本文選題：α-GalCer + NKT　； 參考：《復(fù)旦大學(xué)》2013年博士論文

【摘要】：結(jié)核桿菌是導(dǎo)致人類(lèi)死亡的重要病原體之一,全世界每年大約有800萬(wàn)新發(fā)活動(dòng)性結(jié)核,200萬(wàn)因?yàn)榻Y(jié)核病而死亡,1/3的人口被結(jié)核桿菌感染,其中5-10%發(fā)展為活動(dòng)性結(jié)核,這個(gè)比例在HIV感染者中更高。已經(jīng)成為發(fā)展中國(guó)家的重要公共衛(wèi)生問(wèn)題。BCG疫苗被證明是有效的,它可以預(yù)防兒童時(shí)期的重癥結(jié)核,比如結(jié)核性腦膜炎和粟粒性結(jié)核,其有效率為1/3435和1/9314。因此在TB高發(fā)區(qū),BCG疫苗在小兒出生后即得到接種。但是大量研究表明,BCG疫苗對(duì)于肺結(jié)核的預(yù)防效果受到質(zhì)疑,因此迫切需要更深入的探討機(jī)體結(jié)核桿菌的免疫應(yīng)答以便開(kāi)發(fā)出新的疫苗。NKT細(xì)胞是是一類(lèi)T細(xì)胞免疫調(diào)節(jié)亞群,其數(shù)量和功能與自身免疫疾病和感染性疾病密切相關(guān)。NKT細(xì)胞作為高效的免疫效應(yīng)和調(diào)節(jié)細(xì)胞,其功能多樣,但最重要的功能是抑制Mtb以及其他致病性分支桿菌。在NKT細(xì)胞的胞漿中存在已預(yù)先存在的IFN-γmRNA,如NKT細(xì)胞表面的受體接受刺激信號(hào)后,則可以立即分泌IFN-γ,其分泌水平是CD4+細(xì)胞分泌水平的200倍。體內(nèi)NKT細(xì)胞受到刺激后可以誘導(dǎo)DC細(xì)胞的成熟并加快遞呈可溶性抗原,從而促使CD8+T細(xì)胞發(fā)揮細(xì)胞毒性作用。Mtb感染小鼠或者分支桿菌細(xì)胞壁提取物注射入小鼠體內(nèi)可以誘導(dǎo)NKT細(xì)胞對(duì)Mtb產(chǎn)生強(qiáng)烈應(yīng)答,例如,將牛BCG注射入小鼠后的8天,肺組織中的NKT細(xì)胞比原先增長(zhǎng)6倍；感染后30天,NKT細(xì)胞數(shù)量才開(kāi)始下降,并且其表面的NK1.1表達(dá)也同時(shí)下調(diào)。以往研究告訴我們NKT細(xì)胞識(shí)別脂質(zhì)抗原,包括結(jié)核桿菌的脂質(zhì),在小鼠實(shí)驗(yàn)中已被證明能夠成功誘導(dǎo)保護(hù)性免疫,有實(shí)驗(yàn)證實(shí)將結(jié)核桿菌的細(xì)胞壁脫蛋白以后,再注入小鼠體內(nèi),NKT細(xì)胞可以協(xié)助肉芽腫的形成。NKT細(xì)胞不僅能針對(duì)宿主結(jié)核桿菌感染產(chǎn)生保護(hù)力,甚至對(duì)HIV也具有殺傷活力,而研究證實(shí)HIV感染后,將大大增加機(jī)體對(duì)外源性和內(nèi)源性結(jié)核桿菌的感染。因此在如何激活或者強(qiáng)化NKT細(xì)胞的活性是一個(gè)重要課題,這也是本研究著重解決的問(wèn)題,同時(shí)也是本研究的創(chuàng)新所在,因此本研究分為兩部分：第一部分：研究TB患者體內(nèi)NKT細(xì)胞的數(shù)量與功能在此本部分中,我們將探討TB患者與健康志愿者體內(nèi)NKT細(xì)胞的數(shù)量及其功能的差異,其判斷依據(jù)是NKT細(xì)胞的百分比、IL-4和IFN-γ胞內(nèi)細(xì)胞因子的分泌量。通過(guò)本部分研究,我們發(fā)現(xiàn)對(duì)于活動(dòng)性TB患者來(lái)說(shuō),其體內(nèi)的NKT細(xì)胞的百分比相對(duì)于健康對(duì)照者來(lái)說(shuō)是顯著下降的,第二部分：a-GalCer對(duì)結(jié)核桿菌感染Balb/c小鼠體內(nèi)NKT細(xì)胞功能的影響,我們的研究結(jié)果顯示：健康志愿者體內(nèi)NKT細(xì)胞在受到刺激后,其IFN-y分泌量上升,IL-4分泌未出現(xiàn)明顯改變,TB患者體內(nèi)NKT細(xì)胞在受到刺激后,其IFN-y分泌量上升,IL-4分泌未出現(xiàn)明顯改變,IL-4分泌未出現(xiàn)明顯改變,健康志愿者體內(nèi)NKT細(xì)胞數(shù)量顯著高于TB患者體內(nèi)NKT細(xì)胞的數(shù)量,在NKT細(xì)胞功能上,TB患者顯著低于健康志愿者,二者具有統(tǒng)計(jì)學(xué)差異,在受到藥物刺激后,患者體內(nèi)IFN-y陽(yáng)性,tetramer+的NKT細(xì)胞顯著高于健康對(duì)照組,IL-4陽(yáng)性,tetramer+的NKT細(xì)胞顯著低于健康對(duì)照組,T-SPOT對(duì)于TB的臨床診斷具有重要指導(dǎo)作用。我們發(fā)現(xiàn)在不同給藥劑量,不同給藥途徑的情況下,α-GalCer活化NKT細(xì)胞的能力存在顯著差異。在尾靜脈給藥途徑中,合適給藥劑量為1000ng/10ul；在滴鼻給藥途徑中,我們發(fā)現(xiàn)各劑量組之間無(wú)顯著性差異。在給藥劑量確定的情況下(1000ng/100ul靜脈給藥,1000ng/10ul滴鼻)靜脈給藥途徑時(shí),血清,支氣管肺泡灌洗液,肺勻漿液中IFN-γ分泌量有顯著差異,血清中為最高；采用滴鼻給藥途徑時(shí),血清,支氣管肺泡灌洗液,肺勻漿液中IFN-γ分泌量有顯著差異,肺勻漿液中為最高。通過(guò)本研究我們發(fā)現(xiàn)：無(wú)論何種給藥方式,α-GalCer單獨(dú)用藥作用都不明顯；我們首次應(yīng)用α-GalCer聯(lián)合RFP給藥,結(jié)果顯示該藥物組合可治療Mtb感染小鼠,與RFP單獨(dú)給藥組比較具有統(tǒng)計(jì)學(xué)差異,靜脈給藥作用更明顯；本研究結(jié)果提示：在治療后肺局部的各種炎癥因子(Th1細(xì)胞因子和Th2細(xì)胞因子)總體趨勢(shì)是下降的；我們發(fā)現(xiàn)α-GalCer滴鼻聯(lián)合RFP給藥能夠顯著降低小鼠肺組織局部的炎癥反應(yīng)。
[Abstract]:Mycobacterium tuberculosis is one of the important pathogens causing human death. There are about 800 million new active tuberculosis in the world every year and 2 million of them die from tuberculosis. The population of 1/3 is infected by TB bacilli, and 5-10% develops into active tuberculosis, which is higher among HIV infected people. It has become an important public health question in developing countries. The.BCG vaccine has been proved to be effective. It can prevent severe tuberculosis in children, such as tuberculous meningitis and miliary tuberculosis. The effective rate is 1/3435 and 1/9314. in the high incidence area of TB, and the BCG vaccine is inoculated after the birth of children. However, a large number of studies have shown that the effect of the BCG vaccine on tuberculosis is questioned, therefore, the effect of the vaccine on the prevention of tuberculosis is questioned. It is urgent to explore the immune response of Mycobacterium tuberculosis in order to develop new vaccine.NKT cells is a kind of T cell immunomodulating subgroup. Its quantity and function are closely related to autoimmune diseases and infectious diseases, which are closely related to.NKT cells as effective immune effects and regulating cells, their functions are diverse, but the most important function of the cells. It is the inhibition of Mtb and other pathogenic mycobacteria. The presence of pre existing IFN- gamma mRNA in the cytoplasm of NKT cells, such as the receptors on the surface of the NKT cells, can immediately secrete IFN- gamma, which is 200 times the level of the secretory level of CD4+ cells. In vivo NKT cells can induce the maturation of DC cells and add to the maturation of the DC cells. Express is soluble antigen, which causes CD8+T cells to play cytotoxic effect,.Mtb infected mice or Mycobacterium tumefaciens cell wall extract injection into mice can induce NKT cells to produce a strong response to Mtb, for example, 8 days after the injection of bovine BCG into mice, the NKT cells in the lung tissue are 6 times more than that of the original, and 30 days after infection, NKT The number of cells began to decline and the expression of NK1.1 on its surface decreased simultaneously. Previous studies have shown that NKT cells identified lipid antigens, including the lipid of Mycobacterium tuberculosis. In mice, it has been proved to be able to successfully induce protective immunity. The experiment proved that the cell wall of tuberculosis bacillus was deproteinized and then injected into the mice, NK T cells can assist granulomatosis in the formation of.NKT cells not only for the protection of the host tuberculosis infection, but also to the activity of HIV, and the study confirms that HIV infection will greatly increase the organism's infection to exogenous and endogenous Mycobacterium tuberculosis. Therefore, it is important to activate or strengthen the activity of NKT cells. This is also a problem that this study focuses on, and it is also the innovation of this study. Therefore, this study is divided into two parts: the first part: the study of the number and function of NKT cells in TB patients in this part, we will explore the difference between the number and function of NKT cells in the TB patients and healthy volunteers, and the judgment depends on the difference of the number and function of the cells in the body of the healthy volunteers. According to the percentage of NKT cells, the secretion of cytokine in IL-4 and IFN- gamma cells. By this part, we found that the percentage of NKT cells in the active TB patients was significantly lower than that of the healthy controls, and the second part: a-GalCer had the function of NKT cells in Balb/c mice infected with nuclear bacillus. The results showed that after the stimulation of NKT cells in healthy volunteers, the secretion of IFN-y increased and the secretion of IL-4 did not change obviously. The secretion of IFN-y in the NKT cells in the TB patients increased, the secretion of IL-4 was not obviously changed, the secretion of IL-4 did not change obviously, and the NKT fine in the healthy volunteers was fine. The number of cells was significantly higher than that of NKT cells in TB patients. In the function of NKT cells, the TB patients were significantly lower than those of the healthy volunteers. The two were statistically different. After the stimulation of the drug, the IFN-y positive in the patients, tetramer+ NKT cells were significantly higher than those of the healthy control group, IL-4 positive, and tetramer+ NKT cells were significantly lower than those of the healthy control group, T -SPOT has an important guiding role in the clinical diagnosis of TB. We found that there is a significant difference in the ability of alpha -GalCer to activate NKT cells in different doses and different routes of administration. The appropriate dosage is 1000ng/10ul in the route of drug delivery in the caudal vein; we found no significant difference between the different dose groups. Sex differences. When the dosage of the drug was determined (1000ng/100ul intravenous administration, 1000ng/10ul dripping), the secretion of IFN- gamma in serum, bronchoalveolar lavage fluid and lung homogenate was significantly different, and the highest in serum. The secretion of IFN- gamma in the blood, bronchoalveolar lavage fluid and lung homogenate was used when the nasal drip route was used. There were significant differences in pulmonary homogenate. We found that the effect of -GalCer alone was not obvious in any way of administration; we first used alpha -GalCer combined with RFP, and the result showed that the combination of this drug could treat Mtb infected mice, compared with the RFP alone group. More evidently, the results of this study suggest that the overall trend of various inflammatory factors (Th1 cytokine and Th2 cytokine) in the lung region after treatment is decreased; we have found that alpha -GalCer dripping with RFP can significantly reduce the local inflammatory response in the lung tissue of mice.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R52

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