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钾离子通道Kv1.3在钛离子影响T细胞内钙离子浓度的作用研究

发布时间:2019-01-08 18:45
【摘要】:目的:通过观察钛离子与T细胞作用后细胞膜电位、胞内钙离子变化及Kv 1.3通道表达情况之间的关系,探讨Kv 1.3通道在钛离子影响T细胞内钙离子浓度中的作用。方法:1、体外培养Jurkat T细胞,MTT法检测不同浓度Kv 1.3通道阻滞剂Sh K-Dap22对T细胞增殖活性的影响。2、根据是否用植物血凝素PHA活化将Jurkat T细胞分为PHA(+)及PHA(-)两个大组,两组细胞按加入钛离子浓度的不同分别设立对照组、低、中及高浓度组,相对应以0μmol/L,25μmol/L,50μmol/L,100μmol/L的钛离子进行干预,实时荧光定量多聚酶链反应(Real-time PCR)检测Kv 1.3通道的mRNA的表达情况,流式细胞术检测细胞膜电位及钙离子浓度的变化。同时观察加入ShK-Dap22阻断剂后T细胞膜电位、钙离子浓度及Kv1.3通道的mRNA的表达情况的变化。结果:1、Sh K-Dap22对T细胞增殖具有抑制效应,在0-10 nmol/L时抑制率随浓度的升高而增大,当大于等于10nmol/L时,抑制率无明显差异(P0.05)。2、PHA(+)组,低、中、高三个浓度组的钛离子可以使T细胞Kv 1.3通道mRNA的相对表达量上调、膜电位去极化及胞内Ca~(2+)浓度上升(P0.05),加入ShK-Dap22后,这种上调量与ShK-Dap22(-)组相比有明显降低趋势,但与对照组比较有差异(P0.05)。PHA(-)组,低、中、高三个浓度组的钛离子可以使T细胞Kv 1.3通道mRNA的相对表达量上调、膜电位去极化及胞内Ca~(2+)浓度上升(P0.05),加入ShK-Dap22后,中、高浓度组的mRNA的相对表达量、膜电位去极化及胞内Ca~(2+)浓度高于对照组(P0.05),而低浓度组mRNA的相对表达量与对照组比较差异无统计学意义(P0.05)。且低、中浓度组之间的T细胞膜电位去极化及胞内Ca~(2+)浓度比较也无统计学差异(P0.05)。结论:1、Kv 1.3阻滞剂ShK-Dap22能抑制体外培养的T细胞生长,当Sh K-Dap22超过10nmol/L时抑制率趋于稳定。2、钛离子可通过T细胞膜上钾离子通道Kv 1.3升高胞内钙离子浓度。
[Abstract]:Aim: to investigate the relationship between titanium ion and T cell membrane potential, intracellular calcium and the expression of Kv 1.3 channel, and to explore the role of Kv 1.3 channel in the effect of titanium ion on the intracellular calcium concentration of T cells. Methods: 1. Jurkat T cells were cultured in vitro. The effects of Kv 1.3 channel blocker Sh K-Dap22 on the proliferation of T cells were detected by MTT assay. Jurkat T cells were divided into two groups: PHA () and PHA (-) according to whether they were activated by phytohemagglutinin (PHA). The cells in the two groups were divided into two groups according to the different concentration of titanium ion: control group, low, medium and high concentration groups, corresponding to 0 渭 mol/L,. Titanium ions of 25 渭 mol/L,50 渭 mol/L,100 渭 mol/L were used to intervene, real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of mRNA in Kv 1.3 channel, flow cytometry was used to detect the changes of cell membrane potential and calcium concentration. At the same time, the changes of T cell membrane potential, calcium concentration and mRNA expression of Kv1.3 channel were observed after the addition of ShK-Dap22 blocker. Results: 1Sh K-Dap22 had inhibitory effect on T cell proliferation, and the inhibition rate increased with the increase of concentration at 0-10 nmol/L, but there was no significant difference when the concentration was greater than 10nmol/L (P0.05). 2 the inhibition rate of Sh K-Dap22 group was lower than that of PHA (P0.05) group. The relative expression of Kv 1.3 channel mRNA was up-regulated, membrane potential depolarization and intracellular Ca~ (2) concentration were increased (P0.05), and ShK-Dap22 was added to T cells. Compared with the ShK-Dap22 (-) group, the upregulation showed a significant decrease trend, but there was a significant difference between the two groups (P0.05). PHA (-). The relative expression of Kv 1.3 channel mRNA was up-regulated, membrane potential depolarization and intracellular Ca~ (2) concentration were increased (P0.05). After adding ShK-Dap22, the relative expression of mRNA in middle and high concentration group was increased. The membrane potential depolarization and intracellular Ca~ (2) concentration were higher than those in the control group (P0.05), while the relative expression of mRNA in the low concentration group was not significantly different from that in the control group (P0.05). There was no significant difference in T cell membrane potential depolarization and intracellular Ca~ (2) concentration between middle and middle concentration groups (P0.05). Conclusion: 1 Kv1.3 blocker ShK-Dap22 can inhibit the growth of T cells cultured in vitro, and the inhibitory rate of Kv1.3 blocker tends to be stable when Sh K-Dap22 exceeds 10nmol/L. Titanium ion can increase intracellular calcium concentration through potassium channel Kv 1.3 on T cell membrane.
【学位授予单位】:广西医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R783.6

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