【摘要】： 流感病毒是一種對人類健康和公共衛(wèi)生威脅很大的病毒,每年全球有很多人由于流感病毒感染致病或死亡。其中老年人對流感病毒的抵抗力尤為低下,更易受到流感病毒感染及其誘發(fā)的并發(fā)癥導致的住院及死亡。因而迫切需要研究能夠特別適合老年人應用的安全有效的流感疫苗。闡明免疫衰老的原因,從而啟示和惠及其他老年疫苗的開發(fā)和應用。 本研究利用昆蟲桿狀病毒表達系統(tǒng)共表達H1N1流感病毒A/Perto Rico/8/34的HA,NA和M1蛋白,在體外組裝成流感病毒樣顆粒(Virus-like particles, VLPs)。經鑒定和純化的VLPs保持了與流感病毒粒子相似的形態(tài)大小以及表面糖蛋白的正常生物學活性。低劑量VLPs免疫的成年及老年小鼠與滅活流感病毒(Inactivated influenza virus, IIV)相比顯示相同的免疫原性,均能夠保護被免疫小鼠在致死流感病毒攻擊中存活。但是老年小鼠的保護效果明顯劣于成年小鼠,表現(xiàn)為老年小鼠體重下降程度大于成年小鼠以及攻毒后體內活躍的高水平的抗原特異性CD8 T細胞反應。增大免疫劑量能顯著加強疫苗在老年小鼠中的免疫保護效果,表現(xiàn)為攻毒中輕微的可以忽略不計的體重下降以及攻毒后與成年小鼠相當?shù)牡退降腃D8 T細胞反應。 雖然流感病毒VLPs能對被免疫老年小鼠提供完全保護,但是其并不能取得像成年小鼠一樣良好的保護效果。這樣的結果激起了我們對免疫衰老機制研究的興趣。與其他文獻報道相似,在實驗中我們發(fā)現(xiàn)老年小鼠體內的調節(jié)性T細胞(T regulatory cells, Treg)含量顯著高于成年小鼠Treg細胞約2倍之多。因為Treg細胞對免疫系統(tǒng)的調節(jié)性作用,我們推測這部分累積的Treg細胞可能是造成老年小鼠接種后疫苗免疫反應性低下的原因之一。因此在免疫小鼠前,我們給小鼠腹腔注射CD25的單克隆抗體以暫時消除Treg細胞。實驗結果非常確實的顯示了消除Treg細胞能夠極大提高老年小鼠的特異性疫苗免疫反應,并對老年小鼠提供完美的免疫保護力。實驗結果證明了老年小鼠體內積累的Treg細胞是造成其免疫反應低下的主要原因之一。 在闡明了老年小鼠免疫衰老的可能機制后,我們對老年小鼠進行了一次免疫攻毒保護試驗。以觀察是否消除Treg細胞能夠提高疫苗一次免疫的保護成功率。實驗結果再一次證實了Treg細胞對調節(jié)老年小鼠免疫反應的重要性:注射過CD25抗體的免疫老年小鼠在大劑量致死流感病毒攻擊中全部存活且體重鮮有下降,而沒有注射過CD25抗體的老年小鼠僅有67%存活,且存活個體有明顯的發(fā)病癥狀和體重降低。 本研究結果證明昆蟲桿狀病毒生產的流感病毒VLPs在老年小鼠中具有和IIV疫苗相等同的免疫保護效力。VLPs具有能夠刺激樹突狀細胞—專職抗原遞呈細胞產生細胞因子的能力。流感病毒VLPs代表了一種新型流感疫苗策略,能夠對成年和老年小鼠提供更有效的免疫保護效力,因而具有非常廣闊的應用前景。本研究同時闡明,小鼠免疫衰老的潛在機制——即Treg細胞的累積性增多是造成老年小鼠免疫力低下的主要原因之一。免疫接種前暫時性消除Treg細胞能夠極大恢復老年小鼠被抑制的免疫反應,增強疫苗免疫誘導的體液和細胞免疫反應。針對Treg細胞的疫苗研發(fā)策略為今后老年疫苗的研究和開發(fā)提供了新思路和可能的靶向及解決方案。
[Abstract]:Influenza virus is a kind of virus that threatens human health and public health. Many people all over the world suffer or die from influenza virus infection every year. Safe and effective influenza vaccines that are especially suitable for the elderly. To clarify the causes of immune aging, and thus inspire and benefit the development and application of other vaccines for the elderly.
In this study, the HA, NA and M1 proteins of H1N1 influenza virus A/Perto Rico/8/34 were co-expressed by insect baculovirus expression system and assembled into influenza virus-like particles (VLPs) in vitro. The VLPs identified and purified maintained the similar morphology and size as influenza virus particles and the normal biological activity of surface glycoproteins. Compared with inactivated influenza virus (IIV), adult and aged mice immunized with low dose of VLPs showed the same immunogenicity and could protect the immunized mice from lethal influenza virus attack. Higher levels of antigen-specific CD8 T cells were active in adult mice and in vivo after challenge. Increasing the dose of the vaccine significantly enhanced the immune protective effect of the vaccine in the aged mice, which showed slight negligible weight loss during attack and low levels of CD8 T cells after challenge.
Although influenza virus VLPs can provide complete protection to immunized aged mice, they can not achieve as good protection as adult mice. This result arouses our interest in the study of the mechanism of immune aging. Because Treg cells regulate the immune system, we speculate that this accumulation of Treg cells may be one of the causes of the decreased immune response in the vaccinated aged mice. Monoclonal antibodies were used to temporarily eliminate Treg cells. The results showed that eliminating Treg cells could greatly improve the specific immune response of the aged mice and provide perfect immune protection for the aged mice. One of the main reasons.
After elucidating the possible mechanism of immune senescence in aged mice, we conducted an immune challenge protection test in aged mice to see if eliminating Treg cells could increase the success rate of vaccine immunization. The immunized mice survived the lethal attack of influenza viruses at high doses with little weight loss, while only 67% of the aged mice without CD25 antibody survived, and the survivors had significant symptoms and weight loss.
The results of this study demonstrated that the influenza virus VLPs produced by insect baculovirus have the same immune protective effect as the IIV vaccine in the aged mice. VLPs have the ability to stimulate the production of cytokines by dendritic cells-specialized antigen-presenting cells. Influenza virus VLPs represent a novel influenza vaccine strategy that can be used for adulthood and development This study also elucidates that the cumulative increase of Treg cells, the underlying mechanism of immune senescence in mice, is one of the major causes of immunodeficiency in older mice. Temporary elimination of Treg cells before vaccination can greatly restore immunity. Inhibited immune responses in aged mice enhance the humoral and cellular immune responses induced by vaccines. Vaccine development strategies for Treg cells provide new ideas and possible targets and solutions for future research and development of vaccines for the aged.
【學位授予單位】：中國農業(yè)科學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392

【共引文獻】

相關期刊論文 前3條

1 夏書宇;;運動對衰老過程中免疫機制的影響[J];武漢體育學院學報;2005年11期

2 劉建民;梁鳳霞;李佳;劉溪泉;唐宏圖;吳松;王華;陳澤斌;;電針強壯穴對衰老模型大鼠T細胞免疫調節(jié)功能的影響[J];針刺研究;2009年04期

3 陳艷珍;;酸奶對老年雌性小鼠免疫功能和游泳耐力的影響[J];營養(yǎng)學報;2007年03期

相關博士學位論文 前6條

1 金紅姝;獨活及其醇提物延緩腦老化機制的實驗研究[D];遼寧中醫(yī)學院;2001年

2 宋淑霞;益氣補腎方藥對自然衰老和腎虛致衰老小鼠免疫衰老的防治研究[D];河北醫(yī)科大學;2002年

3 馮仁田;衰老的免疫學研究：自然衰老的免疫系統(tǒng)變化和氧化應激對免疫系統(tǒng)的影響[D];中國協(xié)和醫(yī)科大學;2000年

4 馬世彬;免疫老化中CD28分子的變化及調控機理[D];中國協(xié)和醫(yī)科大學;2003年

5 樊樹芳;H5N1亞型禽流感病毒弱毒活疫苗株構建及在不同動物模型上免疫效力的評價[D];中國農業(yè)科學院;2008年

6 李新明;微波固態(tài)合成低聚糖及其對小鼠抗氧化和免疫活性研究[D];江南大學;2008年

相關碩士學位論文 前4條

1 劉勝利;慢性憤怒應激對大鼠腦老化的影響及其神經內分泌機制[D];河南中醫(yī)學院;2010年

2 史進方;T細胞亞群及相關免疫分子在生理性衰老過程中的調節(jié)性表達及其意義[D];蘇州大學;2005年

3 葉藝;A型流感病毒冷適應株的拯救及免疫原性的初步研究[D];西北農林科技大學;2008年

4 杜三軍;熱量限制及姜黃對大鼠血清及脾組織勻漿IgA、IgG、IgM水平及免疫器官指數(shù)的影響[D];河北醫(yī)科大學;2010年

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