein (Slit2/ROBO1) signaling pathway in high glucose-induced epithelial-mesenchymal transdifferentiation (EMT) and its mechanism. Methods  Human renal tubular epithelial cells (HK-2) were cultured in vitro and subjected to high glucose concentration and time gradient experiments. First, for concentration gradient experiment, the sample was randomly divided into normal group, control group 1, control group 2, high glucose group 1, high glucose group 2. While for high glucose time gradient experiment, the sample was randomly divided into normal group, control group, high glucose 24 h group, high glucose 36 h group and high glucose 48 h group. Western blotting was used to detect the expression changes of Slit2, ROBO1, α-smooth muscle actin (α-SMA) and fibronectin in HK-2 cells, and then the optimal high glucose stimulation concentration and time were screened out. Slit2 over-expressed plasmid and negative control plasmid were transfected into HK-2 cells to verify the successful transfection, the cells were then randomly divided into normal group, control group, high glucose group, high glucose empty group and high glucose Slit2 group. The total protein was extracted after stimulation with optimal high glucose concentration and time, and Western blotting was then performed to detect the change in expression of fibronectin and α-SMA. Results  In the high glucose concentration gradient experiment, the expression of Slit2 declined significantly in high glucose group 1(0.647±0.048) and high glucose group 2(0.210±0.023) than in the normal group (1.000±0.050); the expression of ROBO1 declined significantly in high glucose group 1(0.703±0.041) and high glucose group 2(0.303±0.022) than in the normal group (1.000±0.057); while the expression of fibronectin increased significantly in high glucose group 1(1.953±0.042) and high glucose group 2(2.997±0.078) than in the normal group (0.990±0.059), and the expression of α-SMA increased significantly in high glucose group 1(1.767±0.012) and high glucose group 2(2.427±0.059) than in the normal group (1.033±0.067), all the differences were of statistical significance(P<0.05). Compared with the high glucose group 1, the expressions of Slit2 and ROBO1 decreased, and of fibronectin and α-SMA increased significantly in the high glucose group 2(P<0.05). In the high glucose time gradient experiment, compared with the normal group, the expressions of Slit2 in high glucose 36 h group and high glucose 48 h group decreased (0.943±0.032 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 decreased (1.000±0.058 vs. 0.600±0.023, 0.227±0.028, respectively). Compared with the normal group, the expression of fibronectin increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (0.970±0.040 vs. 1.247±0.052, 1.733±0.084, 2.780±0.090, respectively), and the expression of α-SMA increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (1.033±0.067 vs. 1.277±0.041, 1.767±0.120, 2.537±0.078, respectively), and the difference was statistically significant (P<0.05). Compared with high glucose 24 h group, the expression of Slit2 declined significantly in high glucose 36 h group and high glucose 48 h group(0.893±0.034 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 declined significantly (0.930±0.025 vs. 0.600±0.023, 0.227±0.028, respectively), the expressions of fibronectin and α-SMA increased significantly with statistical significance (P<0.05). Compared with high glucose 36 h group, the expression of Slit2 and ROBO1 declined significantly, and the expression of fibronectin and α-SMA increased significantly in high glucose 48 h group (P<0.05). In the high glucose environment, and achieving Slit2 overexpression and negative control plasmid transfection, the expression of fibronectin increased significantly in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.760±0.012, 2.667±0.027, 1.460±0.034, respectively) than in normal group (1.000±0.058); the expression of α-SMA increased also in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.487±0.048, 2.557±0.037, 1.270±0.017, respectively) than in normal group (1.000±0.050) with statistical significance (P<0.05). Compared with the high glucose+empty group, the expression of fibronectin and α-SMA declined significantly in the high glucose+Slit2 group(P<0.05). Conclusion  The decreased expression of Slit2 and ROBO1 is involved in the high glucose-induced renal tubular EMT. Overexpression of Slit2 may significantly inhibit the high glucose-induced EMT.

基金项目:
国家自然科学基金(81370821、81600583)
作者简介:
参考文献:
服务与反馈:
文章下载】【加入收藏

copyright ©《解放军医学杂志》编辑部
地址:北京市海淀区复兴路22号甲3号人民军医出版社   邮编:100036
电话:0201-882929-8021(军线)    010-51927306 (传真)
京ICP备06014771-2