旋毛虫有效抗原成分对炎症性肠病免疫干预研究
发布时间：2018-08-17 15:22
【摘要】：炎症性肠病(IBD)是一种病因不明的自身免疫性疾病,包括溃疡性肠炎(UC)和克罗恩病(CD)是肠道慢性、复发性病症。自身免疫失衡是IBD发病的直接诱因。环境和遗传因素在IBD的免疫中起重要作用。当环境因素作用于有遗传易感性的个体时,就可能引发机体免疫系统功能失调而发病,IBD在卫生条件较好的发达国家发病率较高,而在卫生不好、条件差的国家发病率反而较低。单纯用环境和遗传因素很难解释这种差异的,因为欠发达地区移民到发达地区的人群IBD发病率较高。这些结果说明,环境因素对IBD的发生是重要的。即而产生了IBD“卫生假说”的理论,即IBD的发生是在缺少对肠道粘膜刺激较少的社会。线虫感染较多国家CD和UC的发病率低,说明肠道寄生虫的感染有效的解释了这个问题。前期工作中以旋毛虫(T.spiralis)作为干预小鼠肠炎模型产生较好的治疗作用。尽管寄生虫对IBD的治疗有益,寄生虫感染所引发的生物安全性问题一直受人关注。活体寄生虫持续感染人体不易被排出体外,可能会影响胃肠的功能,并导致病人心理上难以接受这种治疗。因此,在维持寄生虫生命周期的条件下,利用寄生虫进行大规模治疗并不可行。寄生虫提取物可以与活虫具有相同的治疗效果,不但可以避免感染活虫的隐患,还能使病人更容易接受。旋毛虫有效抗原成分可避免活虫治疗IBD的不利因素,同时保留了旋毛虫对IBD的免疫调控作用,理论上是符合临床治疗要求的生物制剂。 本研究选择已鉴定好的旋毛虫有效抗原成分,即ZH68旋毛虫成虫抗原基因(丝氨酸蛋白酶);WM5旋毛虫肌幼虫抗原基因(丝氨酸蛋白酶抑制剂);WN10旋毛虫新生幼虫抗原基因(半胱氨酸蛋白酶抑制剂);T668旋毛虫新生幼虫抗原基因(丝氨酸蛋白酶),构建可溶性蛋白表达载体,并对抗原基因表达产物进行纯化,目的是获得纯度较高的基因表达产物。其方法是利用镍琼脂糖凝胶FF色谱分离带有His标签的重组蛋白pET-22b-T668、pET-22b-ZH68、pET-22b-WM5和pET-22b-WN10。将纯化浓缩的四种可溶性蛋白在无菌条件下经腹腔注射BALB/c鼠,以生理盐注射水作为对照。三次免疫后用TNBS诱导动物模型,观察诱导CD模型指标的变化情况,包括小鼠的平均体重、生存率、DAI评分、结肠宏观评分和微观损伤评分,检测炎症指标MPO、SOD活性,探讨四种可溶性蛋白对动物模型的治疗效应。实时定量RT-PCR检测各组模型鼠肠粘膜、肠系膜淋巴结及脾脏中IFN-γ、IL-10、IL-12、IL-4、IL-17和TGF-βmRNA的表达含量,用ELISA法检测各组小鼠结肠(LPMC)和脾脏中IFN-γ、IL-12、TGF-β、IL-4、IL-10和IL-17的分泌水平,从而动态研究肠道粘膜免疫和细胞免疫作用关系;并通过流式细胞仪检测(FCM)各组脾细胞内CD4+ CD25+ Foxp3+ Treg细胞数量变化。从细胞水平阐述Treg对机体免疫方面的作用,客观论述蛋白对IBD模型鼠的免疫作用机制。 旋毛虫抗原基因WM5、WN10、Zh68和T668克隆入原核表达载体pET-22b中进行表达。经鉴定四种抗原基因未发生突变,具有完整的开放阅读框,基因全长分别是1315bp、1352bp、1372bp和1609bp,切除信号肽后表达成熟蛋白分子量理论为37.7 kDa、46.9 kDa、47 kDa、49 kDa,表达的融合蛋白的大小与理论相符。表达产物通过镍琼脂糖凝胶柱进行亲和层析分离纯化,SDS-PAGE和Western-Blot检测蛋白质具有良好的免疫原性和反应原性。 旋毛虫有效抗原成分对肠炎模型干预效应结果显示,Zh68和T668蛋白免疫后TNBS诱导鼠模型组平均体重和生存率显著高于盐水注射后造模组,(p0.05),各项临床症状均轻于盐水注射后造模组,DAI评分明显降低(p0.05)。预先免疫Zh68和T668蛋白小鼠于TNBS诱导造模后3d及7d与生理盐水模型组相比在结肠宏观损伤和微观损伤评价上都有所改善(p0.05),粘膜损伤轻微,结肠壁增厚较少、粘连范围较窄,溃疡面浅或临近正常组织,炎性细胞浸润范围较小、减弱,水肿范围较小,MPO值降低,差异显著(p0.05)。SOD评价值显示,盐水注射造模组3d后明显高于蛋白免疫试验组(p0.05),7d后差异不显著(p㧐0.05)。WM5和WN10蛋白免疫造模组与盐水注射未造模组之间没有明显的差异。实时定量RT-PCR结果显示:ZH68和T668蛋白免疫后造模组3d结肠中IFN-γ、IL-12和IL-17 mRNA的表达量显著低于盐水注射造模组(p0.05),而IL-4、IL-10和TGF-βmRNA的表达显著升高(p0.05)。各组蛋白免疫后造模组肠系膜淋巴结中IFN-γ、IL-12和TGF-βmRNA的表达量对盐水注射后造模组没有显著变化,ZH68和T668蛋白后造模组IL-17和IL-10 mRNA的表达量显著下降(p0.05),IL-4 mRNA的表达量显著升高(p0.05)。ZH68和T668蛋白免疫后造模组小鼠在造模脾脏中IL-17、IL-10 mRNA的表达量对模型组的表达显著下降(p0.05),其它细胞因子表达没有显著性差异(p0.05)。ELISA结果显示:ZH68和T668蛋白免疫造模组3d及7d后的IL-10和TGF-β小鼠结肠组织LPMC产生水平显著高于盐水注射造模组(p0.05),而IFN-γ、IL-17和3d后的IL-12 LPMC产生水平显著低于盐水注射造模组(p0.05),7d后的IL-12水平和3d后的IL-4 LPMC产生水平没有变化(p0.05),7d后的IL-4 LPMC产生水平显著高于盐水注射造模组(p0.05)。ZH68和T668蛋白免疫造模组3d及7d后小鼠脾脏淋巴细胞的IL-4、IL-10和TGF-β表达显著高于盐水注射后造模组(p0.05),IL-17产生水平显著低于盐水注射造模组(p0.05),3d后脾脏淋巴细胞产生IFN-γ的水平及第7dIL-12的产生水平与模型组相比未见明显差异(p0.05),7d后脾脏淋巴细胞产生IFN-γ的水平及第3d IL-12的水平显著低于盐水注射造模组(p0.05)。实时定量RT-PCR和ELISA结果显示:WM5和WN10蛋白免疫后造模组变化不显著(p0.05),各蛋白免疫造模组与盐水注射未造模组相比上述显著差异基础上的细胞因子表达呈现相反的结果。流式细胞检测显示:ZH68和T668蛋白免疫后造模组7d脾淋巴细胞表达CD4+ CD2+ Foxp3+ Treg细胞数占CD4+T淋巴细胞的百分率明显低于盐水注射后造模组(p0.05),WM5和WN10蛋白免疫后造模组脾淋巴细胞表CD4+ CD2+ Foxp3+ Treg细胞数占CD4+T淋巴细胞的百分率与盐水注射后造模组相比没有显著变化(p0.05);而3d疾病活动期各组没有明显差异(p0.05)。 旋毛虫有效抗原成分免疫后造模小鼠均表现出对TNBS诱导结肠炎模型具有较好的治疗效应,其可能的机制是:有效抗原成分恢复IBD模型鼠Th1/Th2正常的平衡;抑制了机体的免疫反应和免疫细胞和细胞因子发挥超强的免疫调剂作用,从而使机体免疫失衡恢复到正常水平。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 孙树民;王学林;郭恒;刘明远;;旋毛虫抗原基因T668重组蛋白对鼠结肠炎的保护效应[J];中国兽医学报;2014年03期
相关硕士学位论文 前1条
1 王蕾;限食和多不饱和脂肪酸对DSS诱导的大鼠结肠炎Nrf2氧化通路的影响[D];山西医科大学;2014年
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