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(jié)合病毒抗原和腫瘤抗原,從而激發(fā)抗原特異性細(xì)胞毒性T細(xì)胞(CTL)應(yīng)答和體液免疫應(yīng)答,而高劑量(100μg)gp96會下調(diào)CTL應(yīng)答。但其機(jī)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(dá)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(xì)胞,染色后經(jīng)流式細(xì)胞儀分析和通過ELISPOT、Tetramer實(shí)驗(yàn)檢測,結(jié)果,低免疫劑量(10μg)gp96能引起HBV特異的CTL應(yīng)答,隨gp96的免疫劑量的增加,特異的CTL應(yīng)答下調(diào),而調(diào)節(jié)性T細(xì)胞(Regulatory T cells,Treg)的數(shù)目升高。同時,將Treg細(xì)胞(CD4+CD25+)與效應(yīng)性CD8+細(xì)胞或CD4+CD25-細(xì)胞進(jìn)行細(xì)胞體外抑制實(shí)驗(yàn)研究,結(jié)果Treg細(xì)胞能夠抑制效應(yīng)性T細(xì)胞增殖。而N355能引發(fā)CTL應(yīng)答,卻不能激活Treg。實(shí)驗(yàn)進(jìn)一步給小鼠注射低劑量的環(huán)磷酰胺(Cyclophosphamide, CTX)抑制Treg細(xì)胞,結(jié)果導(dǎo)致CTL細(xì)胞數(shù)量增加。在此基礎(chǔ)上,我們應(yīng)用免疫組化實(shí)驗(yàn)研究了慢性重癥乙型肝炎患者的肝臟組織中g(shù)p96的表達(dá)變化與CTL的相關(guān)性,結(jié)果慢性重癥乙肝病人肝組織中CTL明顯增強(qiáng),且肝組織中g(shù)p96的表達(dá)含量要明顯高于慢性乙肝和肝癌病人。以上研究結(jié)果表明,高劑量的gp96通過上調(diào)Treg細(xì)胞來下調(diào)CTL應(yīng)答。該研究為進(jìn)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(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.11

【參考文獻(xiàn)】

相關(guān)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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本文編號：2213962