模拟HCV HVR1免疫7肽对人不同免疫细胞分泌细胞因子影响的初步研究

发布时间：2018-05-13 23:13

本文选题：丙型肝炎病毒 + 模拟肽　；参考：《中国人民解放军军医进修学院》2008年博士论文

【摘要】： 目前针对慢性丙型肝炎病毒(HCV)感染尚无切实有效的办法,以诱导机体产生细胞免疫为主的HCV治疗新疫苗,可通过重建感染者针对HCV的免疫应答,调动自身的免疫机能来清除病毒,减少非特异性炎症对肝脏的损害的作用。有可能是将来治疗慢性HCV感染的重要方法。本研究的课题是建立在我室长期对中国人群常见的HCV感染毒株(1b、2a)包膜蛋白E2高变区1(HVR1)序列变异规律的研究基础上,根据保守位点合成的一系列长度不等的HCVHVR1模拟表位。之前的研究中,已经验证了其中一个含有7个氨基酸的多肽(7P)能够诱导小鼠免疫应答。本课题主要是在此基础上,针对人不同免疫细胞,研究7P对人不同免疫细胞分泌细胞因子的影响,并初步探讨其机制。课题共分为三个部分。 (一)7P对正常人免疫细胞分泌细胞因子的影响将7P与正常人的外周血单核细胞((PBMCs)共同孵育培养,然后通过检测免疫细胞内细胞因子分泌的改变来研究其免疫变化。结果表明,应用7P刺激正常人PBMCs后,分泌IFN-γ的细胞频数无论在NK细胞、NKT细胞以及CD4~+CD8~-、CD4~-CD8~+T淋巴细胞均明显增高;而分泌IL-4的细胞频数则变化不明显。分泌IL-10的细胞频数在CD4+CD8-、CD4-CD8+T淋巴细胞表现为升高,在NK细胞、NKT细胞表现为下降;而分泌TNF-α的细胞频数在CD4+CD8-、CD4-CDS+T淋巴细胞表现为下降,在NK细胞、NKT细胞表现为升高。 (二)7P刺激免疫细胞分泌细胞因子改变的初步研究首先将7P与正常人的外周血单核细胞((PBMCs)共同孵育培养,然后检测抗原呈递细胞(DC)成熟及细胞因子变化情况。结果表明应用7P刺激DC细胞时,成熟细胞表型的mDC细胞的频数明显下降;成熟的细胞表型的pDC细胞的频数未发生变化,代表mDC分泌功能的IL-12未能检测出来。提示7P体外刺激有可能够降低成熟mDC的表达,未能促进IL-12的分泌;但对pDC表达的影响尚不完全明确。其后采用细胞分选技术,将抗原提呈细胞从PBMCs中分离出去,剩余的成分中包括免疫反应的效应细胞T细胞和NK、NKT细胞,将其和7P共同孵育培养后检测细胞内细胞因子染色。结果表明,当应用磁珠分选之后其与7P共同孵育培养,能够得到与分选之前基本相同的结果。提示7P的这种对细胞因子分泌的调节可能是直接作用于效应细胞,同时对于抗原提呈细胞(DC)也具有直接或间接的作用,而这种作用也可能间接地影响效应细胞的细胞因子分泌。 (三)7P对HCV感染患者免疫细胞分泌细胞因子影响的初步研究将HCV感染患者的PBMCs和7P共同孵育培养后检测细胞内细胞因子染色,来分析7P对HCV感染患者的免疫细胞的作用。与第一部分结果相比,INF-γ在NK及NKT细胞,IL-10在CD4~+CD8~-T细胞及CD4~-CD8~+T细胞的变化是一致的;而TNF-α在NKT细胞出现了相反的变化,即下降。同时,我们也注意到其他未发生具有统计学意义变化的部分,其变化的主要方向也往往和之前的实验结果趋向一致。
[Abstract]:At present, there is no effective and effective method for chronic hepatitis C virus (HCV) infection, in order to induce the organism to produce a new HCV vaccine based on cell immunization. It can be used to reconstruct the immune response to HCV by reconstructing the infected person and mobilize its own immune function to remove the virus and reduce the damage to the liver by non specific inflammation. It may be the future. An important method for the treatment of chronic HCV infection is based on the study of a series of HCVHVR1 mimic epitopes based on the conservative loci of a series of HCVHVR1 mimic epitopes synthesized by the conservative loci, based on a long term study on the variation of the hypervariable region 1 (HVR1) sequence of the envelope protein E2 of the common HCV infection virus (1b, 2a) in the Chinese population. One of these peptides (7P) which contains 7 amino acids can induce immune response in mice. On this basis, the subject is to study the effect of 7P on the secretion of cytokines from different immune cells in human, and the mechanism is preliminarily discussed. The subject is divided into three parts.
(1) effect of 7P on cytokines secreted by normal human immune cells
7P and normal human peripheral blood mononuclear cells (PBMCs) were incubated together, and then the changes of cytokine secretion in the immune cells were detected to study their immune changes. The results showed that the frequency of IFN- gamma secreted by 7P stimulated the normal human PBMCs, both in NK cells, NKT cells, and CD4~+CD8~-, and CD4~-CD8~+T lymphocytes were all clear. The frequency of secreting IL-4 was not obvious. The frequency of secreting IL-10 was in CD4+CD8-, the expression of CD4-CD8+T lymphocyte was elevated and the NKT cells were decreased in NK cells, while the number of TNF- alpha cells in CD4+CD8-, CD4-CDS+T lymphocyte decreased, and NKT cells in NK cells were elevated.
(two) a preliminary study on the changes of cytokines secreted by 7P stimulated immune cells.
First, 7P was incubated with normal human peripheral blood mononuclear cells (PBMCs), and then the maturation of antigen presenting cell (DC) and the change of cytokine were detected. The results showed that the frequency of mDC cells in the mature cell phenotype decreased obviously when 7P was used to stimulate DC cells, and the frequency of the mature cell phenotype of pDC cells did not change, representing mDC. The secretory function of IL-12 was not detected. It was suggested that in vitro stimulation of 7P could reduce the expression of mature mDC and failed to promote the secretion of IL-12, but the effect on the expression of pDC was not completely clear. Then the cell sorting technique was used to separate the antigen presenting cells from PBMCs, and the remaining components included the immune response cell T finely. Cell and NK, NKT cells were incubated with 7P to detect cytokine staining in cells. The results showed that when the magnetic beads were reincubated with 7P, the same results were obtained before the separation, suggesting that the regulation of the secretion of cytokines from 7P may be directly acting on the effector cells and at the same time Antigen presenting cells (DC) also play a direct or indirect role, which may indirectly affect the secretion of cytokines in effector cells.
(three) effect of 7P on cytokines secreted by immune cells in HCV infected patients
HCV infected patients' PBMCs and 7P were incubated together to detect cytokine staining in cells to analyze the effect of 7P on the immune cells of patients with HCV infection. Compared with the results of the first part, INF- gamma in NK and NKT cells, IL-10 in CD4~+CD8~-T cells and CD4~-CD8~+T cells, and TNF- alpha appeared on the opposite side of the cells. At the same time, we also noticed that there were no other parts of statistical significance, and the main direction of the change tended to be consistent with the previous experimental results.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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