高劑量熱休克蛋白gp96及其氨基端片段對調(diào)節(jié)性T細胞介導CTL反應的影響
發(fā)布時間：2018-08-30 18:42
【摘要】：以往的研究表明,熱休克蛋白gp96能特異地結合病毒抗原和腫瘤抗原,從而激發(fā)抗原特異性細胞毒性T細胞(CTL)應答和體液免疫應答,而高劑量(100μg)gp96會下調(diào)CTL應答。但其機制尚不是十分清楚。本研究按照三步法提取和純化了小鼠熱休克蛋白gp96,同時利用大腸桿菌表達和純化了gp96氨基端355氨基酸片段(N355),然后分別以gp96和N355作為佐劑,與乙肝病毒核心蛋白表位HBcAg87-95聯(lián)合免疫BALB/c小鼠,第3次免疫后7天取免疫小鼠脾細胞,染色后經(jīng)流式細胞儀分析和通過ELISPOT、Tetramer實驗檢測,結果,低免疫劑量(10μg)gp96能引起HBV特異的CTL應答,隨gp96的免疫劑量的增加,特異的CTL應答下調(diào),而調(diào)節(jié)性T細胞(Regulatory T cells,Treg)的數(shù)目升高。同時,將Treg細胞(CD4+CD25+)與效應性CD8+細胞或CD4+CD25-細胞進行細胞體外抑制實驗研究,結果Treg細胞能夠抑制效應性T細胞增殖。而N355能引發(fā)CTL應答,卻不能激活Treg。實驗進一步給小鼠注射低劑量的環(huán)磷酰胺(Cyclophosphamide, CTX)抑制Treg細胞,結果導致CTL細胞數(shù)量增加。在此基礎上,我們應用免疫組化實驗研究了慢性重癥乙型肝炎患者的肝臟組織中gp96的表達變化與CTL的相關性,結果慢性重癥乙肝病人肝組織中CTL明顯增強,且肝組織中gp96的表達含量要明顯高于慢性乙肝和肝癌病人。以上研究結果表明,高劑量的gp96通過上調(diào)Treg細胞來下調(diào)CTL應答。該研究為進一步研制通用的高效新型免疫佐劑和疫苗的遞送系統(tǒng)以及將gp96作為免疫調(diào)節(jié)劑用于癌癥和傳染性疾病的治療提供參考依據(jù)。
[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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.11
本文編號：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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.11
【參考文獻】
相關期刊論文 前2條
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
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