CC趋化因子受体5在疾病中的作用研究暨胸腺素α原cDNA多态性研究

发布时间：2018-01-09 20:15

本文关键词：CC趋化因子受体5在疾病中的作用研究暨胸腺素α原cDNA多态性研究　出处：《第二军医大学》2007年博士论文　论文类型：学位论文

【摘要】： CC趋化因子受体5 (cc chemokine receptor 5,CCR5)为细胞膜蛋白,是趋化因子受体的一种亚型。CCR5主要表达在单核细胞、T细胞等白细胞上,最近的研究表明其为活化Thl细胞的表面标志物,提示CCR5与自身免疫性疾病的发生发展有关。我们通过研究CCR5在实验性自身免疫性心肌炎(experimental autoimmune myocarditis, EAM)小鼠模型以及滤泡性甲状腺癌的表达情况试图发现CCR5在疾病发生发展过程中所发挥的作用。我们采用了RT-PCR、免疫组化、流式细胞检测等经典的方法,并成功构建了抗体封闭和细胞过继等实验动物模型,从分子、细胞以及动物模型等不同层次证明了CCR5在EAM小鼠以及滤泡状甲状腺癌的重要性。我们发现CCR5在EAM小鼠模型的外周血中表达含量明显上升,并且在炎性心肌组织中浸润的炎性细胞多数呈CCR5阳性表达;CCR5阳性细胞过继小鼠其心肌炎发病程度明显高于T细胞过继组,而CCR5阴性过继小鼠发病程度则明显降低;以CCR5单克隆抗体封闭EAM小鼠其发病率明显下降。近来的研究表明趋化因子受体参与肿瘤的生长和分化,且与肿瘤的转移有着密切的联系。为研究CCR5在滤泡状甲状腺癌的发生或发展中所起的作用,我们通过对滤泡性甲状腺癌中CCR5的表达情况的分析,发现在滤泡状甲状腺癌组织中,CCR5表达阳性率明显高于甲状腺腺瘤和正常甲状腺组织(PO.OO1),且滤泡状甲状腺癌患者血清血中CCL3, CCL5的浓度显著高于对照组(P0.O1)。提示CCR5在滤泡状甲状腺癌发病进程中发挥着重要的作用。此外通过对人胸腺素α原cDNA测序,分析了胸腺素α原的序列多态性。我们利用RT-PCR技术从外周血及脐带血中扩增胸腺素原cDNA,纯化后与克隆载体pMD18-T连接,经克隆测序,通过与标准序列比对,分析其多态性。序列分析结果表明,克隆的ProTα基因的核苷酸序列并不一致。与已报道的胸腺素α原基因进行比较,发现存在两种变异。(1)107位单核苷酸变异,以及110-121位和191-205位的核苷酸片段缺失;(2)306位单核苷酸缺失,多见于60-80年龄组。故我们认为人胸腺素α原cDNA序列存在多态性,我们发现其存在两种变异序列,但此两种基因变异并未影响到N-端前28个肽,并未影响到胸腺素α原的功能。该结果为胸腺素a原的结构、功能和演化研究提供了信息。
[Abstract]:CC chemokine receptor 5 chemokine receptor 5 (CCR5) is a membrane protein. CCR5 is a subtype of chemokine receptor. CCR5 is mainly expressed on monocyte T cells and other leukocytes. Recent studies have shown that CCR5 is a surface marker for activating Thl cells. It is suggested that CCR5 may be associated with the development of autoimmune diseases. We studied the role of CCR5 in experimental autoimmune myocarditis. Experimental autoimmune myocarditis. EAM mouse model and the expression of follicular thyroid carcinoma try to find out the role of CCR5 in the development of the disease. We used RT-PCR, immunohistochemistry. Flow cytometry and other classical methods, and successfully constructed antibody blocking and cell adoptions of experimental animal models, from the molecules. The importance of CCR5 in EAM mice and follicular thyroid carcinoma was demonstrated at different levels such as cells and animal models. We found that the expression of CCR5 in peripheral blood of EAM mice was significantly increased. And most of the infiltrating inflammatory cells in the inflammatory myocardium showed CCR5 positive expression. The degree of myocarditis in CCR5 positive adoptive mice was significantly higher than that in T cell adoptive mice, but the degree of CCR5 negative adoptive mice was significantly lower. The incidence of CCR5 monoclonal antibody in EAM mice decreased significantly. Recent studies have shown that chemokine receptors are involved in tumor growth and differentiation, and are closely related to tumor metastasis. To study the role of CCR5 in the occurrence or development of follicular thyroid carcinoma. By analyzing the expression of CCR5 in follicular thyroid carcinoma, we found that it was found in follicular thyroid carcinoma. The positive rate of CCR5 expression was significantly higher than that of thyroid adenoma and normal thyroid tissue, and CCL3 in serum of follicular thyroid carcinoma patients. The concentration of CCL5 was significantly higher than that of the control group (P0. 01), which suggested that CCR5 plays an important role in the pathogenesis of follicular thyroid carcinoma. In addition, the sequence polymorphism of human thymosin 伪 proto cDNA was analyzed by sequencing. We amplified thymogen cDNA from peripheral blood and umbilical cord blood by RT-PCR technique. After purification, it was ligated with the clone vector pMD18-T, then cloned and sequenced. The polymorphism was analyzed by alignment with the standard sequence. The nucleotide sequence of the cloned ProT 伪 gene was not consistent with that of the reported thymosin 伪 gene. And deletion of nucleotide fragments at 110-121 and 191-205; The deletion of single nucleotide was found in the 60-80 age group, so we believe that there is polymorphism in the human thymosin 伪 proto cDNA sequence, and we found that there are two variant sequences. However, these two mutations did not affect the function of the first 28 peptides and the function of prothymosin 伪, which provided information for the study of the structure, function and evolution of prothymosin a.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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