【摘要】： 前言 自從1997年Jones和Pascual等人分別用PLG(poly-DL-lactide-co-glycolide)微粒和減毒傷寒桿菌(Salmonilla typhimurium)為運(yùn)載體(vectors)制備口服DNA疫苗以來,已經(jīng)有多篇報(bào)道對口服DNA疫苗在預(yù)防或治療感染、腫瘤和過敏性疾病中的作用進(jìn)行了研究,并取得了較好的效果。但口服DNA疫苗在腸黏膜的表達(dá)部位及細(xì)胞尚不清楚。 腸道黏膜表面是大多數(shù)致病菌進(jìn)人機(jī)體的主要通道。接種黏膜疫苗以激發(fā)免疫效應(yīng)細(xì)胞的活化是抗感染的主要手段之一。腸黏膜免疫系統(tǒng)中參與免疫應(yīng)答的細(xì)胞遍布于腸黏膜內(nèi),其中M細(xì)胞是回腸派氏淋巴結(jié)(Peyer's patch,PP)表面濾泡相關(guān)上皮(follicle-associated epithelium,FAE)中的一種特殊的上皮細(xì)胞,存在于淋巴濾泡上皮之間,與腸上皮細(xì)胞緊密排列在一起,形成上皮屏障,并與其“口袋”中的各種淋巴細(xì)胞緊密接觸。腸腔中的大部分病原體是由M細(xì)胞轉(zhuǎn)運(yùn)至其下的免疫細(xì)胞,從而引起局部的黏膜免疫反應(yīng),乃至系統(tǒng)免疫反應(yīng)。由此可見,M細(xì)胞在腸道黏膜免疫反應(yīng)中起著非常重要的作用。腸上皮細(xì)胞形成規(guī)則的微絨毛,而M細(xì)胞形成不規(guī)則的微皺褶,因此又稱作微皺褶細(xì)胞(Microfold cell)。M細(xì)胞呈圓頂狀,細(xì)胞基底部向頂部(腸腔面)深陷,形成一中央腔。M細(xì)胞的中央腔很大,含有豐富的淋巴細(xì)胞,其頂部及周邊胞質(zhì)很薄,胞質(zhì)中含有大量的吞飲小泡,M細(xì)胞的基底膜常常是不連續(xù)的,可允許淋巴細(xì)胞自由穿過。M細(xì)胞的這些特殊結(jié)構(gòu)及定位大大縮短了含有抗原或病原微生物的吞飲小泡跨越M細(xì)胞的距離,有利于抗原快速進(jìn)人上皮下淋巴組織。目前的研究表明,小腸絨毛上皮M細(xì)胞是攝取腸道細(xì)菌的重要途徑,還有報(bào)道小腸黏膜內(nèi)DC可以攝取抗原物質(zhì)。 抗原85復(fù)合體(Ag85)是BCG合成的能夠刺激機(jī)體產(chǎn)生細(xì)胞免疫和體液免疫的多種成分之一,國外已有研究者將其試用于免疫生物治療。比利時(shí)布魯塞爾巴斯德研究所Huygen研究組多年來在對Ag85進(jìn)行的研究中發(fā)現(xiàn),接種Ag85蛋白主要引起機(jī)體細(xì)胞免疫功能增強(qiáng),他們還觀察到攜帶Ag85基因的的質(zhì)粒DNA疫苗具有:(1)經(jīng)肌肉注射給動(dòng)物(小鼠),主要引起Th1樣的應(yīng)答,產(chǎn)生IL-2、IFN-γ和TNF-α,經(jīng)基因槍注射則主要產(chǎn)生Th2應(yīng)答和抗體產(chǎn)生水平增加;(2)肌肉注射治療膀胱癌,近80%的病人外周血和脾臟中T淋巴細(xì)胞增殖,產(chǎn)生IL-2和IFN-γ的量增加,未見明顯的Th2細(xì)胞活化和抗體產(chǎn)生增加;(3)可誘導(dǎo)長壽命的T細(xì)胞產(chǎn)生;(4)可降低遲發(fā)型超敏反應(yīng)的強(qiáng)度。裸DNA疫苗經(jīng)肌肉注射,其目的基因一般在肌細(xì)胞內(nèi)表達(dá),誘導(dǎo)機(jī)體免疫應(yīng)答的產(chǎn)生,但經(jīng)口服DNA疫苗在消化道細(xì)胞內(nèi)表達(dá)定位目前尚無報(bào)道。 到目前為止,已有的一些研究表明口服DNA疫苗能誘導(dǎo)全身性細(xì)胞和體液免疫應(yīng)答增強(qiáng)。我們在近期的初步實(shí)驗(yàn)中發(fā)現(xiàn)口服DNA疫苗進(jìn)入腸道后,在腸黏膜吸收過程中將對IEL產(chǎn)生效應(yīng)作用,促進(jìn)黏膜免疫功能增強(qiáng),有益于衰老和感染的上皮細(xì)胞的清除與更新,保持黏膜局部結(jié)構(gòu)和功能的完整性。同時(shí),有關(guān)其是否產(chǎn)生負(fù)效應(yīng)也有待進(jìn)一步研究。我們推論口服DNA疫苗所產(chǎn)生的全身性免疫效應(yīng)實(shí)際是腸道局部黏膜免疫應(yīng)答后的一系列反應(yīng)。 在上述研究的基礎(chǔ)上,本實(shí)驗(yàn)檢測了Ag85A基因的表達(dá)產(chǎn)物在腸道局部黏膜上皮細(xì)胞、M細(xì)胞和DC中的分布,為闡明口服DNA疫苗誘導(dǎo)腸道局部黏膜免疫反應(yīng)的機(jī)制及臨床經(jīng)口接種DNA疫苗提供理論和實(shí)驗(yàn)依據(jù)。 實(shí)驗(yàn)材料和方法 一、實(shí)驗(yàn)動(dòng)物 6～8周齡雌性C57BL/6小鼠,購自中科院上海實(shí)驗(yàn)動(dòng)物中心。 二、主要試劑 自制DNA疫苗pCDNA3.1+/Ag85A;Lipofectamine TM 2000購自Invitrogen公司,chicken anti-Ag85A IgY購自Prosci公司,HRP-goat-anti-chicken IgY,FITC-goat-anti-chicken IgY購自Gene公司,Purified Armenian Hamster-anti-mouseCD11c購自BD Pharmingen公司,Texas Red conjugated Goat Anti-Armenian HamsterIgG購自Jackson ImmunoResearch Laboratories,TRITC conjugated UEA-1購自Vector Laboratories,無內(nèi)毒素型超純質(zhì)粒DNA純化試劑盒購自Promega公司,BSA,DAB顯色試劑盒購自北京中杉公司。 三、實(shí)驗(yàn)方法 應(yīng)用基因工程技術(shù)擴(kuò)增全長Ag85A基因序列,經(jīng)測序和同源性分析后將其亞克隆入真核表達(dá)載體pCDNA3.1+,鑒定正確后將重組質(zhì)粒轉(zhuǎn)化感受態(tài)大腸桿菌DH5α,擴(kuò)增抽提無內(nèi)毒素重組質(zhì)粒,進(jìn)一步用脂質(zhì)體包裹制成口服重組DNA疫苗。將C57BL/6小鼠隨機(jī)分為2組,即生理鹽水組和脂質(zhì)體包裹重組質(zhì)粒組。分別將生理鹽水和脂質(zhì)體包裹pCDNA3.1+—Ag85A以灌胃方式投給各組小鼠,共免疫3次,每次間隔14天,末次免疫后14天處死小鼠,取腸,免疫組化、免疫熒光檢測Ag85A在腸黏膜上皮細(xì)胞、派氏淋巴結(jié)、腸道局部M細(xì)胞及樹突狀細(xì)胞的表達(dá)情況。 結(jié)果 Ag85A重組DNA疫苗在小鼠腸道局部黏膜上皮細(xì)胞、M細(xì)胞及派氏淋巴結(jié)樹突狀細(xì)胞均有表達(dá)。Ag85A在靠近小鼠小腸黏膜固有層的上皮細(xì)胞中表達(dá)的強(qiáng)度比靠近腸腔側(cè)的上皮細(xì)胞表達(dá)的強(qiáng)度高,Ag85A在小鼠小腸黏膜M細(xì)胞中表達(dá)強(qiáng)度非常高。 討論 腸道是接觸抗原物質(zhì)最多的器官,腸道相關(guān)淋巴組織主要包括派氏淋巴結(jié)、腸系膜淋巴結(jié),然而,腸道內(nèi)發(fā)揮免疫效應(yīng)的效應(yīng)細(xì)胞并不僅限于此,而是遍布于腸黏膜內(nèi)。M細(xì)胞是攝取腸道內(nèi)抗原物質(zhì)的主要細(xì)胞,分布于小腸派氏淋巴結(jié)和絨毛上皮。目前的研究表明,小腸絨毛上皮M細(xì)胞是攝取腸道細(xì)菌的重要途徑,還有報(bào)道小腸黏膜內(nèi)DC可以攝取抗原物質(zhì)。我們的實(shí)驗(yàn)表明,M細(xì)胞還可以攝取質(zhì)脂體包裹的DNA疫苗,而在小腸黏膜內(nèi)DC中未見到DNA疫苗的表達(dá)。 DC是專職抗原提呈細(xì)胞,其主要功能是攝取、加工處理和提呈抗原。DC包括成熟和未成熟兩種形式,未成熟的DC主要分布在非淋巴器官和血液中,發(fā)揮哨兵作用。當(dāng)病原體進(jìn)入時(shí),未成熟的DC能夠攝取并將其提呈給吞噬細(xì)胞。未成熟DC攝取、加工處理抗原能力強(qiáng),而提呈抗原激發(fā)免疫應(yīng)答能力弱,成熟DC攝取、加工處理抗原能力弱,而提呈抗原、啟動(dòng)免疫應(yīng)答能力強(qiáng)。我們在實(shí)驗(yàn)中未見到小腸黏膜內(nèi)DC表達(dá)DNA疫苗編碼的蛋白,而在派氏淋巴結(jié)內(nèi)部分DC中觀察到了DNA疫苗的表達(dá)。 本實(shí)驗(yàn)檢測了口服Ag85A DNA疫苗在小鼠腸道局部的表達(dá),在此基礎(chǔ)上,我們將進(jìn)一步研究口服DNA疫苗對小鼠腸道分泌型IgA(sIgA)產(chǎn)生情況的影響,從而更加全面地闡釋口服DNA疫苗誘導(dǎo)的局部黏膜免疫應(yīng)答。 結(jié)論 口服脂質(zhì)體包裹的DNA疫苗可以誘導(dǎo)特異性黏膜免疫應(yīng)答,其機(jī)制可能為疫苗進(jìn)入腸道后先被M細(xì)胞攝取,再轉(zhuǎn)運(yùn)到樹突狀細(xì)胞,經(jīng)加工處理提呈給T、B細(xì)胞,發(fā)揮特異性黏膜免疫應(yīng)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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.11

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