KCNQ1在尿酸盐晶体引起单核巨噬细胞分泌IL-1β中的作用研究
发布时间：2018-10-19 16:39
【摘要】：目的:探讨钾离子通道蛋白KCNQ1在尿酸盐晶体刺激人单核巨噬细胞分泌IL-1β中可能的作用。方法:以浓度为60μg/ml的丙二醇甲醚醋酸酯(Phorbol-12-myristate-13-acetate,PMA)诱导人急性单核细胞白血病细胞系(human acute monocytic leukemia cell line,THP-1),诱导时间为72h,得到人单核巨噬细胞。荧光定量PCR检测人单核巨噬细胞中KCNQ1辅基KCNE1是否表达,及相对于KCNQ1的表达量。将实验分成4组:对照组、尿酸盐晶体组、尿酸盐晶体和通道激活剂(ML-277,20nmol/ml)组、尿酸盐晶体和通道阻滞剂(Chromanol 293B,100nmol/ml)组。各实验组中尿酸盐晶体的量均为:100μg/ml。在每组加入相应阻滞剂或抑制剂预处理30min后,期间每10分钟晃动一次,以保持作用充分。之后,实验组加入等量尿酸盐晶体。6h后,应用ELISA法检测上清液中成熟IL-1β的浓度,应用荧光定量PCR检测细胞内IL-1β前体、IL-18前体、TNF-α的表达量。刺激2h后,去上清,用10%的硝酸溶解消化细胞,应用原子吸收分光光度计检测每孔细胞内钾离子的量;应用ATP试剂盒检测细胞内ATP的浓度。结果:辅基KCNE1在该细胞中同时表达,且表达量明显高于KCNQ1.尿酸盐晶体刺激6h后,上清液中成熟IL-1β的浓度,通道激活剂组与尿酸盐晶体组相比明显升高(p0.01),而该通道阻滞剂组与尿酸盐晶体组相比无明显差异。细胞内IL-1β前体、IL-18前体、TNF-α表达量,各实验组间无明显差异。尿酸盐晶体刺激2h后,细胞内钾离子浓度,激活剂组与尿酸盐晶体组相比,细胞内钾离子浓度进一步下降(p0.01),阻滞剂组略高于尿酸盐晶体组,但在目前实验条件下,我们没有观察到这一差异的统计学意义。实验组细胞内ATP的含量明显低于对照组,各实验组之间无明显差异。结论:1.正常状态下,KCNQ1钾离子通道在尿酸盐晶体刺激人单核巨噬细胞分泌IL-1β的过程中可能发挥有限作用,但当其过度激活时,可以明显增加成熟IL-1β的释放。2.因KCNQ1的结构异常使得KCNE1辅基对其抑制性调控作用减弱在成熟IL-1β分泌中的作用值得进一步研究。
[Abstract]:Aim: to investigate the role of potassium channel protein (KCNQ1) in the secretion of IL-1 尾 by human mononuclear macrophages stimulated by uric acid crystals. Methods: human mononuclear macrophages were obtained from 60 渭 g/ml propanediol methyl ether acetate (Phorbol-12-myristate-13-acetate,PMA) induced human acute monocytic leukemia cell line (human acute monocytic leukemia cell line,THP-1 for 72 h. Fluorescence quantitative PCR was used to detect the expression of KCNQ1 counit KCNE1 in human mononuclear macrophages and its expression relative to KCNQ1. The experiment was divided into four groups: control group, uric acid crystal and channel activator (ML-277,20nmol/ml) group, uric acid crystal and channel blocker group (Chromanol 293BX 100nmol / ml). The amount of uric acid crystals in each experimental group was 100 渭 g / ml. After pretreatment of 30min with corresponding blockers or inhibitors in each group, sloshing was made every 10 minutes to maintain full effect. After 6 hours, the concentration of mature IL-1 尾 in the supernatant was detected by ELISA assay, and the expression of IL-1 尾 precursor, IL-18 precursor and TNF- 伪 in the supernatant was detected by fluorescence quantitative PCR. After 2 hours of stimulation, the supernatant was removed, the digestion cells were dissolved with 10% nitric acid, the amount of potassium ions in each hole was detected by atomic absorption spectrophotometer, and the concentration of ATP in the cells was detected by ATP kit. Results: covalent KCNE1 was expressed at the same time in the cell, and the expression level was significantly higher than that in KCNQ1.. After stimulation for 6 h, the concentration of mature IL-1 尾 in the supernatant was significantly higher in the channel activator group than in the uric acid crystal group (p0.01), but there was no significant difference between the channel blocker group and the uric acid crystal group. There was no significant difference in the expression of IL-1 尾, IL-18 precursor and TNF- 伪 among the experimental groups. After being stimulated by uric acid crystal for 2 hours, the concentration of intracellular potassium ion in activator group decreased further (p0.01), the concentration of potassium ion in activator group was slightly higher than that in uric acid crystal group (p0.01), but under the present experimental conditions, We have not observed the statistical significance of this difference. The content of ATP in the experimental group was significantly lower than that in the control group, and there was no significant difference among the experimental groups. Conclusion: 1. In normal state, KCNQ1 potassium channel may play a limited role in the process of stimulation of IL-1 尾 secretion by human mononuclear macrophages, but when it is over-activated, it can obviously increase the release of mature IL-1 尾. 2. Due to the abnormal structure of KCNQ1, the inhibitory effect of KCNE1 counits on IL-1 尾 secretion is weakened, and the role of KCNE1 counit in the secretion of mature IL-1 尾 is worthy of further study.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.7
本文编号：2281718
[Abstract]:Aim: to investigate the role of potassium channel protein (KCNQ1) in the secretion of IL-1 尾 by human mononuclear macrophages stimulated by uric acid crystals. Methods: human mononuclear macrophages were obtained from 60 渭 g/ml propanediol methyl ether acetate (Phorbol-12-myristate-13-acetate,PMA) induced human acute monocytic leukemia cell line (human acute monocytic leukemia cell line,THP-1 for 72 h. Fluorescence quantitative PCR was used to detect the expression of KCNQ1 counit KCNE1 in human mononuclear macrophages and its expression relative to KCNQ1. The experiment was divided into four groups: control group, uric acid crystal and channel activator (ML-277,20nmol/ml) group, uric acid crystal and channel blocker group (Chromanol 293BX 100nmol / ml). The amount of uric acid crystals in each experimental group was 100 渭 g / ml. After pretreatment of 30min with corresponding blockers or inhibitors in each group, sloshing was made every 10 minutes to maintain full effect. After 6 hours, the concentration of mature IL-1 尾 in the supernatant was detected by ELISA assay, and the expression of IL-1 尾 precursor, IL-18 precursor and TNF- 伪 in the supernatant was detected by fluorescence quantitative PCR. After 2 hours of stimulation, the supernatant was removed, the digestion cells were dissolved with 10% nitric acid, the amount of potassium ions in each hole was detected by atomic absorption spectrophotometer, and the concentration of ATP in the cells was detected by ATP kit. Results: covalent KCNE1 was expressed at the same time in the cell, and the expression level was significantly higher than that in KCNQ1.. After stimulation for 6 h, the concentration of mature IL-1 尾 in the supernatant was significantly higher in the channel activator group than in the uric acid crystal group (p0.01), but there was no significant difference between the channel blocker group and the uric acid crystal group. There was no significant difference in the expression of IL-1 尾, IL-18 precursor and TNF- 伪 among the experimental groups. After being stimulated by uric acid crystal for 2 hours, the concentration of intracellular potassium ion in activator group decreased further (p0.01), the concentration of potassium ion in activator group was slightly higher than that in uric acid crystal group (p0.01), but under the present experimental conditions, We have not observed the statistical significance of this difference. The content of ATP in the experimental group was significantly lower than that in the control group, and there was no significant difference among the experimental groups. Conclusion: 1. In normal state, KCNQ1 potassium channel may play a limited role in the process of stimulation of IL-1 尾 secretion by human mononuclear macrophages, but when it is over-activated, it can obviously increase the release of mature IL-1 尾. 2. Due to the abnormal structure of KCNQ1, the inhibitory effect of KCNE1 counits on IL-1 尾 secretion is weakened, and the role of KCNE1 counit in the secretion of mature IL-1 尾 is worthy of further study.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.7
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