内质网应激对T细胞活化诱导细胞凋亡的影响

发布时间：2018-03-21 15:57

本文选题：内质网应激(ER　切入点：stress)　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:内质网应激(Endoplasmic reticulum stress,ER stress)是细胞凋亡途径之一,其引起的凋亡与许多疾病的发病机制有关,如神经退行性疾病、新陈代谢疾病、癌症。T细胞的活化诱导细胞凋亡(Activation induced cell death,AICD)是当抗原清除后,机体调控活化T细胞发生主动凋亡,以维持机体免疫稳态的重要机制。本实验初步探讨了内质网应激对T细胞AICD的影响,为进一步深入了解内质网应激在特异性细胞免疫中的调控机制提供初步的理论依据。方法:1.内质网应激对T细胞活化诱导凋亡的调控。(1)小鼠T淋巴瘤细胞株EL-4细胞用Con-A结合IL-2和CD3抗体体外诱导AICD。(2)从C57BL/6小鼠中取出脾脏,研磨,并破红细胞后得到脾脏的单细胞悬液。计数后以2×10~5细胞加入到96孔板中,用Con-A结合IL-2和CD3抗体体外诱导CD4~+T细胞的AICD。(3)经过Con-A和IL-2活化的脾细胞在转移到CD3抗体包被的96孔板培养时,加入内质网应激诱导剂二硫苏糖醇(dithiothreitol,DTT)或内质网应激抑制剂4-苯基丁酸(4-phenylbutyric acid,4-PBA)后,继续培养24h。(4)用流式细胞术分析脾细胞中各组T细胞中凋亡细胞(Annexin-V~+7-AAD-和Annexin-V~+7-AAD~+)的百分数。检测内质网应激对于CD4~+T细胞AICD过程的调控。2.T细胞定向敲除Sel1L小鼠模型建立(1)应用Cre-lox P重组系统构建条件性基因敲除小鼠,将Lck-Cre杂合子小鼠与Sel1L~(fl/fl)纯合子小鼠雌雄合笼繁殖,应用聚合酶链式反应以及琼脂糖电泳法进行表达Cre重组酶以及Sel1L基因loxp位点鉴定,筛选出实验所需小鼠。(2)Western Blotting法检测在实验小鼠中脾脏T细胞中Sel1L蛋白的表达水平。3.内质网应激对T细胞定向敲除Sel1L小鼠T细胞活化诱导凋亡的调控。(1)分别从Sel1L基因敲除和野生型小鼠中取出脾脏,研磨收集细胞破红后计数,得到脾脏细胞的单细胞悬液。用Con-A结合IL-2和CD3抗体体外诱导AICD。(2)对两种小鼠体外诱导AICD的过程中同样加入DTT或4-PBA处理后,用流式细胞术分析两种小鼠中各组总T细胞中凋亡细胞(Annexin-V~+7-AAD-和Annexin-V~+7-AAD~+)的百分数。结果:(1)小鼠T淋巴瘤细胞株EL-4细胞和C57BL/6小鼠的脾细胞在体外成功建立T细胞的AICD模型,与对照组相比,诱导组T细胞凋亡水平上调明显。同时也发现了随着CD3抗体浓度的增加,EL-4细胞发生AICD的比例增加。(2)小鼠脾细胞进行AICD诱导时加入DTT或4-PBA后,发现内质网应激的激活,可导致CD4~+T细胞的凋亡明显上调,甚至死亡,相反,内质网应激的抑制,CD4~+T细胞中发生凋亡的细胞明显低于DTT组和对照组。(3)Lck~(Cre)×Sel1L~(fl/fl)即T细胞特异性敲除Sel1L基因的纯合子小鼠被繁殖成功并准确鉴定。Lck~(Cre)×Sel1L~(fl/fl)小鼠脾脏T细胞的Sel1L基因的蛋白表达水平与Lck~+/~+×Sel1L~(fl/fl)即野生型小鼠相比显著降低。(4)Sel1L基因敲除小鼠(KO)与野生型小鼠(WT)的脾细胞进行AICD诱导时加入DTT或4-PBA后,在Sel1L基因敲除小鼠(KO)发现内质网激活剂或阻断剂的加入,对总T细胞的AICD无明显影响。而WT组在内质网应激激活时,总T细胞的凋亡明显上调。相反,内质网应激的抑制,总T细胞凋亡的细胞明显低于DTT组和对照组。结论:T细胞的活化诱导凋亡过程中,内质网激活剂或阻断剂的加入分别使T细胞的凋亡水平升高或降低。由此可见,内质网应激在T细胞AICD过程起着重要的作用。通过在Sel1L基因敲除和野生型小鼠脾细胞诱导AICD过程中,加入内质网激活剂或阻断剂的结果显示,Sel1L分子是内质网应激调控T细胞AICD的重要组分。
[Abstract]:Objective: endoplasmic reticulum stress (Endoplasmic reticulum stress, ER stress) is one of the pathways of apoptosis, pathogenesis induced apoptosis and many diseases, such as neurodegenerative diseases, disease The new supersedes the old. activation induced cell death, cancer cell.T (Activation induced cell death, AICD) when the antigen is removed, the body control the activation of T cells active apoptosis, in order to maintain an important mechanism for immune homeostasis. This study investigates the effect of endoplasmic reticulum stress on T cells of AICD, in order to further understand the mechanism of ER stress induced in specific cellular immunity in providing a primary theoretical basis. Methods: 1. regulation of apoptosis induced by endoplasmic reticulum stress activation for T cells. (1) of mouse T lymphoma cell line EL-4 cells induced by AICD. IL-2 combined with Con-A and CD3 antibodies in vitro (2) taken out from C57BL/6 mouse spleen, grinding, and broken red blood cells The cell suspension was obtained after the spleen cell. After counting to 2 * 10~5 cells were added to the 96 Kong Banzhong, combined with Con-A IL-2 and CD3 antibody in vitro CD4~+T cells induced by AICD. (3) through the activation of Con-A and IL-2 in spleen cells transferred to CD3 antibody coated 96 well plate culture, with endoplasmic reticulum stress two inducer dithiothreitol (dithiothreitol, DTT) or the endoplasmic reticulum stress inhibitor 4- (4-phenylbutyric acid, 4-PBA phenyl butyrate) after cultured for 24h. (4) analysis of apoptotic cells in each group of T cells in spleen cells by flow cytometry (Annexin-V~+7-AAD- and Annexin-V~+7-AAD~+). The percentage of detection of endoplasmic reticulum stress in the regulation of.2.T the directional cell CD4~+T cell process of AICD knockout Sel1L mice model was established by Cre-lox (1) to construct P recombinant system conditional knockout mice, Lck-Cre heterozygous mice (fl/fl) and Sel1L~ homozygous mice were mated breeding, The application of polymerase chain reaction and agarose gel electrophoresis of Cre recombinase and Sel1L loxP gene expression identification, screening out the required mice. (2) detection of Western Blotting Sel1L in mice spleen cells T protein.3. expression level of endoplasmic reticulum stress on T cell apoptosis induced by knockdown of directional regulation of activated Sel1L in mice T cells. (1) from the Sel1L gene knockout and wild-type mice removed the spleen cells were collected after grinding, broken red blood count, spleen cell suspensions from single cells. Con-A combined with IL-2 and CD3 antibodies in vitro induced by AICD. (2) of two mice in vitro induced by AICD in the same DTT or join after 4-PBA treatment, analysis of two kinds of apoptotic cells in mice were all T cells by flow cytometry (Annexin-V~+7-AAD- and Annexin-V~+7-AAD~+). Results: the percentage (1) of mouse T lymphoma cell line EL-4 and C57BL/6 AICD model mice spleen cells successfully established T cells in vitro, compared with the control group, T group induced apoptosis level was obviously increased. At the same time also found that with the increase of CD3 antibody concentrations, EL-4 cells increased the proportion of AICD. (2) of mouse spleen cells induced by AICD when added to the DTT or 4-PBA. Found that activation of endoplasmic reticulum stress, can induce the apoptosis of CD4~+T cells was significantly increased, and even death, on the contrary, inhibition of endoplasmic reticulum stress, apoptosis of CD4~+T cells was significantly lower than that of DTT group and control group. (3) Lck~ (Cre) * Sel1L~ (fl/fl) T cell specific knockout mice homozygous for Sel1L the gene was successful reproduction and accurate identification of.Lck~ (Cre) * Sel1L~ (fl/fl) Sel1L gene of mouse spleen T cells protein expression level and Lck~+/~+ * Sel1L~ (fl/fl) is significantly lower than wild type mice. (4) Sel1L gene knockout mice and wild type (KO) (WT) rat spleen cells induced by AICD addition of DTT or 4-PBA, in Sel1L gene knockout mice (KO) found that endoplasmic reticulum activator or blocking agent, has no obvious effect on the total T cells. AICD and group WT in the endoplasmic reticulum stress activation, apoptosis of T cells significantly increased the total. On the contrary, inhibition of endoplasmic reticulum stress and apoptosis of total T cells was significantly lower than that of DTT group and control group. Conclusion: the activation induced apoptosis of T cells, endoplasmic reticulum activators or inhibitors were added to the apoptosis level of T cells increased or decreased. Thus, the endoplasmic reticulum stress plays an important in T cells AICD process. Through the knockout and wild-type mice spleen cells induced by Sel1L gene in AICD in the process of adding endoplasmic reticulum activators or inhibitors showed that Sel1L molecule is an important group of T cells AICD endoplasmic reticulum stress regulation.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R363

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