【摘要】： 目的自然殺傷T(natural killer T cells,NKT)細(xì)胞是一類(lèi)能夠識(shí)別非經(jīng)典的MHCⅠ類(lèi)分子CD1d所遞呈的糖脂抗原的特殊類(lèi)型的T淋巴細(xì)胞,同時(shí)表達(dá)NK和T細(xì)胞表面標(biāo)志NK1.1和TCR。NKT細(xì)胞經(jīng)內(nèi)源性配體鞘糖脂iGb3(isoglobotrihexosylceramide )和外源性糖脂配體α-半乳糖神經(jīng)酰胺(α-GalCer)活化后能夠分泌IFN-γ、IL-4和IL-13等多種細(xì)胞因子,在抗腫瘤、抗病毒感染、自身免疫耐受和自身免疫性疾病中發(fā)揮著重要作用。但由于激活后的NKT細(xì)胞能夠同時(shí)分泌Th1和Th2兩型細(xì)胞因子,影響了iGb3或α-GalCer對(duì)免疫相關(guān)疾病的治療效果,因而限制了其臨床應(yīng)用。因此,若能發(fā)現(xiàn)有效的途徑,積極調(diào)控兩型細(xì)胞因子的相對(duì)產(chǎn)生量,糖脂分子將會(huì)在臨床應(yīng)用中發(fā)揮更廣泛的作用。 研究發(fā)現(xiàn)對(duì)α-GalCer的鞘鞍醇鏈進(jìn)行截短所得的類(lèi)似物OCH,可通過(guò)降低糖脂分子-CD1d復(fù)合物的穩(wěn)定性和增強(qiáng)糖脂分子-TCR的親和力來(lái)實(shí)現(xiàn)IL-4的優(yōu)勢(shì)分泌。而另有研究表明將連接糖基部分與另外兩條鏈的O原子用C原子代替后產(chǎn)生的α-GalCer類(lèi)似物α-C-GalCer,可以通過(guò)增強(qiáng)糖脂分子各部分之間連接的穩(wěn)定性而延長(zhǎng)了與CD1d和NKT細(xì)胞作用的時(shí)間來(lái)實(shí)現(xiàn)IFN-γ的優(yōu)勢(shì)分泌。提示,針對(duì)性改造糖脂,可以誘導(dǎo)NKT細(xì)胞Th1/ Th2型細(xì)胞因子的分泌。雖然有眾多學(xué)者設(shè)計(jì)不同的位點(diǎn)對(duì)糖脂行各種修飾改造,并分析改造后的糖脂活化NKT細(xì)胞方面功能和結(jié)構(gòu)的差異,但目前缺乏對(duì)于內(nèi)源性配體iGb3進(jìn)行改造的研究。 鑒于此,我們對(duì)iGb3和α-GalCe進(jìn)行修飾,獲得了一批化學(xué)修飾的iGb3和α-GalCe類(lèi)似物,在前期工作中,我們初步篩選出兩種類(lèi)似物4-HO-iGb3和4'''-dh-iGb3具有優(yōu)勢(shì)誘導(dǎo)小鼠肝臟NKT細(xì)胞Th1型細(xì)胞因子的分泌能力。本研究采用四聚體染色法進(jìn)一步篩選并確定經(jīng)過(guò)化學(xué)修飾的iGb3類(lèi)似物細(xì)胞因子的優(yōu)勢(shì)分泌,并在此基礎(chǔ)上,深入探討糖脂/APC/TCR間的相互作用以及細(xì)胞微環(huán)境等一些影響NKT細(xì)胞因子分泌的重要因素,為糖脂類(lèi)分子的設(shè)計(jì)和臨床應(yīng)用提供新的策略和依據(jù)。 方法(1)流式細(xì)胞術(shù)法檢測(cè)iGb3類(lèi)似物和α-GalCer類(lèi)似物對(duì)小鼠肝臟、脾臟中NKT細(xì)胞的數(shù)量和胞內(nèi)細(xì)胞因子IFN-γ和IL-4的表達(dá)情況,以及轉(zhuǎn)錄因子STAT1和STAT6的STAT1磷酸化水平。(2)體外誘導(dǎo)小鼠骨髓來(lái)源樹(shù)突裝細(xì)胞的分化和成熟。(3) ELISA法檢測(cè)化學(xué)修飾的iGb3類(lèi)似物作用后小鼠血清中和細(xì)胞培養(yǎng)上清中細(xì)胞因子IFN-γ和IL-4的分泌水平。(4)實(shí)時(shí)定量PCR檢測(cè)小鼠脾臟淋巴細(xì)胞中IFN-γ、IL-4、GATA-3和T-bet的基因表達(dá)情況。(5) QSAR軟件分析糖脂分子與NKT細(xì)胞上TCR受體以及APC細(xì)胞上CD1d分子的親和力高低。(6)蛋白質(zhì)免疫印跡檢測(cè)iGb3類(lèi)似物對(duì)小鼠脾臟淋巴細(xì)胞轉(zhuǎn)錄因子T-bet和GATA-3的蛋白表達(dá)情況。 結(jié)果 一、化學(xué)修飾的iGb3類(lèi)似物4’’’-dh-iGb3優(yōu)勢(shì)誘導(dǎo)小鼠肝臟和脾臟NKT細(xì)胞分泌IFN-γ 予C57BL/6小鼠腹腔注射iGb3及其類(lèi)似物,發(fā)現(xiàn)與iGb3刺激組相比,化學(xué)修飾的iGb3類(lèi)似物4’’’-dh-iGb3雖然并不增加肝脾NKT細(xì)胞的比例和數(shù)量,但可明顯增強(qiáng)肝脾NKT細(xì)胞胞內(nèi)IFN-γ的表達(dá)、分泌及血清中IFN-γ水平,而對(duì)Th2類(lèi)細(xì)胞因子IL-4的表達(dá)和分泌無(wú)明顯影響。用骨髓來(lái)源的DC負(fù)載糖脂,體外刺激小鼠的NKT細(xì)胞系,檢測(cè)NKT細(xì)胞胞內(nèi)細(xì)胞因子表達(dá)和上清細(xì)胞因子含量,亦得到了類(lèi)似的結(jié)果。 二、4′′′-dh-iGb3可增強(qiáng)糖脂與CD1d、糖脂與NKT細(xì)胞TCR之間的結(jié)合力和穩(wěn)定性 運(yùn)用QSAR軟件,在計(jì)算機(jī)上模擬糖脂、抗原遞呈細(xì)胞的CD1d分子以及NKT細(xì)胞的TCR分子的空間結(jié)構(gòu),并進(jìn)行分子結(jié)構(gòu)之間的對(duì)接,發(fā)現(xiàn)在iGb3鞘鞍醇鏈引入HO可以形成一個(gè)疏水鍵,與原來(lái)酰基鏈上現(xiàn)有的HO一同直接地深入到CD1d分子形成的疏水性抗原結(jié)合口袋中,而增強(qiáng)了與CD1d分子上氨基酸Asp80(C)之間的親和力;對(duì)糖基進(jìn)行脫氧處理,使糖脂在與TCR分子氨基酸Lys167結(jié)合的空間位置由糖脂的此側(cè)轉(zhuǎn)變?yōu)樘侵谋藗?cè),使糖脂與TCR之間的結(jié)合分?jǐn)?shù)由原來(lái)的3.3顯著提高到4.9,增加了4'''-dh-iGb3與NKT細(xì)胞TCR的結(jié)合能力。即:修飾后的4’’’-dh-iGb3具有較iGb3更強(qiáng)的與抗原遞呈細(xì)胞CD1d分子結(jié)合的親和力及與NKT細(xì)胞TCR結(jié)合的穩(wěn)定性。 三、4’’’-dh-iGb3能夠上調(diào)Th1類(lèi)細(xì)胞因子相關(guān)轉(zhuǎn)錄因子STAT1的磷酸化和T-bet的表達(dá)水平 予C57BL/6小鼠腹腔注射iGb3及其類(lèi)似物或體外刺激小鼠的NKT細(xì)胞系,觀察NKT細(xì)胞調(diào)控Th1/Th2類(lèi)細(xì)胞因子表達(dá)相關(guān)轉(zhuǎn)錄因子的表達(dá)情況,發(fā)現(xiàn)與iGb3相比,4’’’-dh-iGb3能夠上調(diào)NKT細(xì)胞調(diào)控Th1類(lèi)細(xì)胞因子產(chǎn)生的轉(zhuǎn)錄因子STAT1的磷酸化和T-bet的表達(dá),而調(diào)節(jié)Th2類(lèi)細(xì)胞因子產(chǎn)生的轉(zhuǎn)錄因子STAT6的磷酸化和GATA-3的表達(dá)差異不顯著。 結(jié)論(1) iGb3類(lèi)似物4’’’-dh-iGb3能夠誘導(dǎo)小鼠NKT細(xì)胞優(yōu)勢(shì)分泌Th1類(lèi)細(xì)胞因子IFN-γ。(2) 4’’’-dh-iGb3可提高糖脂與CD1d、糖脂與NKT細(xì)胞TCR之間的穩(wěn)定性和親和力,從而促進(jìn)NKT細(xì)胞的活化和Th1類(lèi)細(xì)胞因子的分泌。(3) 4’’’-dh-iGb3可增強(qiáng)調(diào)控Th1/Th2分化的轉(zhuǎn)錄因子STAT1的磷酸化和T-bet的表達(dá),從而促進(jìn)NKT細(xì)胞IFN-γ的表達(dá)和分泌。(4) 4’’’-dh-iGb3可以作為新型免疫增強(qiáng)劑用于抗腫瘤和感染性疾病的治療。
[Abstract]:Objective Natural killer T cells (NKT) are a special type of T lymphocytes capable of recognizing glycolipid antigens presented by non-classical MHC class I molecule CD1d and expressing NK and T cell surface markers NK1.1 and TCR.NKT cells via endogenous ligand sheath glycolipid iGb3 (isoglobotrihexosylceramide) and exogenous glycolipid binding. Activated human alpha-galactose ceramide (alpha-GalCer) can secrete many cytokines, such as IFN-gamma, IL-4 and IL-13, which play an important role in anti-tumor, anti-virus infection, autoimmune tolerance and autoimmune diseases. However, NKT cells can secrete both Th1 and Th2 cytokines, affecting iGb3 or alpha-GalCer. Glucolipid molecules will play a more extensive role in clinical application if effective ways can be found to actively regulate the relative production of two types of cytokines.
It was found that OCH, an analogue obtained by truncating the saddle alcohol chain of alpha-GalCer, could achieve the predominant secretion of IL-4 by lowering the stability of glycolipid molecule-CD1d complex and enhancing the affinity of glycolipid molecule-TCR. The analogue alpha-C-GalCer can prolong the time of interaction with C D1d and NKT cells by enhancing the stability of the links between the various parts of glycolipid molecules to achieve the predominant secretion of IFN-gamma. It is suggested that targeted modification of glycolipids can induce the secretion of Th1/Th2 cytokines in NKT cells. The functional and structural differences of NKT cells activated by glycolipids and lipids after modification were analyzed. However, there is no study on the modification of endogenous ligand iGb3.
In view of this, we modified iGb3 and alpha-GalCe, and obtained a number of chemically modified iGb3 and alpha-GalCe analogues. In the previous work, we screened two analogues 4-HO-iGb3 and 4''-dh-iGb3, which had the predominant ability to induce the secretion of Th1 cytokines in mouse liver NKT cells. The predominant secretion of cytokines from chemically modified iGb3 analogues was selected and determined. On this basis, the interaction between glycolipids/APC/TCR and some important factors affecting the secretion of NKT cytokines, such as cell microenvironment, were discussed in depth, which provided a new strategy and basis for the design and clinical application of glycolipids.
Methods (1) The number of NKT cells and the expression of intracellular cytokines IFN-gamma and IL-4 in liver and spleen of mice were detected by flow cytometry. (2) Differentiation and maturation of mouse bone marrow-derived dendritic cells were induced in vitro. (3) ELISA assay. The secretion levels of cytokines IFN-gamma and IL-4 in serum and cell culture supernatant of mice treated with chemically modified iGb3 analogues were measured. (4) The gene expression of IFN-gamma, IL-4, GATA-3 and T-bet in splenic lymphocytes of mice was detected by real-time quantitative PCR. (5) QSAR software was used to analyze TCR receptors on glycolipids and NKT cells and CD1d on APC cells. (6) The expression of T-bet and GATA-3 in spleen lymphocytes of mice was detected by Western blot.
Result
First, the predominance of chemically modified iGb3 analogue 4'''-dh-iGb3 induces the secretion of IFN-gamma by mouse liver and spleen NKT cells
Intraperitoneal injection of iGb3 and its analogues into C57BL/6 mice showed that chemically modified iGb3 analogue 4'''-dh-iGb3 did not increase the proportion and number of NKT cells in the liver and spleen, but significantly increased the expression of IFN-gamma in the NKT cells of the liver and spleen, the secretion of IFN-gamma and the level of IFN-gamma in the serum, and the expression of Th2 cytokine IL-4. The expression of cytokines in NKT cells and the content of cytokines in supernatant were detected by stimulating NKT cell lines in vitro with bone marrow derived DC loaded with glucose and lipid.
Two, 4 '' -dh-iGb3 can enhance the binding strength and stability between glycolipid and CD1d, glycolipid and NKT cell TCR.
QSAR software was used to simulate the spatial structure of glycolipid, CD1d molecule of antigen-presenting cells and TCR molecule of NKT cells on a computer. It was found that introducing HO into the saddle alcohol chain of iGb3 could form a hydrophobic bond, which could directly penetrate into the hydrophobic formation of CD1d molecule along with the existing HO in the original acyl chain. The affinity between the glycolipid and the amino acid Asp80 (C) on the C D1d molecule was enhanced by binding the sex antigen in the pocket, and the space position of the glycolipid binding to the amino acid Lys167 on the TCR molecule was changed from the one side of the glycolipid to the other side of the glycolipid by deoxidizing the glycolipid, so that the binding fraction between the glycolipid and the TCR was increased from 3.3 to 4.9, and the binding fraction between the glycoli The binding ability of 4''''-dh-iGb3 to TCR of NKT cells is that the modified 4'''-dh-iGb3 has stronger affinity to CD1d molecule of antigen-presenting cells and stability to TCR of NKT cells than that of iGb3.
3,4'''-dh-iGb3 up-regulates the phosphorylation of Th1 cytokine-related transcription factor STAT1 and the expression of T-bet
After intraperitoneal injection of iGb3 and its analogues into C57BL/6 mice or stimulation of NKT cell lines in vitro, the expression of Th1/Th2 cytokine-related transcription factors regulated by NKT cells was observed. Compared with iGb3, 4'''-dh-iGb3 could up-regulate the phosphorylation of STAT1 and T-bet of Th1 cytokines produced by NKT cells. The phosphorylation of STAT6 and the expression of GATA-3, which regulate the production of Th2 cytokines, were not significantly different.
Conclusion (1) iGb3 analogue 4'''-dh-iGb3 can induce the predominant secretion of Th1 cytokines IFN-gamma. (2) 4''-dh-iGb3 can enhance the stability and affinity between glucose and lipid and TCR of NKT cells, thereby promoting the activation of NKT cells and the secretion of Th1 cytokines. The phosphorylation of STAT1 and the expression of T-bet, which control the differentiation of Th1/Th2, promote the expression and secretion of IFN-gamma in NKT cells. (4) 4'''-dh-iGb3 can be used as a new immunopotentiator in the treatment of tumor and infectious diseases.
【學(xué)位授予單位】：山東省醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

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3 萬(wàn)建R，

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