【摘要】：炎癥性腸病(IBD)是一種病因不明的自身免疫性疾病,包括潰瘍性腸炎(UC)和克羅恩病(CD)是腸道慢性、復(fù)發(fā)性病癥。自身免疫失衡是IBD發(fā)病的直接誘因。環(huán)境和遺傳因素在IBD的免疫中起重要作用。當(dāng)環(huán)境因素作用于有遺傳易感性的個(gè)體時(shí),就可能引發(fā)機(jī)體免疫系統(tǒng)功能失調(diào)而發(fā)病,IBD在衛(wèi)生條件較好的發(fā)達(dá)國(guó)家發(fā)病率較高,而在衛(wèi)生不好、條件差的國(guó)家發(fā)病率反而較低。單純用環(huán)境和遺傳因素很難解釋這種差異的,因?yàn)榍钒l(fā)達(dá)地區(qū)移民到發(fā)達(dá)地區(qū)的人群IBD發(fā)病率較高。這些結(jié)果說(shuō)明,環(huán)境因素對(duì)IBD的發(fā)生是重要的。即而產(chǎn)生了IBD“衛(wèi)生假說(shuō)”的理論,即IBD的發(fā)生是在缺少對(duì)腸道粘膜刺激較少的社會(huì)。線蟲(chóng)感染較多國(guó)家CD和UC的發(fā)病率低,說(shuō)明腸道寄生蟲(chóng)的感染有效的解釋了這個(gè)問(wèn)題。前期工作中以旋毛蟲(chóng)(T.spiralis)作為干預(yù)小鼠腸炎模型產(chǎn)生較好的治療作用。盡管寄生蟲(chóng)對(duì)IBD的治療有益,寄生蟲(chóng)感染所引發(fā)的生物安全性問(wèn)題一直受人關(guān)注�；铙w寄生蟲(chóng)持續(xù)感染人體不易被排出體外,可能會(huì)影響胃腸的功能,并導(dǎo)致病人心理上難以接受這種治療。因此,在維持寄生蟲(chóng)生命周期的條件下,利用寄生蟲(chóng)進(jìn)行大規(guī)模治療并不可行。寄生蟲(chóng)提取物可以與活蟲(chóng)具有相同的治療效果,不但可以避免感染活蟲(chóng)的隱患,還能使病人更容易接受。旋毛蟲(chóng)有效抗原成分可避免活蟲(chóng)治療IBD的不利因素,同時(shí)保留了旋毛蟲(chóng)對(duì)IBD的免疫調(diào)控作用,理論上是符合臨床治療要求的生物制劑。 本研究選擇已鑒定好的旋毛蟲(chóng)有效抗原成分,即ZH68旋毛蟲(chóng)成蟲(chóng)抗原基因(絲氨酸蛋白酶);WM5旋毛蟲(chóng)肌幼蟲(chóng)抗原基因(絲氨酸蛋白酶抑制劑);WN10旋毛蟲(chóng)新生幼蟲(chóng)抗原基因(半胱氨酸蛋白酶抑制劑);T668旋毛蟲(chóng)新生幼蟲(chóng)抗原基因(絲氨酸蛋白酶),構(gòu)建可溶性蛋白表達(dá)載體,并對(duì)抗原基因表達(dá)產(chǎn)物進(jìn)行純化,目的是獲得純度較高的基因表達(dá)產(chǎn)物。其方法是利用鎳瓊脂糖凝膠FF色譜分離帶有His標(biāo)簽的重組蛋白pET-22b-T668、pET-22b-ZH68、pET-22b-WM5和pET-22b-WN10。將純化濃縮的四種可溶性蛋白在無(wú)菌條件下經(jīng)腹腔注射BALB/c鼠,以生理鹽注射水作為對(duì)照。三次免疫后用TNBS誘導(dǎo)動(dòng)物模型,觀察誘導(dǎo)CD模型指標(biāo)的變化情況,包括小鼠的平均體重、生存率、DAI評(píng)分、結(jié)腸宏觀評(píng)分和微觀損傷評(píng)分,檢測(cè)炎癥指標(biāo)MPO、SOD活性,探討四種可溶性蛋白對(duì)動(dòng)物模型的治療效應(yīng)。實(shí)時(shí)定量RT-PCR檢測(cè)各組模型鼠腸粘膜、腸系膜淋巴結(jié)及脾臟中IFN-γ、IL-10、IL-12、IL-4、IL-17和TGF-βmRNA的表達(dá)含量,用ELISA法檢測(cè)各組小鼠結(jié)腸(LPMC)和脾臟中IFN-γ、IL-12、TGF-β、IL-4、IL-10和IL-17的分泌水平,從而動(dòng)態(tài)研究腸道粘膜免疫和細(xì)胞免疫作用關(guān)系;并通過(guò)流式細(xì)胞儀檢測(cè)(FCM)各組脾細(xì)胞內(nèi)CD4+ CD25+ Foxp3+ Treg細(xì)胞數(shù)量變化。從細(xì)胞水平闡述Treg對(duì)機(jī)體免疫方面的作用,客觀論述蛋白對(duì)IBD模型鼠的免疫作用機(jī)制。 旋毛蟲(chóng)抗原基因WM5、WN10、Zh68和T668克隆入原核表達(dá)載體pET-22b中進(jìn)行表達(dá)。經(jīng)鑒定四種抗原基因未發(fā)生突變,具有完整的開(kāi)放閱讀框,基因全長(zhǎng)分別是1315bp、1352bp、1372bp和1609bp,切除信號(hào)肽后表達(dá)成熟蛋白分子量理論為37.7 kDa、46.9 kDa、47 kDa、49 kDa,表達(dá)的融合蛋白的大小與理論相符。表達(dá)產(chǎn)物通過(guò)鎳瓊脂糖凝膠柱進(jìn)行親和層析分離純化,SDS-PAGE和Western-Blot檢測(cè)蛋白質(zhì)具有良好的免疫原性和反應(yīng)原性。 旋毛蟲(chóng)有效抗原成分對(duì)腸炎模型干預(yù)效應(yīng)結(jié)果顯示,Zh68和T668蛋白免疫后TNBS誘導(dǎo)鼠模型組平均體重和生存率顯著高于鹽水注射后造模組,(p0.05),各項(xiàng)臨床癥狀均輕于鹽水注射后造模組,DAI評(píng)分明顯降低(p0.05)。預(yù)先免疫Zh68和T668蛋白小鼠于TNBS誘導(dǎo)造模后3d及7d與生理鹽水模型組相比在結(jié)腸宏觀損傷和微觀損傷評(píng)價(jià)上都有所改善(p0.05),粘膜損傷輕微,結(jié)腸壁增厚較少、粘連范圍較窄,潰瘍面淺或臨近正常組織,炎性細(xì)胞浸潤(rùn)范圍較小、減弱,水腫范圍較小,MPO值降低,差異顯著(p0.05)。SOD評(píng)價(jià)值顯示,鹽水注射造模組3d后明顯高于蛋白免疫試驗(yàn)組(p0.05),7d后差異不顯著(p㧐0.05)。WM5和WN10蛋白免疫造模組與鹽水注射未造模組之間沒(méi)有明顯的差異。實(shí)時(shí)定量RT-PCR結(jié)果顯示:ZH68和T668蛋白免疫后造模組3d結(jié)腸中IFN-γ、IL-12和IL-17 mRNA的表達(dá)量顯著低于鹽水注射造模組(p0.05),而IL-4、IL-10和TGF-βmRNA的表達(dá)顯著升高(p0.05)。各組蛋白免疫后造模組腸系膜淋巴結(jié)中IFN-γ、IL-12和TGF-βmRNA的表達(dá)量對(duì)鹽水注射后造模組沒(méi)有顯著變化,ZH68和T668蛋白后造模組IL-17和IL-10 mRNA的表達(dá)量顯著下降(p0.05),IL-4 mRNA的表達(dá)量顯著升高(p0.05)。ZH68和T668蛋白免疫后造模組小鼠在造模脾臟中IL-17、IL-10 mRNA的表達(dá)量對(duì)模型組的表達(dá)顯著下降(p0.05),其它細(xì)胞因子表達(dá)沒(méi)有顯著性差異(p0.05)。ELISA結(jié)果顯示:ZH68和T668蛋白免疫造模組3d及7d后的IL-10和TGF-β小鼠結(jié)腸組織LPMC產(chǎn)生水平顯著高于鹽水注射造模組(p0.05),而IFN-γ、IL-17和3d后的IL-12 LPMC產(chǎn)生水平顯著低于鹽水注射造模組(p0.05),7d后的IL-12水平和3d后的IL-4 LPMC產(chǎn)生水平?jīng)]有變化(p0.05),7d后的IL-4 LPMC產(chǎn)生水平顯著高于鹽水注射造模組(p0.05)。ZH68和T668蛋白免疫造模組3d及7d后小鼠脾臟淋巴細(xì)胞的IL-4、IL-10和TGF-β表達(dá)顯著高于鹽水注射后造模組(p0.05),IL-17產(chǎn)生水平顯著低于鹽水注射造模組(p0.05),3d后脾臟淋巴細(xì)胞產(chǎn)生IFN-γ的水平及第7dIL-12的產(chǎn)生水平與模型組相比未見(jiàn)明顯差異(p0.05),7d后脾臟淋巴細(xì)胞產(chǎn)生IFN-γ的水平及第3d IL-12的水平顯著低于鹽水注射造模組(p0.05)。實(shí)時(shí)定量RT-PCR和ELISA結(jié)果顯示:WM5和WN10蛋白免疫后造模組變化不顯著(p0.05),各蛋白免疫造模組與鹽水注射未造模組相比上述顯著差異基礎(chǔ)上的細(xì)胞因子表達(dá)呈現(xiàn)相反的結(jié)果。流式細(xì)胞檢測(cè)顯示:ZH68和T668蛋白免疫后造模組7d脾淋巴細(xì)胞表達(dá)CD4+ CD2+ Foxp3+ Treg細(xì)胞數(shù)占CD4+T淋巴細(xì)胞的百分率明顯低于鹽水注射后造模組(p0.05),WM5和WN10蛋白免疫后造模組脾淋巴細(xì)胞表CD4+ CD2+ Foxp3+ Treg細(xì)胞數(shù)占CD4+T淋巴細(xì)胞的百分率與鹽水注射后造模組相比沒(méi)有顯著變化(p0.05);而3d疾病活動(dòng)期各組沒(méi)有明顯差異(p0.05)。 旋毛蟲(chóng)有效抗原成分免疫后造模小鼠均表現(xiàn)出對(duì)TNBS誘導(dǎo)結(jié)腸炎模型具有較好的治療效應(yīng),其可能的機(jī)制是:有效抗原成分恢復(fù)IBD模型鼠Th1/Th2正常的平衡;抑制了機(jī)體的免疫反應(yīng)和免疫細(xì)胞和細(xì)胞因子發(fā)揮超強(qiáng)的免疫調(diào)劑作用,從而使機(jī)體免疫失衡恢復(fù)到正常水平。
[Abstract]:Inflammatory bowel disease (IBD) is an autoimmune disease of unknown etiology. Ulcerative enteritis (UC) and Crohn's disease (CD) are chronic, recurrent diseases of the intestine. Imbalance of autoimmunity is a direct cause of IBD. Environmental and genetic factors play an important role in the immunity of IBD. When environmental factors act on individuals with genetic susceptibility, The incidence of IBD is higher in developed countries with better hygiene conditions, but lower in countries with poor hygiene and poor conditions. These results suggest that environmental factors are important for the occurrence of IBD, which leads to the theory that IBD occurs in societies where there is less irritation to the intestinal mucosa. T. spiralis has a better therapeutic effect as an intervention model of enteritis in mice. Although parasites are beneficial to the treatment of IBD, the biological safety problems caused by parasite infection have always been a concern. Thus, large-scale treatment with parasites is not feasible under the condition of maintaining the parasite's life cycle. Parasite extracts can have the same therapeutic effect as live worms, not only avoiding the hidden danger of infecting live worms, but also making patients more receptive. Trichinella spiralis effective antigen components can prevent live worms from treating IBD. Adverse factors, while retaining the immune regulation of Trichinella spiralis on IBD, are theoretically in line with the requirements of clinical treatment of biological agents.
In this study, the identified effective antigen components of Trichinella spiralis were selected, namely, ZH68 adult worm antigen gene (serine protease); WM5 muscle larva antigen gene (serine protease inhibitor); WN10 newborn larva antigen gene (cysteine protease inhibitor); and T668 newborn larva antigen gene (serine protein inhibitor). The aim of this study was to isolate the recombinant proteins pET-22b-T668, pET-22b-ZH68, pET-22b-WM5 and pET-22b-WN10 with his label by nickel agarose gel FF chromatography. BALB/c mice were intraperitoneally injected with protein under aseptic conditions, and physiological salt water was used as control. After three times of immunization, the animal models were induced by TNBS. The changes of CD model indexes were observed, including average body weight, survival rate, DAI score, colon macroscoring and micro-injury score, MPO and SOD activity, and four kinds of inflammation indexes were detected. The expression levels of IFN-gamma, IL-10, IL-12, IL-4, IL-17 and TGF-beta mRNA in intestinal mucosa, mesenteric lymph nodes and spleen were detected by real-time quantitative RT-PCR, and the secretion levels of IFN-gamma, IL-12, TGF-beta, IL-4, IL-10 and IL-17 in colon (LPMC) and spleen were detected by ELISA. The relationship between intestinal mucosal immunity and cellular immunity was studied. The number of CD4+CD25+Foxp3+Treg cells in spleen cells of each group was detected by flow cytometry.
Trichinella spiralis antigen genes WM5, WN10, Zh68 and T668 were cloned and expressed in prokaryotic expression vector pET-22b. No mutation was detected in the four antigen genes, and they had complete open reading frames. The total length of the gene was 1315 bp, 1352 bp, 1372 BP and 1609 bp, respectively. The molecular weight of mature protein expressed after signal peptide excision was 37.7 kDa, 46.9 kDa, 47 kDa, 49 kDa. The expressed fusion protein was purified by affinity chromatography on a nickel agarose gel column. The protein detected by SDS-PAGE and Western-Blot had good immunogenicity and reactivity.
The intervention effect of effective antigen components of Trichinella spiralis on enteritis model showed that the average body weight and survival rate of TNBS-induced mice model group immunized with Zh68 and T668 protein were significantly higher than those of saline injection model group (p0.05). The clinical symptoms were lighter than those of saline injection model group, and the DAI score was significantly lower (p0.05). Pre-immunization with Zh68 and T668 protein was smaller. Compared with the normal saline group, the macroscopic and microscopic damage of colon were improved 3 and 7 days after TNBS induction (p0.05). The mucosal damage was slight, the thickening of colon wall was less, the adhesion range was narrow, the ulcer surface was shallow or adjacent to the normal tissue. The inflammatory cell infiltration range was smaller, the edema range was smaller, the MPO value was lower and the MPO value was worse. There was no significant difference between the WM5 and WN10 protein immunization group and the non-saline injection group. Real-time quantitative RT-PCR showed that the IFN-IFN in colon of the model group immunized with ZH68 and T668 protein was significantly higher than that of the protein immunization group (p0.05) after 3 days. The expression of IFN-gamma, IL-12 and TGF-beta mRNA in mesenteric lymph nodes of the model group after immunization with histone had no significant change in the model group after saline injection (p0.05), while the expression of IL-4, IL-10 and TGF-beta mRNA in the model group after immunization with ZH68 and T668 protein had no significant change in the expression of IFN-gamma, IL-12 and TGF-beta mRNA. After immunization with ZH68 and T668, the expression of IL-17 and IL-10 mRNA in the spleen of model mice decreased significantly (p0.05). The expression of other cytokines was not significantly different (p0.05). The results of ELISA showed that ZH68 and T668 eggs were immunized with ZH68 and T668 proteins. The levels of LPMC production in colon tissue of IL-10 and TGF-beta mice in white immune model group were significantly higher than those in saline injection group (p0.05), while the levels of IL-12 LPMC production in IFN-gamma, IL-17 and 3 days were significantly lower than those in saline injection group (p0.05). The levels of IL-12 after 7 days and IL-4 LPMC production after 3 days had no change (p0.05), and the levels of IL-4 LPMC production after 7 days were significantly lower than those in saline injection group (p0.05). The expression of IL-4, IL-10 and TGF-beta in splenic lymphocytes of mice immunized with ZH68 and T668 protein was significantly higher than that of mice immunized with saline (p0.05). The production of IL-17 was significantly lower than that of mice injected with saline (p0.05). The production of IFN-gamma in splenic lymphocytes of mice immunized with ZH68 and T668 protein was significantly higher than that of mice injected with saline (p0.05). There was no significant difference in the production of IL-12 between the model group and the WM5 and WN10 protein groups (p0.05). The production of IFN-gamma by splenic lymphocytes and the level of IL-12 on the 3rd day after 7 days were significantly lower than that in the saline injection group (p0.05). Real-time quantitative RT-PCR and ELISA results showed that there was no significant difference in the WM5 and WN10 protein immunization group (p0.05). Flow cytometry showed that the percentage of CD4+CD2+Foxp3+Treg cells in splenic lymphocytes of the model group immunized with ZH68 and T668 was significantly lower than that of the model group immunized with saline (p0.05), WM5 and WN10. The percentage of CD4+CD2+Foxp3+Treg cells on the splenic lymphocyte surface in the model group after protein immunization was not significantly different from that in the model group after saline injection (p0.05), but there was no significant difference in the active stage of disease (p0.05).
The model mice immunized with Trichinella spiralis effective antigen components showed good therapeutic effects on TNBS-induced colitis. The possible mechanisms were as follows: the effective antigen components restored the normal balance of Th1/Th2 in IBD model mice; inhibited the immune response of the body and immune cells and cytokines played a super immunomodulatory role, thereby. The body's immune imbalance is restored to normal level.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫樹(shù)民;王學(xué)林;郭恒;劉明遠(yuǎn);;旋毛蟲(chóng)抗原基因T668重組蛋白對(duì)鼠結(jié)腸炎的保護(hù)效應(yīng)[J];中國(guó)獸醫(yī)學(xué)報(bào);2014年03期

相關(guān)碩士學(xué)位論文 前1條

1 王蕾;限食和多不飽和脂肪酸對(duì)DSS誘導(dǎo)的大鼠結(jié)腸炎Nrf2氧化通路的影響[D];山西醫(yī)科大學(xué);2014年

，

本文編號(hào)：2188059