【摘要】：目的:通過觀察鈦離子與T細胞作用后細胞膜電位、胞內(nèi)鈣離子變化及Kv 1.3通道表達情況之間的關(guān)系,探討Kv 1.3通道在鈦離子影響T細胞內(nèi)鈣離子濃度中的作用。方法:1、體外培養(yǎng)Jurkat T細胞,MTT法檢測不同濃度Kv 1.3通道阻滯劑Sh K-Dap22對T細胞增殖活性的影響。2、根據(jù)是否用植物血凝素PHA活化將Jurkat T細胞分為PHA(+)及PHA(-)兩個大組,兩組細胞按加入鈦離子濃度的不同分別設(shè)立對照組、低、中及高濃度組,相對應(yīng)以0μmol/L,25μmol/L,50μmol/L,100μmol/L的鈦離子進行干預,實時熒光定量多聚酶鏈反應(yīng)(Real-time PCR)檢測Kv 1.3通道的mRNA的表達情況,流式細胞術(shù)檢測細胞膜電位及鈣離子濃度的變化。同時觀察加入ShK-Dap22阻斷劑后T細胞膜電位、鈣離子濃度及Kv1.3通道的mRNA的表達情況的變化。結(jié)果:1、Sh K-Dap22對T細胞增殖具有抑制效應(yīng),在0-10 nmol/L時抑制率隨濃度的升高而增大,當大于等于10nmol/L時,抑制率無明顯差異(P0.05)。2、PHA(+)組,低、中、高三個濃度組的鈦離子可以使T細胞Kv 1.3通道m(xù)RNA的相對表達量上調(diào)、膜電位去極化及胞內(nèi)Ca~(2+)濃度上升(P0.05),加入ShK-Dap22后,這種上調(diào)量與ShK-Dap22(-)組相比有明顯降低趨勢,但與對照組比較有差異(P0.05)。PHA(-)組,低、中、高三個濃度組的鈦離子可以使T細胞Kv 1.3通道m(xù)RNA的相對表達量上調(diào)、膜電位去極化及胞內(nèi)Ca~(2+)濃度上升(P0.05),加入ShK-Dap22后,中、高濃度組的mRNA的相對表達量、膜電位去極化及胞內(nèi)Ca~(2+)濃度高于對照組(P0.05),而低濃度組mRNA的相對表達量與對照組比較差異無統(tǒng)計學意義(P0.05)。且低、中濃度組之間的T細胞膜電位去極化及胞內(nèi)Ca~(2+)濃度比較也無統(tǒng)計學差異(P0.05)。結(jié)論:1、Kv 1.3阻滯劑ShK-Dap22能抑制體外培養(yǎng)的T細胞生長,當Sh K-Dap22超過10nmol/L時抑制率趨于穩(wěn)定。2、鈦離子可通過T細胞膜上鉀離子通道Kv 1.3升高胞內(nèi)鈣離子濃度。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R783.6

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