【摘要】：目的:探討鉀離子通道蛋白KCNQ1在尿酸鹽晶體刺激人單核巨噬細(xì)胞分泌IL-1β中可能的作用。方法:以濃度為60μg/ml的丙二醇甲醚醋酸酯(Phorbol-12-myristate-13-acetate,PMA)誘導(dǎo)人急性單核細(xì)胞白血病細(xì)胞系(human acute monocytic leukemia cell line,THP-1),誘導(dǎo)時(shí)間為72h,得到人單核巨噬細(xì)胞。熒光定量PCR檢測人單核巨噬細(xì)胞中KCNQ1輔基KCNE1是否表達(dá),及相對(duì)于KCNQ1的表達(dá)量。將實(shí)驗(yàn)分成4組:對(duì)照組、尿酸鹽晶體組、尿酸鹽晶體和通道激活劑(ML-277,20nmol/ml)組、尿酸鹽晶體和通道阻滯劑(Chromanol 293B,100nmol/ml)組。各實(shí)驗(yàn)組中尿酸鹽晶體的量均為:100μg/ml。在每組加入相應(yīng)阻滯劑或抑制劑預(yù)處理30min后,期間每10分鐘晃動(dòng)一次,以保持作用充分。之后,實(shí)驗(yàn)組加入等量尿酸鹽晶體。6h后,應(yīng)用ELISA法檢測上清液中成熟IL-1β的濃度,應(yīng)用熒光定量PCR檢測細(xì)胞內(nèi)IL-1β前體、IL-18前體、TNF-α的表達(dá)量。刺激2h后,去上清,用10%的硝酸溶解消化細(xì)胞,應(yīng)用原子吸收分光光度計(jì)檢測每孔細(xì)胞內(nèi)鉀離子的量;應(yīng)用ATP試劑盒檢測細(xì)胞內(nèi)ATP的濃度。結(jié)果:輔基KCNE1在該細(xì)胞中同時(shí)表達(dá),且表達(dá)量明顯高于KCNQ1.尿酸鹽晶體刺激6h后,上清液中成熟IL-1β的濃度,通道激活劑組與尿酸鹽晶體組相比明顯升高(p0.01),而該通道阻滯劑組與尿酸鹽晶體組相比無明顯差異。細(xì)胞內(nèi)IL-1β前體、IL-18前體、TNF-α表達(dá)量,各實(shí)驗(yàn)組間無明顯差異。尿酸鹽晶體刺激2h后,細(xì)胞內(nèi)鉀離子濃度,激活劑組與尿酸鹽晶體組相比,細(xì)胞內(nèi)鉀離子濃度進(jìn)一步下降(p0.01),阻滯劑組略高于尿酸鹽晶體組,但在目前實(shí)驗(yàn)條件下,我們沒有觀察到這一差異的統(tǒng)計(jì)學(xué)意義。實(shí)驗(yàn)組細(xì)胞內(nèi)ATP的含量明顯低于對(duì)照組,各實(shí)驗(yàn)組之間無明顯差異。結(jié)論:1.正常狀態(tài)下,KCNQ1鉀離子通道在尿酸鹽晶體刺激人單核巨噬細(xì)胞分泌IL-1β的過程中可能發(fā)揮有限作用,但當(dāng)其過度激活時(shí),可以明顯增加成熟IL-1β的釋放。2.因KCNQ1的結(jié)構(gòu)異常使得KCNE1輔基對(duì)其抑制性調(diào)控作用減弱在成熟IL-1β分泌中的作用值得進(jìn)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- 偽 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- 偽 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.7

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