旋毛蟲有效抗原成分對炎癥性腸病免疫干預研究
發(fā)布時間：2018-08-17 15:22
【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。當環(huán)境因素作用于有遺傳易感性的個體時,就可能引發(fā)機體免疫系統(tǒng)功能失調而發(fā)病,IBD在衛(wèi)生條件較好的發(fā)達國家發(fā)病率較高,而在衛(wèi)生不好、條件差的國家發(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,因為欠發(fā)達地區(qū)移民到發(fā)達地區(qū)的人群IBD發(fā)病率較高。這些結果說明,環(huán)境因素對IBD的發(fā)生是重要的。即而產(chǎn)生了IBD“衛(wèi)生假說”的理論,即IBD的發(fā)生是在缺少對腸道粘膜刺激較少的社會。線蟲感染較多國家CD和UC的發(fā)病率低,說明腸道寄生蟲的感染有效的解釋了這個問題。前期工作中以旋毛蟲(T.spiralis)作為干預小鼠腸炎模型產(chǎn)生較好的治療作用。盡管寄生蟲對IBD的治療有益,寄生蟲感染所引發(fā)的生物安全性問題一直受人關注。活體寄生蟲持續(xù)感染人體不易被排出體外,可能會影響胃腸的功能,并導致病人心理上難以接受這種治療。因此,在維持寄生蟲生命周期的條件下,利用寄生蟲進行大規(guī)模治療并不可行。寄生蟲提取物可以與活蟲具有相同的治療效果,不但可以避免感染活蟲的隱患,還能使病人更容易接受。旋毛蟲有效抗原成分可避免活蟲治療IBD的不利因素,同時保留了旋毛蟲對IBD的免疫調控作用,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有效抗原成分,即ZH68旋毛蟲成蟲抗原基因(絲氨酸蛋白酶);WM5旋毛蟲肌幼蟲抗原基因(絲氨酸蛋白酶抑制劑);WN10旋毛蟲新生幼蟲抗原基因(半胱氨酸蛋白酶抑制劑);T668旋毛蟲新生幼蟲抗原基因(絲氨酸蛋白酶),構建可溶性蛋白表達載體,并對抗原基因表達產(chǎn)物進行純化,目的是獲得純度較高的基因表達產(chǎn)物。其方法是利用鎳瓊脂糖凝膠FF色譜分離帶有His標簽的重組蛋白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菌條件下經(jīng)腹腔注射BALB/c鼠,以生理鹽注射水作為對照。三次免疫后用TNBS誘導動物模型,觀察誘導CD模型指標的變化情況,包括小鼠的平均體重、生存率、DAI評分、結腸宏觀評分和微觀損傷評分,檢測炎癥指標MPO、SOD活性,探討四種可溶性蛋白對動物模型的治療效應。實時定量RT-PCR檢測各組模型鼠腸粘膜、腸系膜淋巴結及脾臟中IFN-γ、IL-10、IL-12、IL-4、IL-17和TGF-βmRNA的表達含量,用ELISA法檢測各組小鼠結腸(LPMC)和脾臟中IFN-γ、IL-12、TGF-β、IL-4、IL-10和IL-17的分泌水平,從而動態(tài)研究腸道粘膜免疫和細胞免疫作用關系;并通過流式細胞儀檢測(FCM)各組脾細胞內(nèi)CD4+ CD25+ Foxp3+ Treg細胞數(shù)量變化。從細胞水平闡述Treg對機體免疫方面的作用,客觀論述蛋白對IBD模型鼠的免疫作用機制。 旋毛蟲抗原基因WM5、WN10、Zh68和T668克隆入原核表達載體pET-22b中進行表達。經(jīng)鑒定四種抗原基因未發(fā)生突變,具有完整的開放閱讀框,基因全長分別是1315bp、1352bp、1372bp和1609bp,切除信號肽后表達成熟蛋白分子量理論為37.7 kDa、46.9 kDa、47 kDa、49 kDa,表達的融合蛋白的大小與理論相符。表達產(chǎn)物通過鎳瓊脂糖凝膠柱進行親和層析分離純化,SDS-PAGE和Western-Blot檢測蛋白質具有良好的免疫原性和反應原性。 旋毛蟲有效抗原成分對腸炎模型干預效應結果顯示,Zh68和T668蛋白免疫后TNBS誘導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),各項臨床癥狀均輕于鹽水注射后造模組,DAI評分明顯降低(p0.05)。預先免疫Zh68和T668蛋白小鼠于TNBS誘導造模后3d及7d與生理鹽水模型組相比在結腸宏觀損傷和微觀損傷評價上都有所改善(p0.05),粘膜損傷輕微,結腸壁增厚較少、粘連范圍較窄,潰瘍面淺或臨近正常組織,炎性細胞浸潤范圍較小、減弱,水腫范圍較小,MPO值降低,差異顯著(p0.05)。SOD評價值顯示,鹽水注射造模組3d后明顯高于蛋白免疫試驗組(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有明顯的差異。實時定量RT-PCR結果顯示:ZH68和T668蛋白免疫后造模組3d結腸中IFN-γ、IL-12和IL-17 mRNA的表達量顯著低于鹽水注射造模組(p0.05),而IL-4、IL-10和TGF-βmRNA的表達顯著升高(p0.05)。各組蛋白免疫后造模組腸系膜淋巴結中IFN-γ、IL-12和TGF-βmRNA的表達量對鹽水注射后造模組沒有顯著變化,ZH68和T668蛋白后造模組IL-17和IL-10 mRNA的表達量顯著下降(p0.05),IL-4 mRNA的表達量顯著升高(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的表達量對模型組的表達顯著下降(p0.05),其它細胞因子表達沒有顯著性差異(p0.05)。ELISA結果顯示: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后小鼠脾臟淋巴細胞的IL-4、IL-10和TGF-β表達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明顯差異(p0.05),7d后脾臟淋巴細胞產(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)。實時定量RT-PCR和ELISA結果顯示: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上的細胞因子表達呈現(xiàn)相反的結果。流式細胞檢測顯示:ZH68和T668蛋白免疫后造模組7d脾淋巴細胞表達CD4+ CD2+ Foxp3+ Treg細胞數(shù)占CD4+T淋巴細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胞表CD4+ CD2+ Foxp3+ Treg細胞數(shù)占CD4+T淋巴細胞的百分率與鹽水注射后造模組相比沒有顯著變化(p0.05);而3d疾病活動期各組沒有明顯差異(p0.05)。 旋毛蟲有效抗原成分免疫后造模小鼠均表現(xiàn)出對TNBS誘導結腸炎模型具有較好的治療效應,其可能的機制是:有效抗原成分恢復IBD模型鼠Th1/Th2正常的平衡;抑制了機體的免疫反應和免疫細胞和細胞因子發(fā)揮超強的免疫調劑作用,從而使機體免疫失衡恢復到正常水平。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
本文編號：2188059
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
【引證文獻】
相關期刊論文 前1條
1 孫樹民;王學林;郭恒;劉明遠;;旋毛蟲抗原基因T668重組蛋白對鼠結腸炎的保護效應[J];中國獸醫(yī)學報;2014年03期
相關碩士學位論文 前1條
1 王蕾;限食和多不飽和脂肪酸對DSS誘導的大鼠結腸炎Nrf2氧化通路的影響[D];山西醫(yī)科大學;2014年
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