TB患者NKT细胞数量与功能的研究及α-GalCer对Mtb感染后小鼠体内NKT细胞功能的影响

发布时间：2018-05-16 07:22

本文选题：α-GalCer + NKT　；参考：《复旦大学》2013年博士论文

【摘要】：结核杆菌是导致人类死亡的重要病原体之一,全世界每年大约有800万新发活动性结核,200万因为结核病而死亡,1/3的人口被结核杆菌感染,其中5-10%发展为活动性结核,这个比例在HIV感染者中更高。已经成为发展中国家的重要公共卫生问题。BCG疫苗被证明是有效的,它可以预防儿童时期的重症结核,比如结核性脑膜炎和粟粒性结核,其有效率为1/3435和1/9314。因此在TB高发区,BCG疫苗在小儿出生后即得到接种。但是大量研究表明,BCG疫苗对于肺结核的预防效果受到质疑,因此迫切需要更深入的探讨机体结核杆菌的免疫应答以便开发出新的疫苗。NKT细胞是是一类T细胞免疫调节亚群,其数量和功能与自身免疫疾病和感染性疾病密切相关。NKT细胞作为高效的免疫效应和调节细胞,其功能多样,但最重要的功能是抑制Mtb以及其他致病性分支杆菌。在NKT细胞的胞浆中存在已预先存在的IFN-γmRNA,如NKT细胞表面的受体接受刺激信号后,则可以立即分泌IFN-γ,其分泌水平是CD4+细胞分泌水平的200倍。体内NKT细胞受到刺激后可以诱导DC细胞的成熟并加快递呈可溶性抗原,从而促使CD8+T细胞发挥细胞毒性作用。Mtb感染小鼠或者分支杆菌细胞壁提取物注射入小鼠体内可以诱导NKT细胞对Mtb产生强烈应答,例如,将牛BCG注射入小鼠后的8天,肺组织中的NKT细胞比原先增长6倍；感染后30天,NKT细胞数量才开始下降,并且其表面的NK1.1表达也同时下调。以往研究告诉我们NKT细胞识别脂质抗原,包括结核杆菌的脂质,在小鼠实验中已被证明能够成功诱导保护性免疫,有实验证实将结核杆菌的细胞壁脱蛋白以后,再注入小鼠体内,NKT细胞可以协助肉芽肿的形成。NKT细胞不仅能针对宿主结核杆菌感染产生保护力,甚至对HIV也具有杀伤活力,而研究证实HIV感染后,将大大增加机体对外源性和内源性结核杆菌的感染。因此在如何激活或者强化NKT细胞的活性是一个重要课题,这也是本研究着重解决的问题,同时也是本研究的创新所在,因此本研究分为两部分：第一部分：研究TB患者体内NKT细胞的数量与功能在此本部分中,我们将探讨TB患者与健康志愿者体内NKT细胞的数量及其功能的差异,其判断依据是NKT细胞的百分比、IL-4和IFN-γ胞内细胞因子的分泌量。通过本部分研究,我们发现对于活动性TB患者来说,其体内的NKT细胞的百分比相对于健康对照者来说是显著下降的,第二部分：a-GalCer对结核杆菌感染Balb/c小鼠体内NKT细胞功能的影响,我们的研究结果显示：健康志愿者体内NKT细胞在受到刺激后,其IFN-y分泌量上升,IL-4分泌未出现明显改变,TB患者体内NKT细胞在受到刺激后,其IFN-y分泌量上升,IL-4分泌未出现明显改变,IL-4分泌未出现明显改变,健康志愿者体内NKT细胞数量显著高于TB患者体内NKT细胞的数量,在NKT细胞功能上,TB患者显著低于健康志愿者,二者具有统计学差异,在受到药物刺激后,患者体内IFN-y阳性,tetramer+的NKT细胞显著高于健康对照组,IL-4阳性,tetramer+的NKT细胞显著低于健康对照组,T-SPOT对于TB的临床诊断具有重要指导作用。我们发现在不同给药剂量,不同给药途径的情况下,α-GalCer活化NKT细胞的能力存在显著差异。在尾静脉给药途径中,合适给药剂量为1000ng/10ul；在滴鼻给药途径中,我们发现各剂量组之间无显著性差异。在给药剂量确定的情况下(1000ng/100ul静脉给药,1000ng/10ul滴鼻)静脉给药途径时,血清,支气管肺泡灌洗液,肺匀浆液中IFN-γ分泌量有显著差异,血清中为最高；采用滴鼻给药途径时,血清,支气管肺泡灌洗液,肺匀浆液中IFN-γ分泌量有显著差异,肺匀浆液中为最高。通过本研究我们发现：无论何种给药方式,α-GalCer单独用药作用都不明显；我们首次应用α-GalCer联合RFP给药,结果显示该药物组合可治疗Mtb感染小鼠,与RFP单独给药组比较具有统计学差异,静脉给药作用更明显；本研究结果提示：在治疗后肺局部的各种炎症因子(Th1细胞因子和Th2细胞因子)总体趋势是下降的；我们发现α-GalCer滴鼻联合RFP给药能够显著降低小鼠肺组织局部的炎症反应。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R52

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