γδT细胞在小鼠自身免疫性葡萄膜炎中保护作用的实验研究

目的:γδT细胞在自身免疫性葡萄膜炎(EAU)中的作用尚不完全清楚。以往认为γδT细胞可能是诱导免疫炎症反应的重要启动因素,但体内研究结果:不一。本文拟探讨γδT细胞在EAU的发病中的致病或保护作用。方法:分离纯化小鼠γδT细胞,体外抗原激活后输注于野生型B6小鼠;用GL3抗体注射法清除野生型B6小鼠体内的γδT细胞;或直接使用γδT细胞基因敲除小鼠,IRBP1-20免疫法制作小鼠EAU模型,观察小鼠眼球炎症的临床评分和病理改变,与野生型B6小鼠的EAU模型相比较。结果:1)EAU时γδT细胞数量明显增加;2)早期活化的γδT细胞主要产生和表达IL-17;3)预先输注活化的γδT细胞后,IRBP1-20诱导的EAU眼球炎症明显减轻;4)GL3清除γδT细胞后,EAU眼球炎症明显加重,γδT细胞基因敲除小鼠的EAU眼球炎症明显加重;5)缺乏γδT细胞的小鼠预先输注γδT细胞后,EAU炎症明显减轻。结论:激活的γδT细胞在EAU发病中可能起到控制免疫炎症不致失控的保护作用,这种保护作用可能是通过产生IL-17实现的。

PI-IBS小鼠肠黏膜γδT细胞表型和功能变化的实验研究

目的:探讨PI-IBS小鼠γδT细胞的表型和功能及其在PI-IBS发病中的作用。方法:旋毛虫感染小鼠,观察肠道炎症、腹壁撤退反射(内脏高敏感性)和结肠传输时间(肠道动力)。分别在感染后第2周和第8周处死动物,取末端回肠和近端结肠组织,免疫荧光组化,激光扫描共聚焦显微镜观察肠黏膜γδT细胞的分布和数量变化。收集PI-IBS小鼠肠道淋巴结和脾脏的淋巴细胞,单克隆抗体免疫磁珠分选法分离和纯化γδT细胞,3HTdR法检测其增殖情况,FACS检测其表面分子CD69、CD62L,ELISA检测细胞培养上清液中IFN-γ及IL-17的表达。结果:感染后第2周,PI-IBS小鼠肠道炎症明显,肠黏膜γδT细胞数量明显增加,明显增殖和活化,产生IL-17明显增加(P<0.01)。感染后第8周,PI-IBS小鼠肠道炎症基本消退,动物的腹壁撤退反射和结肠传输试验明显异常。肠黏膜γδT细胞数量仍然明显高于对照组小鼠,仍然有明显增殖、活化和产生IL-17(P<0.01)。结论:肠道γδT细胞增殖、活化及分泌IL-17可能参与PI-IBS的发病。

小鼠γδT细胞抗原呈递功能的实验研究

目的:证实小鼠γδT细胞抗原也具有呈递功能。方法:观察了高度纯化的小鼠γδT细胞体外激活后MHCⅡ分子及其他共刺激分子的表达与其表面特征性TCR的关系。用自身反应抗原IRBP/MOG特异性T细胞株作为反应细胞,以增殖反应及细胞因子表达作为效应指标,观察γδT细胞的抗原呈递功能。结果:小鼠γδT细胞在激活后可表面表达MHCⅡ分子,并能有效呈递抗原给T细胞,使之发生抗原特异性免疫应答。结论:小鼠γδT细胞具有抗原呈递功能,在启动特异性免疫应答的过程中起重要作用。另一方面,不象人γδT细胞,表达MHCⅡ分子限于新激活的小鼠γδT细胞,后者失去了表面γδTCR。当γδT细胞重新获得表面γδTCR时,其MHCⅡ抗原表达则逐渐减少。

Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用

背景C57BL／6及B10Rm小鼠是实验性自身免疫性葡萄膜炎（EAU）动物模型常用的小鼠种系，其中C57BL／6小鼠免疫后眼部炎症较轻，而B10Rm小鼠免疫后眼部表现为典型的后葡萄膜炎病理改变。目的观察培养的光感受器间维生素A类结合蛋白（IRBP）抗原特异性Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用，研究EAU中Th1及Th17细胞的致病机制。方法选取C57BL／6小鼠、B10Rm小鼠各10只，尾根部及躯干皮下注射200Ix1含有200μgIRBP1—20或IRBP161—180抗原及完全弗氏佐剂（CFA）的乳化液，共均匀注射6个点以免疫动物。流式细胞仪分析B10Rm小鼠模型的眼内浸润T细胞类别。分离培养C57BL／6小鼠淋巴结及脾脏IRBP特异性T细胞，分别加入白细胞介素2（IL-2）或IL-23以适于Th1、Th17细胞生长。将培养至第5天的Th1、Th17细胞分别加入到经干扰素-1（IFN-1）预处理的单层视网膜星形细胞中，观察细胞间的相互作用，检测肿瘤坏死因子-α（TNF—α）的质量浓度。结果B10R111小鼠EAU模型眼球中有大量炎性细胞浸润，流式细胞仪检测证实含有IFN-γ＋、IL-17＋细胞和CD45＋细胞，分别占9．5％、5．1％和41．4％。IRBP1—20刺激后6d，含IL-2和IL-23培养基中IFN-γ＋细胞分别为44．0％、8．0％，IL17＋细胞分别为1．O％、26．O％。Th1、Th17与视网膜星形细胞相互作用24h后，可见视网膜星形细胞死亡脱落，Th17较Th1具有更强的杀伤作用。Th1、Th17细胞分别与星形细胞共培养48h，两种培养基中TNF—Ot的质量浓度分别为（500±10）、（801±24）μg／L，差异有统计学意义（t=-20．36，P=0．00）。结论Th1、Th17对视网膜星形细胞均有杀伤作用，Th17细胞杀伤作用更强，在葡萄膜炎致病过程中发挥更重要的作用。杀伤过程中既有细胞直接接触作用，又有通过细胞因子介导的间接作用。

Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35892-2018 [Issued 6 February 2018 Effective from 1 September 2018]

These Chinese National Guidelines(GB/T 35892-20181)were issued February 06,2018 and became effective September 01,2018.The authors recognized the urgent need for an authentic English translation to inform the international community of the compliance requirements in China.It was appreciated that the final translation must reflect the specialist understanding of those working under the Guideline whilst remaining faithful to the meaning of the original Chinese text.A three-step translation process was therefore determined.Step 1:A professional interpretation service(KL Communications,UK)was commissioned to prepare a literal translation of the Chinese text.Supportive documents were provided which explained specialist terminology.This translation was checked by two bilingual experts.Step 2:A workshop was held in Nanjing in May 2019 to which were invited experts in laboratory animal welfare and ethical use.These included international native English-speaking and Chinese-speaking delegates.The delegates worked in multilingual teams to review sections of the literal translation ahead of the workshop,and to agree an authentic interpretation during the workshop.Step 3:Following the workshop,three bilingual experts(two native Chinese speakers and one native English speaker)reviewed the entire document to ensure consistency of terminology and general accuracy.This document is thus not a“literal translation”but an“accurate interpretation”of the original text.Any challenge of work being performed under these Guidelines should rely on the Chinese text in the first place.However,this translation may be used as mitigating evidence,especially where those performing the work are non-Chinese speakers.

Upregulated adenosine 2A receptor accelerates post-infectious irritable bowel syndrome by promoting CD4+T cells’T helper 17 polarization

BACKGROUND Post-infectious irritable bowel syndrome(PI-IBS)is generally regarded as a functional disease.Several recent studies have reported the involvement of lowgrade inflammation and immunological dysfunction in PI-IBS.T helper 17(Th17)polarization occurs in IBS.Adenosine and its receptors participate in intestinal inflammation and immune regulation.AIM To investigate the role of Th17 polarization of CD4+T cells regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS A PI-IBS model was established by infecting mice with Trichinella spiralis.The intestinal A2AR and CD4+T lymphocytes were detected by immunohistochemistry,and the inflammatory cytokines were detected by enzyme-linked immunoassay.CD4+T lymphocytes present in the animal’s spleen were separated and cultured with or without A2AR agonist and antagonist.Western blotting and real-time quantitative polymerase chain reaction were performed to determine the effect of A2AR on the cells and intestinal tissue.Cytokine production was determined.The protein and mRNA levels of A2AR associated signaling pathway molecules were also evaluated.Furthermore,A2AR agonist and antagonist were injected into the mouse model and the clinical features were observed.RESULTS The PI-IBS mouse model showed increased expression of ATP and A2AR(P<0.05),and inhibition of A2AR improved the clinical features in PI-IBS,including the abdominal withdrawal reflex and colon transportation test(P<0.05).The number of intestinal CD4+T cells and interleukin-17(IL-17)protein levels increased during PI-IBS,which was reversed by administration of the A2AR antagonist(P<0.05).CD4+T cells expressed A2AR and produced IL-17 in vitro,which was regulated by the A2AR agonist and antagonist.The A2AR antagonist increased the production of IL-17 by CD4+T cells via the Janus kinase-signal transducer and activator of transcriptionreceptor-related orphan receptorγsignaling pathway.CONCLUSION The results of the present study suggested that the upregulation of A2AR increases PI-IBS by promoting the Th17 polarization of CD4+T cells.

γδ T细胞和αβ T细胞在体外抗原特异性免疫应答中的协同作用

γδ T细胞和αβT细胞是两种表型和功能不同的T淋巴细胞亚群。为研究两种细胞在抗原特异性免疫应答中是否具有协同作用,并进一步探讨其相互作用的方式,本文利用实验性自身免疫性葡萄膜炎小鼠模型获得脾脏和淋巴结T细胞,用γδ TCR和αβTCR单克隆抗体染色,分别用带有磁珠的抗FITC和抗PE单克隆抗体通过阳性或阴性选择获得高度纯化的γδ T细胞和αβ T细胞,观察不同比例的γδT细胞和αβT细胞在体外共孵育后IRBP1-20抗原特异性增殖和细胞因子表达,并用细胞培养薄膜将二者分开,测定细胞因子表达情况。结果表明,γδ T细胞或αβT细胞(99%纯度)都不能单独发生抗原特异性免疫应答。只有在二者比例为5%γδT细胞和95%αβT细胞时能产生最大的抗原特异性反应。当两种细胞脱离接触时,不能出现抗原特异性反应。γδ T细胞或αβ T细胞的培养上清也不能使γδT细胞或αβT细胞发生抗原特异性反应。本研究提示,小鼠γδT细胞和αβT细胞在抗原特异性免疫应答中具有协同作用,这种协同作用是通过两种细胞相互接触,γδT细胞协助αβT细胞产生免疫应答实现的。