TSP1对高糖诱导的肾小管上皮细胞损伤的影响研究  

Effect of TSP1 on high glucose-induced damage of renal tubular epithelial cells

作　　者：周才芳 曾秀琴[1] 曾庆义[1] Zhou Caifang;Zeng Xiuqin;Zeng Qingyi(Department of Nephropathy, Third People's Hospital of Huizhou City, Huizhou 516000, China)

机构地区：[1]惠州市第三人民医院肾内科

出　　处：《中华细胞与干细胞杂志（电子版）》2019年第2期65-71,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

摘　　要：目的探讨血小板反应蛋白1(TSP1)对高糖诱导的肾小管上皮细胞损伤及炎症因子分泌的影响。方法以肾小管上皮细胞为研究对象,通过转染TSP-1 shRNA(shTSP-1),沉默TSP-1 基因,探讨下调TSP1 对高糖条件下肾小管上皮细胞损伤的影响。肾小管上皮细胞依次分为:对照组(以5.6 mmol/L 葡萄糖培养)、高糖组(以30 mmol/L 葡萄糖培养)、NC +高糖组(对照慢病毒转染,以30 mmol/L 葡萄糖培养)、干扰+高糖组(TSP1 shRNA 慢病毒转染以30mmol/L 葡萄糖培养)。 Realtime PCR 和Western Blot 检测高糖对细胞中TSP1 表达影响,同时检测TSP1 shRNA 干扰效果。DCFH-DA 法检测细胞中活性氧(ROS)水平,硫代巴比妥酸法检测培养液中丙二醛(MDA)含量,ELISA 法检测培养液上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)含量,Annexin V-FITC/PI 双染法检测细胞凋亡,Western Blot 法检测细胞中活化的Caspase-3(c-caspase-3)蛋白水平。两组均数差异比较采用独立样本t 检验,多组均数间差异比较采用单因素方差分析,组间两两比较采用SNK-q 检验。结果高糖组细胞中TSP1mRNA 和蛋白水平高于对照组(P < 0.05)。干扰+高糖组细胞中TSP1 mRNA 和蛋白水平低于高糖组(P < 0.05)。与对照组比较,高糖组细胞中ROS 水平升高,培养液中MDA、TNF-α、IL-8 含量升高[(2.36±0.21)nmol/ml、(45.91±2.87)ng/ml、(25.42±3.26) ng/ ml :(1.05±0.13)nmol/ ml、(20.14±1.36)ng/ml、(12.98±1.63)ng/ml],差异有统计学意义(F = 18.595,F =43.825,F = 21.155,P < 0.05);细胞凋亡率升高(P < 0.05),细胞中c-caspase-3 蛋白水平升高(P <0.05)。与高糖组和NC +高糖组比较,干扰+高糖组细胞中ROS 水平降低,培养液中MDA、TNF-α、IL-8 含量降低[(1.63±0.10)nmol/ml、(34.20±2.06)ng/ml、(18.75±1.62)ng/ ml :(2.36 ± 0.21) nmol/ ml、(45.91±2.87)ng/ml、(25.42±3.26)ng/ml 和(2.30±0.42)nmol/ ml、(46.32±5.24) ng/ml、(26.91±2.74)ng/ml],差异具有统计学意义(F = 18.595,F = 43.825,F =21.155,P < 0Objective To investigate the effects of TSP1 on renal tubular epithelial cell injury and secretion of inflammatory factors induced by high glucose. Methods TSP-1 shRNA (shTSP-1)was transfected into renal tubular epithelial cells to silence TSP-1 gene. The renal tubular epithelial cells were divided into control group(treated with 5.6 mmol/L glucose), high glucose group(treated with 30 mmol/L glucose), NC + high glucose group(treated with 30 mmol/ L glucose, transfected with lentivirus control), interference + high glucose group(treated with 30 mmol/L glucose culture, transfected with TSP1 shRNA lentivirus). Realtime PCR and Western Blot were used to detect the effect of high glucose on the expression of TSP1 and the effect of TSP1 shRNA on the expression of TSP1. The level of ROS in cells was detected by DCFH-DA. The content of MDA in culture medium was detected by thiobarbituric acid. ELISA was used to detect TNF-α and IL-8 in the supernatant of the culture medium. Annexin V-FITC/PI double staining was used to detect apoptosis. Western Blot assay was used to detect c-caspase-3 in cells. Independent sample t test was used to compare the mean difference between the two groups, single factor analysis of variance was used to compare the difference among multiple groups, and SNK-q test was used to compare the two groups. Results The levels of TSP1 mRNA and protein in the high glucose group were higher than those in the control group(P < 0.05). The levels of TSP1 mRNA and protein in the interference + high glucose group were lower than those in the high glucose group (P < 0.05). Compared with the control group, the level of ROS in the high glucose group increased, the contents of MDA, TNF-α and IL-8 increased in culture medium, the rate of apoptosis increased, the level of c-caspase-3 protein in the cells increased [(2.36±0.21)nmol/ml,( 45.91±2.87)ng/ml,( 25.42±3.26)ng/ ml vs (1.05±0.13)nmol/ml,( 20.14±1.36)ng/ml,( 12.98±1.63)ng/ml], the difference has statistical significance (P < 0.05). Compared with high gl

关 键 词：TSP1 肾小管上皮细胞 高糖 炎症因子 损伤 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]