CN109568585B - Cd38抑制剂在制备抗类风湿关节炎药物中的应用 - Google Patents
Cd38抑制剂在制备抗类风湿关节炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及抗类风湿关节炎药物领域，具体涉及CD38抑制剂在制备抗类风湿关节炎药物中的应用。本申请研究表明RA患者血液和滑液中CD38+NK细胞含量、CD38+NK/CD38+NKT比例升高，变化情况与患者DAS28呈正相关，启示了CD38+NK细胞、CD38+NKT细胞有望成为RA的治疗靶点。进一步研究表明，RA中高水平的CD38+NK及CD38+NK/CD38+NKT比值可以降低TGF‑β分泌和激活MNC中CD4+T细胞mTOR通路，抑制CD4+T细胞分化Treg细胞及IL‑10表达、提升IFN‑γ水平和Th1/Th2、Th17/Treg比值，导致抗炎和免疫耐受能力降低，加剧RA病程。
Description
技术领域
本发明涉及抗类风湿关节炎药物领域，具体涉及一种CD38NK细胞抑制剂或CD38NKT激动剂在抗类风湿关节炎领域的应用。
背景技术
类风湿性关节炎(rheumatoid arthritis，RA)是一种以慢性炎症和骨侵蚀为特征的自身免疫性疾病，发病机制尚不十分清楚。现代免疫学认为CD4+Th细胞(CD4+helper Tcells)尤其是Th1/Th2细胞平衡和Th17(interleukin-17-secreting CD4+helper Tcells)/Treg(regulatory T cells)细胞平衡失调是RA免疫紊乱重要的原因。
环腺苷二磷酸核糖水解酶(Cyclic ADP Ribose Hydrolase，cADPRH，CD38)是细胞膜上的一种糖蛋白，在NK细胞、B细胞、T细胞、单核巨噬细胞、树突状细胞(Dentric cell,DC)中都有表达，在多发性骨髓瘤细胞(multiple myeloma cells)中高表达。本申请发明人前期通过转录组学研究发现CD38在RA滑膜组织中与强直性脊柱炎和骨关节炎相比特异性高表达，而且CD38+细胞和CD38+CD56+细胞中比例在RA患者外周血中显著升高，CD38+细胞水平也与患者类风湿因子Rheumatic factor(RF因子)水平呈现显著正相关。有人在CD38敲除小鼠上用牛Ⅱ型胶原诱导性关节炎(collagen-induced arthritis，CIA)，发现CD38敲除后可以明显缓解CIA的发生和发展。上述研究都显示CD38可能对RA发病起着重要作用。
NK细胞是颗粒状的天然淋巴细胞，不表达重排的抗原受体，在人类中通过CD16和CD56的表达来鉴定。NK细胞占外周血单核细胞的5-15％，发挥天然免疫作用，对病原体早期免疫反应中十分重要。据报道，RA滑液中富集大量NK细胞，导致滑液大量产生促炎细胞因子。CD38也可以通过与CD16相互作用，促进NK细胞杀伤功能。NKT样细胞(CD3+CD16+CD56+)是共表达T细胞受体和NK细胞受体的特殊淋巴亚群，可通过产生炎性细胞因子如IFN-γ、穿孔素和颗粒酶发挥抗感染和抗肿瘤的作用。据报道，NKT细胞在RA、系统性红斑狼疮(systemic lupus erythematosus，SLE)、系统性硬化症(systemic sclerosis)、系统性红斑狼疮(systemic lupus erythematosus)、多发性硬化症(multiple sclerosis)和胰岛素依赖的糖尿病(insulin-dependent diabetes mellitus)等多种自身免疫性疾病中表达降低或功能缺失。
目前的研究中，尚未公开过CD38+NK和CD38+NKT在RA和CIA大鼠外周血和关节滑液中的表达水平，同时CD38+NK细胞和CD38+NKT细胞及其平衡对RA的作用和免疫调控机制还属于空白内容，针对CD38+NK细胞与RA作用关系及机制进行研究，有利于为类风湿关节炎药物的开发提供有力的依据。
发明内容
针对现有技术中的问题，本申请对CIA大鼠、健康志愿者、RA患者外周血和滑液中CD38+NK细胞、CD38+NKT细胞的含量进行了检测，经统计学分析表明，RA患者血液和滑液中CD38+NK细胞升高、CD38+NKT细胞含量降低，CD38+NK/CD38+NKT比例升高，变化情况与患者DAS28(类风湿关节炎疾病活动分数计算器)呈正相关，CIA大鼠中CD38+NK/CD38+NKT比值也与CIA炎症指数明显相关，启示了CD38+NK细胞、CD38+NKT细胞有望成为RA的治疗靶点。另一方面，CD38+NK细胞升高、CD38+NKT细胞含量降低，CD38+NK/CD38+NKT比例升高作为类风湿检测靶点用于流式、ELISA、各种检测试剂的设计。
本发明第一方面，提供CD38+NK细胞抑制剂或CD38+NKT细胞激动剂在制备抗类风湿关节炎药物中的应用。
在本申请研究收集RA患者外周血和关节滑液，分别以健康人外周血和骨关节炎(OA)滑液为对照，流式方法检测CD38+各淋巴细胞亚型的占总淋巴细胞的比例，同时本申请研究也建立胶原蛋白诱导大鼠关节炎模型对上述结果进行验证。与健康志愿者组(n＝30)或OA滑液(n＝30)相比，RA患者组外周血(n＝30)和滑液(n＝30)中CD38+NK细胞比例显著上升(P值分别为0.0008和0.0002)，CD38+NKT细胞比例显著降低(P值分别为0.0121和0.0189)。RA血液和滑液CD38+NK/CD38+NKT(分别从从0.62-23，平均值为4.545；从0.942-14.450,平均值3.07)也明显升高(P值分别为0.0004和<0.0001)，并且与患者DAS28呈正相关(P值分别为<0.0001和<0.0001)。CIA大鼠实验也得到类似结果，CD38+NK/CD38+NKT与CIA炎症指数明显相关。以上研究结果充分表明了针对CD38+NK细胞抑制剂或CD38+NKT细胞激动剂有望作为抗类风湿关节炎药物进行应用。
优选的，上述应用中，CD38+NK细胞抑制剂或CD38+NKT细胞激动剂是一种提高Treg、IL-10表达的抗类风湿关节炎药物。
本研究用流式方法从RA滑液和健康人外周血中分离提取CD38+NK细胞和CD38+NKT细胞，将CD38抗体处理后上述细胞与MNC或CD4+T细胞共培养后，MNC细胞中Treg细胞比例和IL-10水平升高。Treg和IL-10与人体自身免疫反应相关，其数量的升高有利于人体免疫力增强，缓解RA病程。
进一步优选的，上述CD38+NK细胞抑制剂或CD38+NKT细胞激动剂是一种降低MNC细胞中Treg、IL-10表达的抗类风湿关节炎药物。
优选的，上述应用中，CD38+NK细胞抑制剂或CD38+NKT细胞激动剂是一种Th1/Th2和Th17/Treg抑制剂的抗类风湿关节炎药物。
Th1/Th2和Treg/Th17作为T细胞亚群中的两个重要的稳态平衡，在机体抗肿瘤免疫、炎症作用机制以及机体免疫稳态中发挥重要作用，Th1/Th2和Th17/Treg比值的降低有利于炎症的恢复和免疫功能的提升，有利于类风湿关节炎的治疗的和恢复。
进一步优选的，上述CD38+NK细胞抑制剂或CD38+NKT细胞激动剂是一种提高CD4+T细胞中Th1/Th2和Th17/Treg比值的抗类风湿关节炎药物。
本发明第二方面，提供CD38+NK细胞抑制剂或CD38+NKT细胞激动剂作为TGF-β激动剂的应用。
TGF-β信号通路参与细胞生长，细胞分化，细胞凋亡，细胞动态平衡等其它细胞功能。本申请研究表明，RA中高水平的CD38+NK及CD38+NK/CD38+NKT比值可以降低TGF-β分泌，同时意味着CD38+NK细胞抑制剂或CD38+NKT细胞激动剂可以作为TGF-β的激动剂应用于生理过程的调节。现有技术中有相关研究表明TGF-β可通过调节mTOR诱导iTreg细胞的生成，TGF-β的激动剂也有望作为一种Treg细胞促进剂用于治疗类风湿性关节炎。
本发明第三方面，提供CD38+NK细胞抑制剂或CD38+NKT细胞激动剂作为mTOR抑制剂的应用。
优选的，上述CD38+NK细胞抑制剂或CD38+NKT细胞激动剂同时还是P70S6抑制剂。
mTOR信号通路促进物质代谢、参与细胞凋亡、自噬，与多种疾病相关，P70S6是其下游信号通路。mTOR的抑制状态下有利于免疫细胞的生成。本申请研究结果表明经CD38抗体处理后，CD4+T细胞中Th1/Th2和Th17/Treg比值降低，磷酸化P70S6、磷酸化mTOR和mTOR蛋白表达水平下调而抑制mTOR信号通路。
本发明第四方面，提供CD38+NK细胞抑制剂或CD38+NKT细胞激动剂作为IFN-γ抑制剂的应用。
优选的，上述提供CD38+NK细胞抑制剂或CD38+NKT细胞激动剂作为MNC中IFN-γ的抑制剂。
进一步优选的，该CD38+NK细胞抑制剂或CD38+NKT细胞激动剂同时还是MNC中IL-2的激动剂。
IL-2、IFN-γ为细胞免疫中的上调免疫，通过炎症反应清除抗原。本申请研究表明CD38抗体可以显著提升MNC中IL-2分泌水平并降低IFN-γ水平，增强机体免疫功能。
优选的，上述CD38+NK细胞抑制剂为CD38抗体。
本发明第五方面，检测CD38+NK细胞、CD38+NKT细胞含量的试剂作为RA诊断试剂的应用。
优选的，上述CD38+NK细胞、CD38+NKT细胞取自外周血和关节滑液。
本申请研究揭示了CD38+NK细胞升高、CD38+NKT细胞含量降低，CD38+NK/CD38+NKT比例升高与RA疾病呈现正相关，启示对CD38+NK细胞、CD38+NKT细胞含量的检测有望成为RA的诊断标准。
本发明的有益效果
1.类风湿关节炎作为一种自身免疫性疾病，其发病机制并不明确。本申请中选取CIA大鼠、健康志愿者及RA患者的外周血及关节滑液作为研究对象，针对CD38+NK细胞、CD38+NKT细胞的含量与疾病关系进行了研究，结论表明：CD38+NK和CD38+NKT比例的表达水平与RA患者DAS28指数正相关，为明确RA患病机制作为了进一步的研究，同时也为RA的诊断提供了新的诊断方法。
2.本申请对CD38+NK和CD38+NKT比例影响RA病程的机制做了进一步的研究，结果表明：RA中高水平的CD38+NK及CD38+NK/CD38+NKT比值可以降低TGF-β分泌和激活MNC中CD4+T细胞mTOR通路，结果抑制CD4+T细胞分化Treg细胞及IL-10表达、提升IFN-γ水平和Th1/Th2、Th17/Treg比值，导致抗炎和免疫耐受能力降低，加剧RA病程。该机制的明确有利于为CD38+NK细胞抑制剂或CD38+NKT细胞激动剂作为抗类风湿关节炎药物进行应用提供依据，以及用药靶点的指导。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。
图1：CD38+NK细胞和CD38+NKT细胞在CIA大鼠外周血淋巴细胞中的变化图；
图1A：正常对照组(n＝24)和CIA组(n＝24)外周血中各淋巴细胞亚型占淋巴细胞(CD45+)的比例；
图1B：正常对照组和CIA组外周血中CD38+细胞亚型占淋巴细胞(CD45+)的比例；
图1C：正常对照组和CIA组外周血中CD38+NK/CD38+NKT比例变化。
图1D：CIA组外周血中CD38+NK/CD38+NKT与疾病活动评分相关性分析。
图2：CD38+NK细胞和CD38+NKT细胞在RA外周血淋巴细胞中的变化图；
图2A：健康人(n＝30)和RA患者(n＝30)外周血中各淋巴细胞亚型占淋巴细胞(CD45+)的比例；
图2B：健康人和RA患者外周血中CD38+细胞亚型占淋巴细胞(CD45+)的比例；
图2C：健康人和RA患者外周血中CD38+NK/CD38+NKT比例变化；
图2D：RA患者外周血中CD38+NK/CD38+NKT与DAS28相关性分析。
图3：CD38(+)NK细胞和CD38(+)NKT细胞在OA和RA滑液的表达变化图；
图3A：OA患者(n＝30)和RA患者(n＝30)滑液中各淋巴细胞亚型占淋巴细胞(CD45+)比例；
图3B：OA患者和RA患者滑液中CD38+细胞亚型占淋巴细胞(CD45+)比例；
图3C：OA患者和RA患者滑液中CD38+NK/CD38+NKT比例变化；
图3D：RA患者滑液中CD38+NK/CD38+NKT与DAS28疾病活动性指数相关性分析。
图4：CD38+NK和CD38+NKT细胞对滑液MNC中淋巴细胞亚群及细胞因子的影响图。
图4A：CD38+NK细胞和CD38+NKT细胞与去除CD16+CD56+CD38+细胞的RA滑液来源的MNC细胞共培养模式图；
图4B：CD38+NK细胞和CD38+NKT细胞对滑液MNC中各淋巴细胞亚型的影响；
图4C：CD38+NK细胞和CD38+NKT细胞滑液MNC共培养后滑液中各细胞因子分泌的影响；
图5：CD38+NK和CD38+NKT对外周血MNC中淋巴细胞亚群及细胞因子的影响图；
图5A：CD38+NK细胞和CD38+NKT细胞对外周血MNC中各淋巴细胞亚型的影响；
图5B：CD38+NK细胞和CD38+NKT细胞与外周血MNC共培养后培养液中各细胞因子分泌的影响。
图6：CD38+NK和CD38+NKT细胞对CD4+T细胞分化Th细胞的作用及分子机制；
图6A：CD38+NK细胞或CD38+NKT细胞与RA滑液来源的CD4+T细胞共培养模式图；
图6B：CD38+NK和CD38+NKT细胞对CD4+T细胞中CD4+IFN-γ+Th1和CD4+IL-4+Th2细胞分化的作用；
图6C：CD38+NK和CD38+NKT细胞对CD4+T细胞中CD4+IL17+Th17和CD4+CD25+Foxp3+Treg分化的作用；
图6D：CD38+NK和CD38+NKT细胞对CD4+T细胞中mTOR信号分子通路的影响；
图6E：CD38+NK和CD38+NKT细胞中CD3、CD28和TGF-β的mRNA的表达水平。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
目前的研究中，尚未公开过CD38+NK和CD38+NKT在RA和CIA大鼠外周血和关节滑液中的表达水平，同时CD38+NK细胞和CD38+NKT细胞及其平衡对RA的作用和免疫调控机制还属于空白内容，针对CD38+NK细胞与RA作用关系及机制进行研究，有利于为类风湿关节炎药物的开发提供有力的依据。因此，本申请对CD38+NK细胞、CD38+NK/CD38+NKT比值与RA的关系与机制进行了深入的研究。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例与对比例详细说明本申请的技术方案。
实施例1
一、材料与方法
1.标本和临床资料
RA患者外周血(n＝30)、RA患者关节滑液(n＝30)和骨关节炎(Osteriarthritis，OA)患者关节滑液(n＝30)在2017年9月至2018年9月收集于山东省千佛山医院骨外科。入选的RA患者均符合1987年美国风湿病学会(American College of Rheumatology,ACR)修订的RA分类标准和2010年美国类风湿学会(ACR)/欧洲抗风湿病联盟(The European LeagueAgainst Rheumatism,EULAR)的分类标准，并排除活动性感染、恶性肿瘤等合并症。健康志愿者(n＝30)外周血来源于山东省千佛山医院查体中心。病例收集和实验设计获得山东省千佛山医院伦理委员会批准【批准号：20160901】，患者签署知情同意授权书。患者临床信息见Table1.
Table 1.Clinical information
2.牛Ⅱ型胶原诱导性关节炎大鼠模型的构建及疾病活动评分。
大鼠适应性饲养1周，将54只大鼠随机分为2组，正常对照组(Normal control,NC,n＝24)，CIA(Collagen-Induced Arthritic，胶原诱导性关节炎)模型组(n＝30)。CIA模型组大鼠每只以0.2ml/只的剂量在大鼠尾根部皮内注射牛II型胶原(美国Chondrex公司，Cat.NO.：150479)和完全弗氏佐剂(美国Sigma公司,Cat NO.：SLBR1681V)等量混合的乳化剂，进行初次免疫。一周后，同样的方法注射牛II型胶原和不完全弗氏佐剂(美国Sigma公司，Cat.NO.：SLBN5308V)等量混合的乳化剂，进行加强免疫。隔天观察毛色、精神状态、活动状态，用游标卡尺测量足趾厚度变化，用电子称测量体重。根据足趾变化，绘制炎症曲线。与正常对照组比较足趾明显肿胀，包括变形和僵直，活动障碍则认为CIA建模成功。用最小显著性差异法(Least Significant Difference，LSD)比较炎症曲线有显著性差异(P＜0.05)，用流式检测外周血血清中各细胞因子的浓度。采用以下标准对关节的炎症程度(疾病活动)进行评分：0分，正常；1分，指爪微红、微肿；2分，整个指爪红肿；3分，严重红肿，包括变形或僵直。每只大鼠最高分为12分。
3.滑液或外周血单个核细胞的分离
取肝素钠抗凝外周血10-20ml，在外周血中加入等体积的PBS(phosphatebuffered solution,磷酸盐缓冲液)稀释混匀；取滑液10-20ml，用透明质酸酶(hyaluronidase，10U/ml)在37℃预处理30min，加入3倍体积的PBS，2500rpm离心10min后，吸弃上清。取50ml EP管加入25ml的淋巴细胞分离液(天津灏洋华科,Cat.No.：LTS1077)，800xg下离心20min。吸取白色环状Mononuclear Cell(MNC)层，获得外周血单个核细胞(peripheral blood mononuclear cells,PBMC)或滑液单个核细胞(Synovial FluidMononuclear Cell,SFMC)。PBS清洗、重悬细胞4℃待用。
4.人滑液淋巴细胞亚群的检测
取20μl CD3-FITC、CD8-PE、CD45-PerCP、CD4-APC、CD3-FITC、CD16+56-PE、CD45-PerCP或CD19-APC流式抗体(ACEA Biosciences Cat.No.8920008)加入管底部，漩涡振荡3秒混匀。分别取100μl滑液分别加入管底部，混匀，轻轻混匀3秒，室温孵育30min。流式细胞仪检测(美国ACEA BIO，Cat.No.NovoCyte D2040R)。
5.人外周血淋巴细胞亚群的检测
取20μl CD3-FITC、CD8-PE、CD45-PerCP、CD4-APC、CD3-FITC、CD16+56-PE、CD45-PerCP或CD19-APC流式抗体(ACEA Biosciences Cat.No.8920008)加入管底部，漩涡振荡3秒混匀。分别取50μl全血分别加入管底部，混匀，室温孵育15min。每个样本管各加450μl稀释好的溶血素，轻轻混匀3秒，室温孵育15min。流式细胞仪检测(美国ACEA BIO，Cat.No.NovoCyte D2040R)。
6.大鼠外周血淋巴细胞亚群的检测
用致命剂量的氯胺酮和赛拉嗪对大鼠进行人道安乐死，在下腔静脉用EDTA采血管采集外周血1-2ml,250xg离心5min离心，吸弃血浆，用红细胞裂解液冰上裂解10-30min。加入1mlPBS(phosphate buffered solution,磷酸盐缓冲液)液,250xg离心5min。吸弃上清，加入1ml PBS重悬细胞，于4℃待用。获得关节腔灌洗液后，加入1ml PBS，250xg下离心5min。洗涤后，吸弃上清，加入100ulPBS重悬，于4℃待用。取以上待测样本100ul加入流式抗体，4℃避光孵育30min，加入500ul PBS 250xg离心5min。吸弃上清,加入100ul PBS重悬，用流式细胞仪上机检测各细胞亚型。用APC anti-rat CD3抗体(美国Biolegend,Cat.No.201413)检测CD3+T细胞，用APC anti-rat CD3抗体、FITC anti-rat CD4抗体(美国Biolegend,Cat.No.203305)检测CD3+CD4+T细胞，用APC anti-rat CD3抗体、PerCP anti-rat CD8a(美国Biolegend,Cat.No.201712)抗体检测CD3+CD8+T细胞，用APC anti-rat CD4抗体(美国Biolegend,Cat.No.201509)、FITC anti-rat CD25抗体(美国Biolegend,Cat.No.202103)、PE anti-rat FOXP3Antibody(美国Biolegend,Cat.No.320007)检测CD4+CD25+Foxp3+Treg细胞，用APC anti-ratCD3抗体、FITC anti-rat CD45RA抗体(美国Biolegend,Cat.No.202305)检测CD3-CD45RA+B细胞，用APC anti-rat CD3抗体、FITC anti-rat CD161抗体(美国Biolegend,Cat.No.B238452)检测CD3-CD161+NK、CD3+CD161+NKT细胞。7.人CD38+淋巴细胞亚型的检测
取待测样本100ul加入流式抗体Anti-human-CD38-percp(美国Biolegend,Cat.No.：356622)、Anti-human CD3FITC/(CD16+CD56)PE Cocktail(美国Biolegend,Cat.No.：319101)、Anti-human-CD3-FITC(美国Biolegend,Cat.No.：300305)、Anti-human-CD19-APC(美国Biolegend,Cat.No.：302211)，4℃孵育30min。加入500ul PBS，200xg下离心5min，吸弃上清，再加入100ul PBS重悬。用流式细胞仪上机检测CD38+T细胞(CD3+CD38+)、CD38+B细胞(CD3-CD19+CD38+)、CD38+NK(CD38+CD3-CD16+CD56+)细胞和CD38+NKT(CD38+CD3+CD16+CD56+)细胞比例。
8.大鼠CD38+淋巴细胞亚型的检测
取待测样本100ul加入流式抗体用PE anti-rat CD38抗体(美国Biolegend,Cat.No.B210722)、FITC anti-rat CD161抗体(美国Biolegend,Cat.No.B238452)，用APCanti-rat CD3抗体(美国Biolegend,Cat.No.201413)、FITC anti-rat CD45RA抗体(美国Biolegend,Cat.No.202305)4℃孵育30min。加入500ul PBS，200xg下离心5min，吸弃上清，再加入100ul PBS重悬。用流式细胞仪上机检测CD38+CD3-CD161+NK、CD38+CD3+CD161+NKT、CD38+CD3+T细胞、CD38+CD3-CD45RA+B细胞比例。
9.人Th1/Th2细胞因子的检测
本实验使用人Th1/Th2细胞因子检测试剂盒(杭州Cell Gene，Cat.NO.20171001)。向各标准品管中加入25ul对应浓度的标准品，样本管中也各加入25ul待检测样本，然后向所有标准品管及样品管中分别加入25ul荧光检测试剂。将孵育好的捕获微球震荡混匀后，向所有标准品管及样品管中分别加入25ul，震荡混匀后室温孵育2.5小时。孵育完成后，每管加入1ml PBS溶液洗涤，200xg5min离心，弃上清。每管加入100ul PBS溶液，震荡重悬后流式细胞仪检测IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ的浓度。
10.人Th1和Th2细胞的检测
本实验使用人Th1/Th2细胞检测试剂盒(Mutiscience,Cat.NO.70-KTH101-25)。对取125μl抗凝血至流式管中，加入125μl不含血清的培养基和1μlPMA/Ionomycin/BFA/Monensin Mixture(250x)。取125μl抗凝血和125μl不含血清的培养基作为对照。37℃孵育4-6小时。用含10％胎牛血清的培养基重悬沉淀，使细胞浓度为1x 107/ml。或取250μl PBMC至流式管中，加入1μlPMA/Ionomycin/BFA/Monensin Mixture(250x)。以只含PBMC的样本作为对照。37℃孵育4-6小时。从样本管和对照管中取100μl细胞悬液至新的流式管中，加入5μl FITC anti-Human CD3和5μl PerCP-Cy5.5anti-Human CD8α抗体，室温避光孵育15分钟。每管加入100μlFIX&PERM Medium A，室温孵育15分钟。用蒸馏水将10x Flow CytometryStaining Buffer稀释为1x，每管加入2ml预冷1x FlowCytometry Staining Buffer，300xg离心5分钟，弃上清。每管加入100μl FIX&PERM Medium B、5μl PE anti-human IFN-γ和5μl APC anti-Human IL-4。室温孵育15分钟。每管加入2ml 1x Flow Cytometry StainingBuffer，300xg离心5分钟，弃上清。每管加入500μl 1x Flow Cytometry Staining Buffer重悬，上机检测CD3+CD8-IFN-γ+Th1细胞、CD3+CD8-IL-4+Th2细胞，并用Th1占淋巴细胞比例与Th2占淋巴细胞比例的比值计算Th1/Th2。
11.人Th17细胞的检测
本实验使用人Th17细胞检测试剂盒(Mutiscience,Cat.NO.70-KTH117-25)。取125μl抗凝血至流式管中，加入125μl不含血清的培养基和1μl PMA/IonomycinMixture(250x)和1μl BFA/Monensin Mixture(250x)，37℃孵育4-6小时。用含10％胎牛血清的培养基重悬沉淀，使细胞浓度为1x 107/ml。或取250μlPBMC至流式管中，加入1μlPMA/IonomycinMixture(250x)和1μlBFA/Monensin Mixture(250x)。37℃孵育4-6小时。从样本管和对照管中取100μl细胞悬液至新的流式管中，加入5ul FITC anti-Human CD3和5μl PerCP-Cy5.5anti-Human CD8α。震荡混匀，室温孵育15分钟。每管加入100μl FIX&PERM Medium A，震荡混匀，室温孵育15min。用蒸馏水将10x Flow Cytometry Staining Buffer稀释为1x，每管加入2ml预冷1x Flow Cytometry Staining Buffer，300x g离心5min，弃上清。每管加入100μl FIX&PERM Medium B和5μl PE anti-Human IL-17A。室温孵育15min。每管加入2ml1x Flow Cytometry Staining Buffer，300x g离心5min，弃上清。每管加入500μl 1x FlowCytometry Staining Buffer重悬，流式细胞仪上机检测CD3+CD8-IL-17A+Th17细胞。
12.人调节性T细胞(Treg)的检测
本实验用人调节性T细胞(Treg)检测试剂盒(Mutiscience,70-KTR101-25)。在流式管中加入100μl人抗凝血，加入5μl FITC anti-Human CD4和5μl APCanti-Human CD25涡旋震荡混匀，室温避光孵育15min。每管加入2ml 1x FCMLysing Solution工作液，涡旋震荡混匀，室温避光孵育15min。室温下300-400xg离心5min，弃去上清。每管加入2ml 1x FlowCytometry Staining Buffer，涡旋震荡混匀。室温下300-400x g下离心5min，弃去上清。每管加入1ml 1xFixation/Permeabilization工作液，涡旋震荡混匀，室温孵育30-60分钟。每管加入2ml 1x Permeabilization Buffer，室温下300-400x g离心5min，弃去上清。用100μl 1x Permeabilization Buffer重悬沉淀。加入5μl PE anti-Human Foxp3或5μl RatPEIgG2a Isotype Control，震荡混匀，室温避光孵育至少30min。每管加入2ml 1xPermeabilization Buffer，室温下300-400x g离心5min，弃去上清。每管加入500μl 1xFlow Cytometry Staining Buffer重悬，流式细胞仪检测CD4+CD25+Foxp3+Treg细胞。
13.大鼠调节性T细胞(Treg)的检测
在流式管中加入100μl人抗凝血，加入5μl APC anti-rat CD4抗体(美国Biolegend,Cat.No.201509)和5μl FITC anti-rat CD25抗体(美国Biolegend,Cat.No.202103)涡旋震荡混匀，室温避光孵育15min。每管加入2ml 1x RBCLysis/FixationSolution(美国Biolegend,Cat.No.422401)工作液，涡旋震荡混匀，室温避光孵育15min。室温下300-400x g离心5min，弃去上清。每管加入2ml 1xCell Staining Buffer(Biolegend,Cat.No.：420201)，涡旋震荡混匀。室温下300-400x g下离心5min，弃去上清。然后用Fixation Buffer(Biolegend,Cat.No.：420801)固定细胞，室温孵育固定20min。350xg下离心5min，弃上清。用Cell StainingBuffer(Biolegend,Cat.No.：420201)洗一遍细胞，350xg，离心5min，弃上清。用Permeabilization Wash Buffer(Biolegend,Cat.No.：421002)和True-NuclearTranscription Factor Buffer Set(Biolegend,Cat.No.：424401)重悬固定的细胞，室温避光20min。加入5μl PE anti-rat FOXP3Antibody(美国Biolegend,Cat.No.320007)或5μl PE Rat IgG2a Isotype Control(美国Biolegend,Cat.No.400507)震荡混匀，室温避光孵育至少30min。每管加入2ml 1xPermeabilizationBuffer(Biolegend,Cat.No.：421002)，室温下300-400x g离心5min，弃去上清。每管加入500μl 1x Flow Cytometry Staining Buffer(Biolegend,Cat.No.：420201)重悬，流式细胞仪检测CD4+CD25+Foxp3+Treg细胞。
14.流式分选制备CD38+NK、CD38+NKT细胞和去除CD38+CD16+CD56+细胞的MNC细胞
取RA患者外周血或滑液，分离MNC。200x g下离心5min，吸弃上清，再加入500ulPBS重悬。加入流式抗体Anti-human-CD38-percp(美国Biolegend,Cat.No.：356622)、Anti-human CD3FITC/(CD16+CD56)PE Cocktail(美国Biolegend,Cat.No.：319101)抗体各10ul，4℃孵育30min。加入PBS后，200x g离心10min收集细胞，PBS重悬待用。建立CD38+、CD16+CD56+阳性门及阴性门，在CD38+和CD16+CD56+细胞群中以CD3+/-区分CD38+NK和CD38+NKT细胞，阴性门中细胞群即为去除CD38+CD16+CD56+细胞的MNC细胞。分选后对样本进行分选纯度的检测。
15.分选CD4+T细胞
本实验使用磁珠分选CD4+T细胞试剂盒(Miltenyi Biotec,Cat.No.：130-045-101)。对按上述方法获得的RA滑液单个核细胞(SFMC)300xg离心10min,吸弃上清。1x10^7细胞加入80μl的缓冲液重悬，再加20μl的CD4磁珠，涡旋震荡仪混匀，4℃孵育15min。再加入2ml的缓冲液，300xg离心10min,吸弃上清，用500μl的缓冲液重悬。将LS柱(LS Separationcolumns)放置MACS磁力架，加0.5ml缓冲液润洗3次。将500μl的标本至分选柱上收集阴性细胞，反复3次。将LS柱从MACS磁力架移开，加1ml缓冲液至分选柱，用配备的活塞快速压细胞至阳性收集管。用流式细胞仪测定新分选CD4+T细胞的纯度。
16.CD38+NK、CD38+NKT细胞与CD4+T细胞或去除CD38+CD16+CD56+细胞的MNC细胞共培养
将分选获得的去除CD38+CD16+CD56+细胞的MNC细胞或CD4+T细胞在CD3/CD28磁珠(挪威Life Technologies AS,Cat.NO.11161D)存在的条件下[21]以2x10^6/ml密度种0.4μm Transwell小室下层(Corning 
Cat.NO.3413)。取CD38+NK或CD38+NKT细胞，以2x10^6/ml密度种板。分别加入用CD38mAb(1μg/ml)(TheraMabs,Cat.No.：945721-28-8)或等量牛血清白蛋白(BSA)(SIGMA,Cat.No.：9048-46-8)处理24h后，200xg离心5min去除CD38mAb后，以2x10^6/ml密度种板铺种于小室(0.4μm)上层中。共培养48h后，收集细胞，流式检测MNC或CD4+T中各淋巴细胞亚型。同时收集上清，流式检测细胞因子(IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ)。
17.Real-time PCR检测
从上述实验小室下层收集CD4+T细胞，WB检测mTOR细胞分子。从上述实验小室上层中收集CD38+NK或CD38+NKT细胞，提取RNA，逆转录提取RNA(FOREGENE公司,RE-0301T),将其逆转录为cDNA(Vazyme公司,R223-01)。实时荧光定量PCR(Thermofisher,StepOnePlusTMReal-Time PCR System)检测CD3γ、CD28、TGF-βmRNA水平的表达。
CD3γ引物序列如下：
Forward primers：5‘GCATTTTCGTCCTTGCTGTTGGG3’，
Reverse primers：5‘GGTCATCTTCTCGATCCTTGAGG3’；
CD28引物：
Forward primers：5‘GAGAAGAGCAATGGAACCATTATC’，
Reverse primers：5‘TAGCAAGCCAGGACTCCACCAA3’；
TGF-β引物：
Forward primers：5‘TACCTGAACCCGTGTTGCTCTC’，
Reverse primers：5‘GTTGCTGAGGTATCGCCAGGAA3’。
Western blotting(WB)检测
收集共培养后的CD4+T细胞，每孔细胞分别加入100ul RIPA裂解液(上海beyotime,Cat NO.P10013B)和1ul PMSF混合，冰上裂解30min，4℃离心机12000r/min低温离心15min。BCA试剂盒(上海beyotime,Cat NO.P0009)测定上清液蛋白浓度。加入上样缓冲液(上海beyotime,Cat NO.P0015L)95℃金属浴10min使蛋白变性，于-80℃保存备用。蛋白样品进行SDS-PAGE凝胶(上海beyotime,CatNO.P0012AC)电泳，完成后将蛋白转印至PVDF膜上(中国BOSTER，CatNO.13G26C36)。5％脱脂奶粉封闭1h后，使用mTOR SubstratesAntibody Sampler Kit(Cell Signaling Technology,Cat.No.：9862)检测mTOR信号通路的变化。试剂盒包括mTOR信号转导至下游底物，包括(mTOR、Phospho-mTOR、p70S6激酶和4E-BP1)和二抗，适用于蛋白质印迹实验法(Western blot，WB)检测其靶标蛋白的内源水平。分别用试剂盒中Phospho-mTOR,mTOR,Phospho-p70S6Kinase(Thr389)(Ser371),Phospho-4E-BP1(Thr37/46)抗体按说明书稀释后4℃过夜孵育，TBS洗三次后用试剂盒中anti-rabbitIgG,HRP-linked Antibody室温孵育2h，洗涤后，凝胶成像系统(Bio-Rad伯乐,CatNO.SYSTEM GelDoc XR+)曝光显影，以GAPDH作为内参，用Image J进行灰度值分析。
二、统计学分析
采用SPSS 17.0软件(Biomedical Computer Programs)进行正态和方差齐性的检验。采用单因素方差分析进行多组间显著性检验，两两比较采用LSD法，相关性分析采用pearson相关分析，|r|≥0.8两变量高度相关；0.8>|r|≥0.5两变量中度相关；0.5>|r|≥0.3两变量低度相关；0.3>|r|两变量无相关；检验标准α＝0.05，P,＜0.05为差异有统计学。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 常晓天
<120> CD38抑制剂在制备抗类风湿关节炎药物中的应用
<130> 2010
<160> 6
<170> PatentIn version 3.3
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tacctgaacc cgtgttgctc tc 22
<210> 6
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<212> DNA
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gttgctgagg tatcgccagg aa 22
Claims (1)
1.CD38+NK细胞抑制剂在制备抗类风湿关节炎药物中的应用，其特征在于，所述CD38+NK细胞抑制剂为CD38抗体，CD38抗体为Cas.No.：945721-28-8。
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| PCT/CN2019/127369 WO2020135318A1 (zh) | 2018-12-29 | 2019-12-23 | Cd38抑制剂在制备抗类风湿关节炎药物中的应用 |
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| CN113893247A (zh) * | 2021-11-12 | 2022-01-07 | 青岛大学附属医院 | 78c或其药物组合物在制备抗类风湿性关节炎产品中的应用 |
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| CN107475193A (zh) * | 2017-08-29 | 2017-12-15 | 常晓天 | Padi4刺激的dc‑cik细胞组合物及应用 |
| WO2018078083A1 (en) * | 2016-10-28 | 2018-05-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating multiple myeloma |
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| US20170115275A1 (en) * | 2015-10-23 | 2017-04-27 | Arizona Board Of Regents On Behalf Of Arizona State University | Engineered substrates for high-throughput generation of 3d models of tumor dormancy, relapse and micrometastases for phenotype specific drug discovery and development |
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| WO2018078083A1 (en) * | 2016-10-28 | 2018-05-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating multiple myeloma |
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