【作者】 杨柳；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2012， 博士

【摘要】 第一部分RasGRP1在湿疹患者外周血单个核细胞及T细胞中的表达及意义目的研究RasGRP1在湿疹患者外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)1中的表达,纯化PBMC中的T细胞,进一步研究RasGRP1在湿疹患者外周血T细胞中的表达,并探讨其意义。方法使用Ficoll法提取湿疹患者及正常人外周血中的PBMC,采用免疫磁珠分选法从PBMC中分选T细胞,验证T细胞的分选纯度。使用RT-PCR及Western Blot方法检测湿疹患者及正常人外周血PBMC和T细胞中RasGRP1的表达。结果使用Ficoll法提取的外周血PBMC的纯度接近1000%,而使用免疫磁珠分选法分选T细胞,其纯度能达到98%以上。在PBMC中,湿疹患者RasGRP1的mRNA和蛋白质的表达水平明显高于正常人,在纯化后的T细胞中,湿疹患者RasGRP1的mRNA和蛋白质的表达水平也明显高于正常人。结论使用免疫磁珠分选外周血PBMC中的T细胞纯度较高。湿疹患者外周血PBMC及T细胞中RasGRP1的表达水平均增高,提示T细胞中RasGRP1的异常表达可能是湿疹发病过程中的一个重要因素。第二部分地塞米松对湿疹患者T细胞中RasGRP1表达的作用及意义目的研究地塞米松对湿疹患者外周血T细胞中RasGRP1的作用,筛选产生作用的最适浓度,并探讨地塞米松对T细胞的影响及意义。方法选取临床上使用地塞米松的重症湿疹患者,抽取其地塞米松治疗前及治疗后的外周血,并纯化T细胞。检测地塞米松治疗前后T细胞内RasGRP1的表达水平。在体外实验中,将地塞米松配成三种不同浓度1×10-5mol/L、1×10-6mol/L及1×10-7mol/L,与未用激素治疗的湿疹患者外周血T细胞共同培养,使用RT-PCR的方法检测T细胞内RasGRP1的mRNA水平。并得出最适合的作用浓度。将此浓度的地塞米松作用于T细胞,观察不同时间点(0h、6h、12h、18h、24h)地塞米松对T细胞内RasGRP1表达的影响。使用MTT的方法检测不同时间点、不同浓度的地塞米松作用于T细胞时,对细胞凋亡的影响。结果未使用地塞米松治疗时,湿疹患者T细胞内RasGRP1的表达高于正常人,使用地塞米松治疗1周后,T细胞内RasGRP1的表达明显下降,接近正常人的表达水平。地塞米松能抑制湿疹患者外周血T细胞RasGRP1的表达,在三种不同浓度的地塞米松中,1×10-Smol/L的浓度对RasGRP1表达的抑制作用最明显。使用浓度为1×104mo1/L的地塞米松对湿疹患者外周T细胞进行不同时间点的刺激,随着时间的延长,地塞米松的抑制作用加强。24h时地塞米松对T细胞内RasGRP1的抑制作用最强。而不同浓度的地塞米松均能促进T细胞的凋亡,这种作用呈时间依赖性。结论地塞米松能抑制湿疹患者外周血T细胞中RasGRP1的表达,并促进T细胞的凋亡。这种抑制作用随着地塞米松浓度的增加而增加,随着时间的延长而加强。湿疹患者在临床治疗时,使用地塞米松治疗1周后,外周血T细胞内RasGRP1的水平下降,接近正常人水平。这可能为激素的抗炎机制增加了新观点。第三部分构建含有H-RasGRP1-shRNA的慢病毒载体及其对T细胞的干扰效应目的以RasGRP1为靶点,构建含有目的基因H-RasGRP1-shRNA的慢病毒载体,转染T细胞对其进行RNA干扰,观察转染后T细胞表达RasGRP1的变化。研究基因沉默RasGRP1后对T细胞内Ras下游信号通路蛋白的影响,以及对细胞凋亡和细胞因子分泌的影响,并探讨其作用机制。方法构建含有目的基因H-RasGRP1-shRNA的慢病毒载体,并检测其生物学滴度。对T细胞进行转染,使用RT-PCR和Western Blot的方法检测转染后不同时间点(12h、24h、48h、72h)T细胞内RasGRP1的表达情况,并使用流式细胞仪检测T细胞的凋亡。使用12-十四酸佛波酯-13-乙酸盐(Phorbol12-myristate13-acetate, PMA)及离子霉素刺激T细胞,观察T细胞内Ras蛋白下游信号通路蛋白p-Erk、Erk、Akt及p-Akt的表达。用ELISA的方法检测T细胞分泌细胞因子IL-2、IL-4及IL-17的情况。使用慢病毒载体转染PMA及离子霉素刺激后的T细胞,观察转染后T细胞内p-Erk、Erk、Akt及p-Akt蛋白表达的变化,以及T细胞分泌细胞因子的变化。结果成功构建H-RasGRP1-shRNA的慢病毒载体,其生物学滴度达到1.0×108TU/ml,符合转染要求。使用慢病毒载体转染T细胞后,T细胞内RasGRP1的mRNA及蛋白水平下降,随着时间的延长(12h、24h、48h、72h),RasGRP1的表达逐渐下降,72h时RasGRP1的表达几乎消失。同时转染后T细胞的凋亡加快。使用PMA及离子霉素对T细胞进行刺激,刺激前,湿疹患者和正常人T细胞内均有p-Erk、p-Akt、Erk及Akt的表达,且湿疹患者的表达高于正常人。使用PMA及离子霉素刺激后,p-Erk、p-Akt、 Erk及Akt的表达水平明显增高,以湿疹患者增加的更为明显。T细胞表达细胞因子IL-2、IL-4及IL-17的水平明显增加。用慢病毒颗粒基因转染PMA及离子霉素刺激后的T细胞,T细胞内p-Erk及p-Akt表达下降,而Erk及Akt的表达无明显变化。T细胞的培养上清液中分泌的细胞因子IL-2、IL-4及IL-17的表达水平下降。结论成功构建含目的基因H-RasGRP1-shRNA的慢病毒载体,其生物学滴度能满足转染要求。慢病毒载体能成功转染T细胞,抑制T细胞内RasGRP1的表达。RasGRP1基因沉默后,T细胞的凋亡增加。当湿疹发生时,T细胞受体活化后可能通过活化RasGRP1的表达,介导Ras蛋白的下游信号分子Erk及Akt的磷酸化,导致T细胞细胞因子释放增加。由此可见,RasGRP1可能成为湿疹治疗中的一个作用靶点,为基因治疗及免疫治疗提供新的思路。更多还原

【Abstract】 Part I The expressions of RasGRPl in peripheral blood lymphocytes and T cells of eczema patientsObjective To study the expressions of RasGRP1in peripheral blood mononuclear cells (PBMCs) of patients with eczema. To purify T cells from PBMC and evaluate the expressions of RasGRP1in T cells of eczema patients. Furthermore, to explore its significance.Methods Human PBMCs in eczema patients and the normal subjects were extracted by Ficoll extractionl. T cells were separated from PBMCs by means of immunomagnetic beads. The purity of separation was validated and the expressions of RasGRP1in T cells of eczema patients and the normal subjects were detected by RT-PCR and Western Blot.Results The purity of PBMC was close to100%by Ficoll extraction, while the purity of T cell could reach more than98%by immunomagnetic separation. In PBMCs and the purified T cells, the expressions of RasGRP1in eczema patients were higher than those in the normal subjects.Conclusions T cells with high purity could be acquired by the use of immunomagnetic separation. The expressions of RasGRP1in both PBMC and T cells of eczema patients were higher than the normal subjects. Aberrant expressions of RasGRP1might contribute to the pathogenesis of eczema. Part II The effects of dexamethasone on the expressions of RasGRPl in T cells of eczema patientsObjective To study the effects of dexamethasone on the expression of RasGRP1in T cells of eczema patients. To select the optimum concentration of dexamethasone and investigate its effects on T cell function.Methods Patients with severe eczema using dexamethasone were selected. The expressions of RasGRP1in T cells before and after dexamethasone therapy were detected. Dexamethasone was dubbed in three different concentrations of1×10-5mol/L,1×10-6mol/L and1×10-7mol/L. Dexamethasone co-cultured with T cells in eczema patients without dexamethasone therapy. The expressions of RasGRP1were detected by RT-PCR. To observe the expressions of RasGRP1in T cells at different time points (Oh,6h,12h,18h,24h) of dexamethasone. After the stimulation of different concentrations of dexamethasone, the apoptosis of T cells were detected by MTT.Results Without dexamethasone therapy, The expressions of RasGRP1in patients with eczema were higher than those in normal subjects. One week later by the use of dexamethasone therapy, the expressions of RasGRP1decreased significantly, which were close to the normal expression levels. Dexamethasone could inhibit the expressions of RasGRP1in eczema patients with three different concentrations of dexamethasone.1×10-5mol/L concentration had the best inhibitory effect on the expressions of RasGRPl. When using the concentration of1×10-5mol/L to inhibit T cell, the expressions of RasGRP1decreased with time prolonging. The inhibitory effect of dexamethasone was strongest after24h. Different concentrations of dexamethasone could promote apoptosis of T cells. And the apoptosis in a concentration of1×10-5mol/L dexamethasone was the strongest.Conclusions Dexamethasone could inhibit the expression of RasGRP1in peripheral blood T cells of the patients with eczema and promote the apoptosis of T cells. The effects of inhibition increased with the increase in the concentration of dexamethasone and time. One week After the use of dexamethasone, the level of RasGRP1in peripheral blood T cells of eczema patients decreased to the normal levels. This may be a new mechanism for anti-inflammatory effects of dexamethasone. Part Ⅲ Construction of lentiviral vectors containing H-RasGRPl-shRNA and its effects on T cellsObjective To construct lentiviral vectors containing the gene of H-RasGRP1-shRNA and transfect T cells. To explore the expressions of RasGRP1in T cells after RNA interference. To study the effects on Ras downstream signaling proteins, apoptosis and cytokine secretion after silencing RasGRP1gene. And to investigate the mechanisms in T cells.Methods Lentiviral vectors containing the gene of H-RasGRP1-shRN A were constructed and the viral titer1.0×108TU/ml was acquired. When T cells were transfected by lentiviral vectors, the expressions of RasGRP1in T cells were detected by RT-PCR and Western Blot methods at different time points (12h,24h,48h,72h). Apoptosis of T cells were detected by flow cytometry. After PMA and ionomycin stimulated T cells, the expression of Ras protein downstream signaling pathways-p-Erk, Erk, Akt and p-Akt were observed. To detect T cell cytokine secretion of IL-2, IL-4and IL-17by the method of ELISA. Lentiviral vectors were transfected into PMA and ionomycin stimulated T cells. The expressions of p-Erk, Erk, Akt and p-Akt protein in transfected T cells, as well as T cells cytokines were detected.Results H-RasGRP1-shRNA lentiviral vectors were constructed successfully and its biological titer reached1.0×108TU/ml, which could be used for gene delivery. After transfection of T cells by lentiviral vectors, the expression of RasGRP1decreased in the mRNA and protein levels. With the extension of time (12h,24h,48h,72h), RasGRP1expression levels gradually decreased and expression of RasGRP1almost disappeared at72h. Apoptosis of T cells accelerated obviously. The expressions of p-Erk, Erk, Akt and p-Akt were found in T cells of both eczema patients and the normal subjects. And the expressions in eczema patients were higher than those in the normal subjects. After the stimulation PMA and ionomycin on T cells, the expressions of p-Erk, Erk, Akt and p-Akt were significantly increased, especially in eczema patients. In addition, the levels of T cell cytokine IL-2, IL-4and IL-17were significantly increased. After lentiviral vectors transfected PMA and ionomycin-stimulated T cells, the expressions of p-Erk and p-Akt decreased, while the expression of Erk and Akt did not change significantly. The levels of cytokines IL-2, IL-4and IL-1in T cell culture supernatants were on the decrease.Conclusions Lentiviral vectors containing H-RasGRP1-shRNA had been constructed successfully, which could meet the requirement of transfection. Lentiviral vectors could be successfully transfected to T cells and inhibit the expressions of RasGRP1and apoptosis in T cells. When the eczema occurs, T cell receptor activation may be mediated by the activation of RasGRP1expression. The downstream signaling molecule of Ras protein-Erk and Akt phosphorylates, leading to the increasing of T cell cytokine. Therefore, RasGRP1may be a possible target in eczema treatment. It provides a novel idea for gene therapy and immune therapy.更多还原