本文選題：內(nèi)質(zhì)網(wǎng)應(yīng)激(ER　切入點(diǎn)：stress)　出處：《江蘇大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ER stress)是細(xì)胞凋亡途徑之一,其引起的凋亡與許多疾病的發(fā)病機(jī)制有關(guān),如神經(jīng)退行性疾病、新陳代謝疾病、癌癥。T細(xì)胞的活化誘導(dǎo)細(xì)胞凋亡(Activation induced cell death,AICD)是當(dāng)抗原清除后,機(jī)體調(diào)控活化T細(xì)胞發(fā)生主動(dòng)凋亡,以維持機(jī)體免疫穩(wěn)態(tài)的重要機(jī)制。本實(shí)驗(yàn)初步探討了內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)T細(xì)胞AICD的影響,為進(jìn)一步深入了解內(nèi)質(zhì)網(wǎng)應(yīng)激在特異性細(xì)胞免疫中的調(diào)控機(jī)制提供初步的理論依據(jù)。方法:1.內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)T細(xì)胞活化誘導(dǎo)凋亡的調(diào)控。(1)小鼠T淋巴瘤細(xì)胞株EL-4細(xì)胞用Con-A結(jié)合IL-2和CD3抗體體外誘導(dǎo)AICD。(2)從C57BL/6小鼠中取出脾臟,研磨,并破紅細(xì)胞后得到脾臟的單細(xì)胞懸液。計(jì)數(shù)后以2×10~5細(xì)胞加入到96孔板中,用Con-A結(jié)合IL-2和CD3抗體體外誘導(dǎo)CD4~+T細(xì)胞的AICD。(3)經(jīng)過(guò)Con-A和IL-2活化的脾細(xì)胞在轉(zhuǎn)移到CD3抗體包被的96孔板培養(yǎng)時(shí),加入內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)劑二硫蘇糖醇(dithiothreitol,DTT)或內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-苯基丁酸(4-phenylbutyric acid,4-PBA)后,繼續(xù)培養(yǎng)24h。(4)用流式細(xì)胞術(shù)分析脾細(xì)胞中各組T細(xì)胞中凋亡細(xì)胞(Annexin-V~+7-AAD-和Annexin-V~+7-AAD~+)的百分?jǐn)?shù)。檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)于CD4~+T細(xì)胞AICD過(guò)程的調(diào)控。2.T細(xì)胞定向敲除Sel1L小鼠模型建立(1)應(yīng)用Cre-lox P重組系統(tǒng)構(gòu)建條件性基因敲除小鼠,將Lck-Cre雜合子小鼠與Sel1L~(fl/fl)純合子小鼠雌雄合籠繁殖,應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)以及瓊脂糖電泳法進(jìn)行表達(dá)Cre重組酶以及Sel1L基因loxp位點(diǎn)鑒定,篩選出實(shí)驗(yàn)所需小鼠。(2)Western Blotting法檢測(cè)在實(shí)驗(yàn)小鼠中脾臟T細(xì)胞中Sel1L蛋白的表達(dá)水平。3.內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)T細(xì)胞定向敲除Sel1L小鼠T細(xì)胞活化誘導(dǎo)凋亡的調(diào)控。(1)分別從Sel1L基因敲除和野生型小鼠中取出脾臟,研磨收集細(xì)胞破紅后計(jì)數(shù),得到脾臟細(xì)胞的單細(xì)胞懸液。用Con-A結(jié)合IL-2和CD3抗體體外誘導(dǎo)AICD。(2)對(duì)兩種小鼠體外誘導(dǎo)AICD的過(guò)程中同樣加入DTT或4-PBA處理后,用流式細(xì)胞術(shù)分析兩種小鼠中各組總T細(xì)胞中凋亡細(xì)胞(Annexin-V~+7-AAD-和Annexin-V~+7-AAD~+)的百分?jǐn)?shù)。結(jié)果:(1)小鼠T淋巴瘤細(xì)胞株EL-4細(xì)胞和C57BL/6小鼠的脾細(xì)胞在體外成功建立T細(xì)胞的AICD模型,與對(duì)照組相比,誘導(dǎo)組T細(xì)胞凋亡水平上調(diào)明顯。同時(shí)也發(fā)現(xiàn)了隨著CD3抗體濃度的增加,EL-4細(xì)胞發(fā)生AICD的比例增加。(2)小鼠脾細(xì)胞進(jìn)行AICD誘導(dǎo)時(shí)加入DTT或4-PBA后,發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激的激活,可導(dǎo)致CD4~+T細(xì)胞的凋亡明顯上調(diào),甚至死亡,相反,內(nèi)質(zhì)網(wǎng)應(yīng)激的抑制,CD4~+T細(xì)胞中發(fā)生凋亡的細(xì)胞明顯低于DTT組和對(duì)照組。(3)Lck~(Cre)×Sel1L~(fl/fl)即T細(xì)胞特異性敲除Sel1L基因的純合子小鼠被繁殖成功并準(zhǔn)確鑒定。Lck~(Cre)×Sel1L~(fl/fl)小鼠脾臟T細(xì)胞的Sel1L基因的蛋白表達(dá)水平與Lck~+/~+×Sel1L~(fl/fl)即野生型小鼠相比顯著降低。(4)Sel1L基因敲除小鼠(KO)與野生型小鼠(WT)的脾細(xì)胞進(jìn)行AICD誘導(dǎo)時(shí)加入DTT或4-PBA后,在Sel1L基因敲除小鼠(KO)發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)激活劑或阻斷劑的加入,對(duì)總T細(xì)胞的AICD無(wú)明顯影響。而WT組在內(nèi)質(zhì)網(wǎng)應(yīng)激激活時(shí),總T細(xì)胞的凋亡明顯上調(diào)。相反,內(nèi)質(zhì)網(wǎng)應(yīng)激的抑制,總T細(xì)胞凋亡的細(xì)胞明顯低于DTT組和對(duì)照組。結(jié)論:T細(xì)胞的活化誘導(dǎo)凋亡過(guò)程中,內(nèi)質(zhì)網(wǎng)激活劑或阻斷劑的加入分別使T細(xì)胞的凋亡水平升高或降低。由此可見(jiàn),內(nèi)質(zhì)網(wǎng)應(yīng)激在T細(xì)胞AICD過(guò)程起著重要的作用。通過(guò)在Sel1L基因敲除和野生型小鼠脾細(xì)胞誘導(dǎo)AICD過(guò)程中,加入內(nèi)質(zhì)網(wǎng)激活劑或阻斷劑的結(jié)果顯示,Sel1L分子是內(nèi)質(zhì)網(wǎng)應(yīng)激調(diào)控T細(xì)胞AICD的重要組分。
[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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R363

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相關(guān)期刊論文 前2條

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本文編號(hào)：1644550