摘要
目的观察脂多糖作用下大鼠腹膜间皮细胞表达干扰素诱导蛋白(IP)-10的情况及核因子(NF)KB信号通路在其中的作用。方法分离及培养大鼠腹膜间皮细胞。于脂多糖不同浓度(0、10、100、1 000、10 000 ng/ml)作用3 h及脂多糖(100 ng/ml)作用不同时间点(0、1、3、6、12、24、48 h)收集细胞;脂多糖(100 ng/ml)或BAY 11-7085(一种IκBα的磷酸化抑制剂)不同浓度(5μmol/L)预处理2 h加脂多糖,作用3 h后收集细胞;采用RT-PCR方法检测IP-10 mRNA表达。采用蛋白印迹检测NF-κB p65(p65)蛋白表达,采用ELISA方法测定IP-10蛋白的表达。结果与常规培养基对照组相比,10 ng/ml脂多糖组IP-10 mRNA表达显著升高(P <0.05), 1 000 ng/ml脂多糖组最高,但与10 ng/ml脂多糖组相比,差异无统计学意义(P>0.05)。脂多糖(100 ng/ml)作用下,IP-10 mRNA表达从1 h开始升高,3 h达到高峰,之后逐渐降低。常规培养的大鼠腹膜间皮细胞结构性表达p-p65蛋白;加入脂多糖后,p-p65蛋白表达显著增加,其中30 min至1 h表达最强,之后逐渐降低,至2 h仍显著高于常规培养组(P <0. 05)。加入5μmol/L BAY11-7085后,脂多糖诱导的IP-10基因和蛋白表达显著降低(P<0.05)。结论脂多糖以时间依赖和浓度依赖模式上调IP-10的表达。NF-κB信号途径参与调节脂多糖诱导的大鼠腹膜间皮细胞IP-10的表达。
Objective To observe the expression of interferon induced protein （IP） -10 and the role of nuclear factor-kappaB （NF-κB） signaling pathway in rat peritoneal mesothelial cells （RPMCs） un- der the action of lipopolysaccharide （LPS）. Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and cultured under defined in vitro conditions. The cells were exposed respectively to dif- ferent concentrations of LPS （0, 10, 100, 1 000, 10 000 ng/ml） for 3 h or treated with LPS （ 100 ng/ml） for different time points （0, 1, 3, 6, 12, 24, 48 h）. For observing the effect of LPS on the expression of p-p65 and p65, the RPMCs were treated with LPS （100 ng/ml） for different time points （0, 15, 30, 60, 120 rain）. For observing the effect of BAYll-7085 on the expression of IP-10 mRNA, the RPMCs were treated by LPS or pretreated with BAY11-7085 （5 μmol/L） for 2 h, then treated with LPS for another 3 h, respectively. Expression of IP-10 mRNA was examined by reverse transcription-polymerase chain reaction （RT-PCR）. Expression of NF-κB and p-NF-κB protein was detected by Western blot. The secretion of IP- 10 was determined by enzyme-linked immunosorbent assay （ELISA）. Results Compared with the control group, stimulation of RPMCs with 10 ng/ml LPS resulted in a significant increase in the expression of IP-10 mRNA （P 〈 0. 05）. 1 000 ng/ml LPS has the strongest effect on IP-10 expression compared with that of 10 ng/ml and 100 ng/ml LPS. Treatment with 100 ng/ml LPS resulted in time-dependent increase in the gene level of IP-10, with the peak at 3 h. However, after that time point, the gene level of them was gradually attenuated. Following treatment with LPS （ 100 ng/ml） , the level of p-NF-κB began to increase at 15 rain, gradually reached the peak at 1 hour, and then decreased. But the level of which at 2 h is still significant higher than that of medium control. 5 μmol/L BAY1 1-7085 significantly decreased the up-regulation of IP- 10 induced by LPS. Conclusions LPS enhanced the expression of IP-10 on RPMCs in a concentration-de- pendent and a time-dependent manner. LPS induced expression of IP-IO depended on the NF-κB signal transduction pathway.
作者
张云芳
冯俊霞
赵世莉
徐嘉琪
李红艳
Zhang Yunfang;Feng Junxia;Zhao Shill; Xu Jiaqi; Li Hongyan(Department of Nephrology, Huadu District People＇s Hospital, Guangzhou 510800, Chin)
出处
《中国医师杂志》
CAS
2018年第7期990-994,998,共6页
Journal of Chinese Physician
基金
广东省自然基金(2016A030313420)
广东省科技计划项目(粤科规财【2015】110号0024)
广州市医学重点学科建设项目(2017-2020)
花都区医疗卫生一般科研项目(16-HDWS-015
17-HDWS-001)~~
