口服Ag85A DNA疫苗在小鼠肠道局部的表达

发布时间：2018-08-26 11:21

【摘要】： 前言自从1997年Jones和Pascual等人分别用PLG(poly-DL-lactide-co-glycolide)微粒和减毒伤寒杆菌(Salmonilla typhimurium)为运载体(vectors)制备口服DNA疫苗以来,已经有多篇报道对口服DNA疫苗在预防或治疗感染、肿瘤和过敏性疾病中的作用进行了研究,并取得了较好的效果。但口服DNA疫苗在肠黏膜的表达部位及细胞尚不清楚。肠道黏膜表面是大多数致病菌进人机体的主要通道。接种黏膜疫苗以激发免疫效应细胞的活化是抗感染的主要手段之一。肠黏膜免疫系统中参与免疫应答的细胞遍布于肠黏膜内,其中M细胞是回肠派氏淋巴结(Peyer's patch,PP)表面滤泡相关上皮(follicle-associated epithelium,FAE)中的一种特殊的上皮细胞,存在于淋巴滤泡上皮之间,与肠上皮细胞紧密排列在一起,形成上皮屏障,并与其“口袋”中的各种淋巴细胞紧密接触。肠腔中的大部分病原体是由M细胞转运至其下的免疫细胞,从而引起局部的黏膜免疫反应,乃至系统免疫反应。由此可见,M细胞在肠道黏膜免疫反应中起着非常重要的作用。肠上皮细胞形成规则的微绒毛,而M细胞形成不规则的微皱褶,因此又称作微皱褶细胞(Microfold cell)。M细胞呈圆顶状,细胞基底部向顶部(肠腔面)深陷,形成一中央腔。M细胞的中央腔很大,含有丰富的淋巴细胞,其顶部及周边胞质很薄,胞质中含有大量的吞饮小泡,M细胞的基底膜常常是不连续的,可允许淋巴细胞自由穿过。M细胞的这些特殊结构及定位大大缩短了含有抗原或病原微生物的吞饮小泡跨越M细胞的距离,有利于抗原快速进人上皮下淋巴组织。目前的研究表明,小肠绒毛上皮M细胞是摄取肠道细菌的重要途径,还有报道小肠黏膜内DC可以摄取抗原物质。抗原85复合体(Ag85)是BCG合成的能够刺激机体产生细胞免疫和体液免疫的多种成分之一,国外已有研究者将其试用于免疫生物治疗。比利时布鲁塞尔巴斯德研究所Huygen研究组多年来在对Ag85进行的研究中发现,接种Ag85蛋白主要引起机体细胞免疫功能增强,他们还观察到携带Ag85基因的的质粒DNA疫苗具有:(1)经肌肉注射给动物(小鼠),主要引起Th1样的应答,产生IL-2、IFN-γ和TNF-α,经基因枪注射则主要产生Th2应答和抗体产生水平增加;(2)肌肉注射治疗膀胱癌,近80%的病人外周血和脾脏中T淋巴细胞增殖,产生IL-2和IFN-γ的量增加,未见明显的Th2细胞活化和抗体产生增加;(3)可诱导长寿命的T细胞产生;(4)可降低迟发型超敏反应的强度。裸DNA疫苗经肌肉注射,其目的基因一般在肌细胞内表达,诱导机体免疫应答的产生,但经口服DNA疫苗在消化道细胞内表达定位目前尚无报道。到目前为止,已有的一些研究表明口服DNA疫苗能诱导全身性细胞和体液免疫应答增强。我们在近期的初步实验中发现口服DNA疫苗进入肠道后,在肠黏膜吸收过程中将对IEL产生效应作用,促进黏膜免疫功能增强,有益于衰老和感染的上皮细胞的清除与更新,保持黏膜局部结构和功能的完整性。同时,有关其是否产生负效应也有待进一步研究。我们推论口服DNA疫苗所产生的全身性免疫效应实际是肠道局部黏膜免疫应答后的一系列反应。在上述研究的基础上,本实验检测了Ag85A基因的表达产物在肠道局部黏膜上皮细胞、M细胞和DC中的分布,为阐明口服DNA疫苗诱导肠道局部黏膜免疫反应的机制及临床经口接种DNA疫苗提供理论和实验依据。实验材料和方法一、实验动物 6～8周龄雌性C57BL/6小鼠,购自中科院上海实验动物中心。二、主要试剂自制DNA疫苗pCDNA3.1+/Ag85A;Lipofectamine TM 2000购自Invitrogen公司,chicken anti-Ag85A IgY购自Prosci公司,HRP-goat-anti-chicken IgY,FITC-goat-anti-chicken IgY购自Gene公司,Purified Armenian Hamster-anti-mouseCD11c购自BD Pharmingen公司,Texas Red conjugated Goat Anti-Armenian HamsterIgG购自Jackson ImmunoResearch Laboratories,TRITC conjugated UEA-1购自Vector Laboratories,无内毒素型超纯质粒DNA纯化试剂盒购自Promega公司,BSA,DAB显色试剂盒购自北京中杉公司。三、实验方法应用基因工程技术扩增全长Ag85A基因序列,经测序和同源性分析后将其亚克隆入真核表达载体pCDNA3.1+,鉴定正确后将重组质粒转化感受态大肠杆菌DH5α,扩增抽提无内毒素重组质粒,进一步用脂质体包裹制成口服重组DNA疫苗。将C57BL/6小鼠随机分为2组,即生理盐水组和脂质体包裹重组质粒组。分别将生理盐水和脂质体包裹pCDNA3.1+—Ag85A以灌胃方式投给各组小鼠,共免疫3次,每次间隔14天,末次免疫后14天处死小鼠,取肠,免疫组化、免疫荧光检测Ag85A在肠黏膜上皮细胞、派氏淋巴结、肠道局部M细胞及树突状细胞的表达情况。结果 Ag85A重组DNA疫苗在小鼠肠道局部黏膜上皮细胞、M细胞及派氏淋巴结树突状细胞均有表达。Ag85A在靠近小鼠小肠黏膜固有层的上皮细胞中表达的强度比靠近肠腔侧的上皮细胞表达的强度高,Ag85A在小鼠小肠黏膜M细胞中表达强度非常高。讨论肠道是接触抗原物质最多的器官,肠道相关淋巴组织主要包括派氏淋巴结、肠系膜淋巴结,然而,肠道内发挥免疫效应的效应细胞并不仅限于此,而是遍布于肠黏膜内。M细胞是摄取肠道内抗原物质的主要细胞,分布于小肠派氏淋巴结和绒毛上皮。目前的研究表明,小肠绒毛上皮M细胞是摄取肠道细菌的重要途径,还有报道小肠黏膜内DC可以摄取抗原物质。我们的实验表明,M细胞还可以摄取质脂体包裹的DNA疫苗,而在小肠黏膜内DC中未见到DNA疫苗的表达。 DC是专职抗原提呈细胞,其主要功能是摄取、加工处理和提呈抗原。DC包括成熟和未成熟两种形式,未成熟的DC主要分布在非淋巴器官和血液中,发挥哨兵作用。当病原体进入时,未成熟的DC能够摄取并将其提呈给吞噬细胞。未成熟DC摄取、加工处理抗原能力强,而提呈抗原激发免疫应答能力弱,成熟DC摄取、加工处理抗原能力弱,而提呈抗原、启动免疫应答能力强。我们在实验中未见到小肠黏膜内DC表达DNA疫苗编码的蛋白,而在派氏淋巴结内部分DC中观察到了DNA疫苗的表达。本实验检测了口服Ag85A DNA疫苗在小鼠肠道局部的表达,在此基础上,我们将进一步研究口服DNA疫苗对小鼠肠道分泌型IgA(sIgA)产生情况的影响,从而更加全面地阐释口服DNA疫苗诱导的局部黏膜免疫应答。结论口服脂质体包裹的DNA疫苗可以诱导特异性黏膜免疫应答,其机制可能为疫苗进入肠道后先被M细胞摄取,再转运到树突状细胞,经加工处理提呈给T、B细胞,发挥特异性黏膜免疫应答。
[Abstract]:Preface
Since 1997, when Jones and Pascual et al. prepared oral DNA vaccines using PLG (poly-DL-lactide-co-glycolide) particles and attenuated typhoid bacilli (Salmonilla typhimurium) as vectors respectively, there have been many reports on the role of oral DNA vaccines in the prevention or treatment of infections, tumors and allergic diseases, and have taken them. It has a good effect. However, the expression sites and cells of oral DNA vaccine in the intestinal mucosa are not clear.
The intestinal mucosal surface is the main channel through which most pathogenic bacteria enter the human body. Inoculation of mucosal vaccines to stimulate the activation of immune effector cells is one of the main means of anti-infection. A special epithelial cell in the follicle-associated epithelium (FAE) that exists between the epithelium of lymphoid follicles and closely aligns with intestinal epithelial cells to form an epithelial barrier and is in close contact with various lymphocytes in their pockets. This shows that M cells play an important role in the intestinal mucosal immune response. Intestinal epithelial cells form regular microvilli, while M cells form irregular microfolds, so they are also called microfold cells. M cells are domed and thin. M cells have a large central cavity with abundant lymphocytes. Their top and peripheral cytoplasm are very thin. There are a lot of swallowing vesicles in the cytoplasm. The basement membrane of M cells is often discontinuous, allowing lymphocytes to pass through these special structures and locations freely. It shortens the distance between swallowing vesicles containing antigens or pathogenic microorganisms and facilitates the rapid entry of antigens into human subepithelial lymphoid tissue.
Antigen 85 complex (Ag85) is one of the components synthesized by BCG that can stimulate cellular and humoral immunity. Researchers abroad have tried to use Ag85 in immunobiotherapy. They also observed that the plasmid DNA vaccine carrying Ag85 gene had: (1) intramuscular injection of Ag85 gene into animals (mice) mainly caused Th1-like responses, producing IL-2, IFN-gamma and TNF-alpha, while gene gun injection mainly produced Th2 response and increased antibody production; (2) intramuscular injection of Ag85 gene in the treatment of bladder cancer, nearly 80% of patients outside the body. T lymphocytes proliferated in peripheral blood and spleen, producing IL-2 and IFN-gamma, but no significant increase in Th2 cell activation and antibody production was observed; (3) long-lived T cells could be induced; (4) delayed type hypersensitivity could be reduced. Nude DNA vaccines were injected intramuscularly to induce the body's immune response. But the expression of oral DNA vaccine in digestive tract cells has not been reported.
Up to now, some studies have shown that oral DNA vaccines can induce enhanced systemic cellular and humoral immune responses. In our recent preliminary experiments, we have found that oral DNA vaccines, after entering the intestinal tract, will have an effect on IEL in the intestinal mucosal absorption process, promote the enhancement of mucosal immune function, beneficial to aging and infectious epithelium. We conclude that the systemic immune effect of oral DNA vaccine is actually a series of reactions after local intestinal mucosal immune response.
On the basis of the above studies, the distribution of Ag85A gene expression products in intestinal mucosal epithelial cells, M cells and DC was detected in this study, which provided theoretical and experimental basis for elucidating the mechanism of local intestinal mucosal immune response induced by oral DNA vaccine and clinical oral DNA vaccination.
Experimental materials and methods
I. Experimental Animals
The 6~8 week old female C57BL/6 mice were purchased from the Shanghai experimental animal center of Chinese Academy of Sciences.
Two, main reagents
Homemade DNA vaccine pCDNA3.1+/Ag85A; Lipofectamine TM 2000 from Invitrogen, chicken anti-Ag85A IgY from Prosci, HRP-goat-anti-chicken IgY, FITC-goat-anti-chicken IgY from Gene, Purified Armenian Hamster-anti-mouseCD11c from BDmingen, Texas Red conjugated Goat Anti-Armenian Hamster IgG was purchased from Jackson ImmunoResearch Laboratories, TRITC conjugated UEA-1 from Vector Laboratories, and non-endotoxin type ultrapure plasmid DNA purification kits from Promega, BSA, DAB color reagent kits from Beijing Zhongshan Company.
Three, the experimental method.
The full-length Ag85A gene sequence was amplified by genetic engineering technology, and subcloned into eukaryotic expression vector pCDNA3.1+. After identification, the recombinant plasmid was transformed into competent E. coli DH5a. The recombinant plasmid without endotoxin was amplified and extracted. The recombinant DNA vaccine was prepared by liposome encapsulation. The mice were randomly divided into two groups: normal saline group and liposome-encapsulated recombinant plasmid group. The mice were immunized with normal saline and liposome-encapsulated pCDNA3.1+-Ag85A by gavage for three times, 14 days apart. The mice were sacrificed 14 days after the last immunization. The expression of M cells and dendritic cells in the lymph nodes and intestine.
Result
Ag85A recombinant DNA vaccine was expressed in local intestinal mucosal epithelial cells, M cells and Payne's lymph node dendritic cells of mice. Ag85A was expressed more strongly in epithelial cells near the lamina propria of intestinal mucosa than in epithelial cells near the lumen of intestine. Ag85A was highly expressed in M cells of intestinal mucosa of mice.
discuss
The intestinal tract is the organ that is most exposed to antigens. The intestinal lymphoid tissues include mainly Pap's lymph nodes and mesenteric lymph nodes. However, the effector cells that exert immune effects in the intestinal tract are not limited to this, but distributed throughout the intestinal mucosa. Current studies have shown that intestinal villous epithelial M cells are an important way of uptake of intestinal bacteria. It has also been reported that DC in intestinal mucosa can uptake antigens. Our experiments show that M cells can also uptake plasmolipid-encapsulated DNA vaccines, but there is no expression of DNA vaccines in intestinal mucosa.
DC is a professional antigen presenting cell whose main functions are uptake, processing and presentation of antigens. DC includes mature and immature forms. Immature DC mainly distributes in non-lymphoid organs and blood, and plays a sentinel role. When pathogens enter, immature DC can uptake and present it to phagocytes. Immature DC uptake, plus. The ability of presenting antigens to elicit immune responses is strong, but the ability of presenting antigens to elicit immune responses is weak, the ability of taking up mature DCs and processing antigens is weak. The ability of presenting antigens and initiating immune responses is strong.
This study examined the local expression of oral Ag85A DNA vaccine in the intestine of mice. On this basis, we will further study the effect of oral DNA vaccine on the production of secretory IgA (sIgA) in the intestine of mice, so as to more comprehensively explain the local mucosal immune response induced by oral DNA vaccine.
conclusion
Oral liposome-encapsulated DNA vaccine can induce specific mucosal immune response. The mechanism may be that the vaccine enters the intestinal tract and is taken up by M cells, then transported to dendritic cells, and then presented to T and B cells by processing.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.11

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