【作者】 田智；

【导师】 汪世平；

【作者基本信息】 中南大学 ， 病原生物学， 2012， 博士

【摘要】 研究背景血吸虫病(schistosomiasis)是一种流行广泛、严重危害人类健康和社会经济发展的人兽共患寄生虫病,我国是日本血吸虫病(schistosomiasis japonica)流行最严重的四个国家之一。日本血吸虫(Schistosoma japonicum, Sj)致病的主要原因是雌虫产出的大量虫卵沉积于肝、肠等组织中,形成虫卵肉芽肿、组织损伤和继发性肝纤维化。肝纤维化的进一步发展导致肝硬化,患者可因上消化道出血、肝性昏迷等严重并发症而致死。因此,阻止虫卵肉芽肿病变的形成,对于控制日本血吸虫病的进程、防止晚期血吸虫病的发生具有重要的意义。目前,临床上采用单一的吡喹酮治疗血吸虫病仍存在一定缺陷,吡喹酮只能杀灭童虫和成虫,而不能阻止肝组织内的虫卵继续分泌抗原,虫卵可溶性抗原(soluble matured egg antigen, SEA)可引起组织内虫卵肉芽肿反应,由于血吸虫病患者发现时多己转为慢性,肝组织内虫卵已经或正在形成虫卵肉芽肿或肝纤维化,有效杀虫治疗后病人依然有转化为晚期的可能,SEA诱导产生的免疫病理反应并不因杀虫治疗而终止。IL-18是目前己知的对血吸虫病肝纤维化具有治疗作用的细胞因子,然而单纯使用细胞因子治疗需连续、大剂量的用药,势必导致毒副作用的增加以及宿主体内细胞因子的严重失衡。因此,我们拟利用实验室前期筛选得到的对日本血吸虫雌虫、未成熟虫卵、成熟虫卵等多个阶段均具有高特异性免疫靶向作用及识别能力的单链抗体(Sj single-chain Fv antibody, SjscFv),将其与对血吸虫性肝纤维化具有治疗作用的细胞因子IL-18融合表达,产生SjscFv-IL18融合蛋白,借助SjscFv的免疫及靶向作用携带IL-18进入虫卵肉芽肿和肝纤维化形成的局部,特异性提高局部免疫作用及IL-18浓度,诱导日本血吸虫感染慢性期从Th2型免疫应答转化为Th1型优势的免疫应答,同时能够极大地降低IL-18对其它正常组织器官的毒副作用,以期达到更好的抗日本血吸虫性病纤维化的效果。研究目的构建真核重组质粒pVAX1/SjscFv-IL18,转染巨噬细胞后,使用SEA刺激后的巨噬细胞上清液处理肝星状细胞(HSCs),观察对HSCs激活、增殖和凋亡的影响,并检测HSCs中与细胞外基质(ECM)沉积相关的基因表达,评估其抗肝纤维化的能力；并在日本血吸虫病肝纤维化小鼠中研究其对虫卵肉芽肿及肝纤维化形成的影响及其作用机制。研究方法1、真核重组质粒pVAX1/SjscFv-IL18的构建；采用SOE-PCR技术构建重组质粒pVAX1/SjscFv-IL18和pVAX1/S/scFv,转化大肠杆菌后,进行大量表达,并通过双酶切和测序鉴定,使用生物信息学方法分析其结构特征。2、pVAX1/SjscFv-IL18对HSCs胶原生成与降解的影响将各组重组真核质粒转染巨噬细胞后,采用SEA刺激巨噬细胞后的上清液处理HSCs,应用实时荧光定量PCR(qPCR)检测胶原相关基因mRNA水平、MTT检测HSCs细胞增殖、流式细胞术检测HSCs凋亡。3、pVAX1/SjscFv-IL18对日本血吸虫病肝纤维化的影响建立日本血吸虫病肝纤维化小鼠模型,将pVAX1/SjscFv-IL18注入小鼠体内,采用]HE、Masson染色方法分别检测肝组织内虫卵肉芽肿的大小、肝组织内胶原沉积的情况,应用ELISA检测小鼠Thl/Th2型细胞因子、IL-18,免疫组化检测肝组织中TGF β1和IL-18, qPCR检测肝脏中胶原相关基因mRNA水平。研究结果1、PCR、双酶切及测序结果显示重组质粒插入序列与目的序列一致,CD-Search证实所构建质粒表达蛋白中包含目的蛋白所需的两个Ig超家族和IL-1超家族保守结构域,说明真核重组质粒pVAX1/SjscFv-IL18构建成功。2. pVAX1/SjscFv-IL18转染巨噬细胞后,采用SEA刺激该巨噬细胞产生的上清液处理HSCs,与对照组相比,能够部分降低HSCs中a-SMA的mRNA水平,并抑制HSCs增殖、促进HSCs凋亡,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达。3、pVAX1/SjscFv-IL18注入日本血吸虫病肝纤维化小鼠后,能够降低肝组织内虫卵肉芽肿的大小,减少肝组织内胶原的沉积,并在体内表达大量的IL18,诱导Thl型优势的免疫反应,减少肝脏中TGF β1表达,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达,且能够在虫卵肉芽肿局部提高IL-18的浓度,从而产生更好的抗日本血吸虫病肝纤维化的作用。结论1、成功构建了真核重组质粒pVAX1/SjscFv-IL18。2、pVAX1/SjscFv-IL18在体外能够通过影响巨噬细胞内细胞因子的分泌进而抑制HSCs中胶原的生成,促进胶原降解。3、pVAX1/SjscFv-IL18能够通过SjscFv的免疫靶向作用提高IL-18的生物学活性,从而抑制虫卵肉芽肿的形成,并产生更好的抗日本血吸虫病肝纤维化的作用。更多还原

【Abstract】 BackgroundSchistosomiasis japonicum is a severe endemic disease mainly prevalent in Central South China; it remains to be a serious zoonosis and seriously endanger people’s health and effect social and economic development of epidemic areas. In the case of Schistosoma japonicum (S. japonicum) infection, the chronic onset of disease is not due to the adult worms but is chiefly related to the T-cell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines. The trapped eggs induce delayed hypersensitive reactions, evoking the formation of inflammatory granuloma and subsequent fibrosis. Liver fibrosis will further develop to cirrhosis, which causes death by supervene upper gastrointestinal hemorrhage or hepatic coma. Therefore, it is very neccesory to regular eggs-induced granuloma formation to prevent occurrence of advanced schistosomiasis japonica.At present, praziquantel still is the only way to treat schistosomiasis in clinic. It could kill the schistosome, but not the eggs trapped in tissue. The eggs could cause granuloma formation by secreting antigen continually, and those schistosomiasis patients already have granuloma formation and subsequent fibrosis. The eggs-induced immune response will not terminate by treated with praziquantel. Interleukin-18(IL-18) is currently known cytokines for ameliorates treatment of schistosomiasis liver fibrosis. However, the approach of cytokines therapy need large amounts of particular cytokine, that will have serous side-effects and cause the immune system imbalance.In our previous work, a S. japonicum single-chain fragment variable (SjscFv) specifically bound to the S.japonicum soluble immature egg antigen (SIEA) of26-28kDa was obtained from immunized mice, and its capability to target S.japonicum SIEA and soluble mature egg antigen (SEA) was confirmed. In present study, we will fuse this SjscFv with IL-18and clone into the eukaryotic vector pVAX1to produce SjscFv-IL18fusion protein, the specific S/scFv will targeting the IL-18to the site where parasitic eggs are embedded in the liver tissues and hepatic fibrosis is induced by SEA. Then the site-specific accumulation of functional IL-18will induce a predominant T-helper1(Thl) reaction, reduce the side-effects in normal tissue and induce more potent antifibrotic activity.ObjectiveThe objective of present study is to fuse SjscFv with IL-18and clone into the eukaryotic vector pVAX1and then transfect into macrophages. After treated with SEA, the supernatant of treated macrophages were used to incubate hepatic stellate cells (HSCs) and further observe the activation, proliferation and apoptosis of HSCs. The changes of extracellular matrix-related genes were used to assess its antifibrotic abilities in vitro. And use it as a therapeutic DNA vaccine to study its effect and mechanism on ameliorates hepatic fibrosis in vivo.Methods1. Identification of pVAX1/SjscFv-IL18plasmidSjscFv and IL-18genes were linked using the splicing overlap extension PCR (SOE-PCR) method with the help of a glycine-rich linker consisting of15amino acids (Gly4Ser)3. The fusion gene SjscFv-IL18was cloned into the eukaryotic expression vector pVAXl which has been double digested with EcoR I and Xho I and transfected into E. coli BL21(DE3), and finally confirmed by double digestion with restriction enzymes and sequencing. Bioinformatics tools were used to predict its encoding protein structures.2. pVAX1/SjscFv-IL18interfere the collagen generation and degradation in vitroAfter recombinant eukaryotic plasmid transfected into macrophage, the supernate of SEA-treated macrophage were used to stimulate HSCs as conditioned media. The mRNA expression of those genes relatived with collagen generation and degradation in HSCs were analyzed by real time quantity PCR (qPCR). The proliferation and apoptotic of HSCs were measured by MTT and flow cytometric, respectively.3. Effects of pVAX1/SjscFv-IL18on the mice liver fibrosis caused by schistosomiasisMice were challenged with S.japonicum cercariae infection on their abdominal skin. Forty-five days later, all infected mice were treated with praziquantel. At the same time, four kinds of plasmid were injected intramuscularly. Twenty wks after challenge, all mice were euthanized. The granuloma volume and collagen contents were estimated by H&E and Masson staining. IL-18and Thl/Th2cytokines level were analyzed by ELISA. TGF β1and IL-18in liver section were detected by immunohistochemistry (IHC). The mRNA expression of those genes relatived with collagen generation and degradation were analyzed by qPCR.Results1. Double digesting and sequencing confirmed the constructure of recombinant plasmid pVAX1/SjscFv-IL18. CD-search indicated that its encoding protein contained two Ig superfamily and one IL-1superfamily conserved domain.2. pVAX1/SjscFv-IL18downregulated the mRNA expression of a-SMA, Col Ⅰ, Col Ⅲ, TIMP-1and upregulated the mRNA expression of MMPs-1in HSCs. It also could suppress proliferation and promote apoptotic in HSCs. 3. pVAX1/SjscFv-IL18could induce dominant Thl cytokines response in vivo. Consistent with the levels of Thl and Th2cytokines, mice vaccinated with pVAX1/SjscFv-IL18developed much less hepatic fibrosis, which was evaluated by average volumn of granuloma and collagen contents. It also could downregulate the mRNA expression of Col I, Col III, TIMP-1and upregulate the mRNA expression of MMPs-1in liver. Furthermore, pVAX1/SjscFv-IL18was more efficient than pVAX1/IL-18for its capability to deliver IL-18towards the site of hepatic fibrosis.Conclusions1. pVAX1/SjscFv-IL18was successfully constructed.2. pVAX1/SjscFv-IL18can reduce collagen in vitro.3. pVAX1/SjscFv-IL18can decrease hepatic fibrosis by delivering IL-18towards the site where the granuloma occur.更多还原