高剂量热休克蛋白gp96及其氨基端片段对调节性T细胞介导CTL反应的影响
发布时间：2018-08-30 18:42
【摘要】：以往的研究表明,热休克蛋白gp96能特异地结合病毒抗原和肿瘤抗原,从而激发抗原特异性细胞毒性T细胞(CTL)应答和体液免疫应答,而高剂量(100μg)gp96会下调CTL应答。但其机制尚不是十分清楚。本研究按照三步法提取和纯化了小鼠热休克蛋白gp96,同时利用大肠杆菌表达和纯化了gp96氨基端355氨基酸片段(N355),然后分别以gp96和N355作为佐剂,与乙肝病毒核心蛋白表位HBcAg87-95联合免疫BALB/c小鼠,第3次免疫后7天取免疫小鼠脾细胞,染色后经流式细胞仪分析和通过ELISPOT、Tetramer实验检测,结果,低免疫剂量(10μg)gp96能引起HBV特异的CTL应答,随gp96的免疫剂量的增加,特异的CTL应答下调,而调节性T细胞(Regulatory T cells,Treg)的数目升高。同时,将Treg细胞(CD4+CD25+)与效应性CD8+细胞或CD4+CD25-细胞进行细胞体外抑制实验研究,结果Treg细胞能够抑制效应性T细胞增殖。而N355能引发CTL应答,却不能激活Treg。实验进一步给小鼠注射低剂量的环磷酰胺(Cyclophosphamide, CTX)抑制Treg细胞,结果导致CTL细胞数量增加。在此基础上,我们应用免疫组化实验研究了慢性重症乙型肝炎患者的肝脏组织中gp96的表达变化与CTL的相关性,结果慢性重症乙肝病人肝组织中CTL明显增强,且肝组织中gp96的表达含量要明显高于慢性乙肝和肝癌病人。以上研究结果表明,高剂量的gp96通过上调Treg细胞来下调CTL应答。该研究为进一步研制通用的高效新型免疫佐剂和疫苗的递送系统以及将gp96作为免疫调节剂用于癌症和传染性疾病的治疗提供参考依据。
[Abstract]:Previous studies have shown that heat shock protein (gp96) can specifically bind virus antigen and tumor antigen to stimulate antigen-specific cytotoxic T cell (CTL) response and humoral immune response, while high dose (100 渭 g) gp96 can down-regulate CTL response. But its mechanism is not very clear. In this study, the amino terminal 355 amino acid fragment (N355) of mouse heat shock protein (gp96,) was expressed and purified by Escherichia coli, and then gp96 and N355 were used as adjuvants respectively. BALB/c mice were immunized with hepatitis B virus core protein epitope (HBcAg87-95). Spleen cells were immunized 7 days after the third immunization. Flow cytometry analysis and ELISPOT,Tetramer assay showed that low dose (10 渭 g) gp96 could induce HBV specific CTL response. The specific CTL response was down-regulated and the number of regulatory T cell (Regulatory T cells,Treg was increased with the increase of gp96 immune dose. At the same time, the inhibition of Treg cells (CD4 CD25) with effectual CD8 cells or CD4 CD25- cells in vitro was studied. The results showed that Treg cells could inhibit the proliferation of effective-T cells. N355 can trigger the CTL response, but not activate the Treg. Mice were further injected with low dose cyclophosphamide (Cyclophosphamide, CTX) to inhibit Treg cells, resulting in an increase in the number of CTL cells. On this basis, we studied the correlation between the expression of gp96 and CTL in liver tissue of patients with chronic severe hepatitis B by immunohistochemistry. The results showed that CTL in liver tissue of patients with chronic severe hepatitis B was significantly increased. The expression of gp96 in liver tissue was significantly higher than that in patients with chronic hepatitis B and liver cancer. These results suggest that high dose gp96 down-regulates CTL response by up-regulating Treg cells. This study provides a reference for the further development of a universal delivery system for novel immune adjuvants and vaccines and the use of gp96 as an immunomodulator for the treatment of cancer and infectious diseases.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.11
本文编号：2213962
[Abstract]:Previous studies have shown that heat shock protein (gp96) can specifically bind virus antigen and tumor antigen to stimulate antigen-specific cytotoxic T cell (CTL) response and humoral immune response, while high dose (100 渭 g) gp96 can down-regulate CTL response. But its mechanism is not very clear. In this study, the amino terminal 355 amino acid fragment (N355) of mouse heat shock protein (gp96,) was expressed and purified by Escherichia coli, and then gp96 and N355 were used as adjuvants respectively. BALB/c mice were immunized with hepatitis B virus core protein epitope (HBcAg87-95). Spleen cells were immunized 7 days after the third immunization. Flow cytometry analysis and ELISPOT,Tetramer assay showed that low dose (10 渭 g) gp96 could induce HBV specific CTL response. The specific CTL response was down-regulated and the number of regulatory T cell (Regulatory T cells,Treg was increased with the increase of gp96 immune dose. At the same time, the inhibition of Treg cells (CD4 CD25) with effectual CD8 cells or CD4 CD25- cells in vitro was studied. The results showed that Treg cells could inhibit the proliferation of effective-T cells. N355 can trigger the CTL response, but not activate the Treg. Mice were further injected with low dose cyclophosphamide (Cyclophosphamide, CTX) to inhibit Treg cells, resulting in an increase in the number of CTL cells. On this basis, we studied the correlation between the expression of gp96 and CTL in liver tissue of patients with chronic severe hepatitis B by immunohistochemistry. The results showed that CTL in liver tissue of patients with chronic severe hepatitis B was significantly increased. The expression of gp96 in liver tissue was significantly higher than that in patients with chronic hepatitis B and liver cancer. These results suggest that high dose gp96 down-regulates CTL response by up-regulating Treg cells. This study provides a reference for the further development of a universal delivery system for novel immune adjuvants and vaccines and the use of gp96 as an immunomodulator for the treatment of cancer and infectious diseases.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.11
【参考文献】
相关期刊论文 前2条
1 ;Significant correlation between expression level of HSP gp96 and progression of hepatitis B virus induced diseases[J];World Journal of Gastroenterology;2004年08期
2 ;Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice[J];World Journal of Gastroenterology;2005年19期
，本文编号：2213962
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2213962.html
最近更新
教材专著
