CN116531396A - 2'-脱氧肌苷在治疗自身免疫性脑炎中的应用 - Google Patents
2'-脱氧肌苷在治疗自身免疫性脑炎中的应用 Download PDFInfo
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Abstract
2'‑脱氧肌苷在治疗自身免疫性脑脊髓炎中的应用,2'‑脱氧肌苷的结构式如下:本发明证明了2'‑脱氧肌苷(DI)抑制淋巴细胞增殖和炎症相关因子的表达。
Description
技术领域
本发明属于生物医药技术领域,具体涉及2′-脱氧肌苷在治疗自身免疫性脑炎中的应用。
背景技术
多发性硬化(multiple sclerosis,MS)是一种中枢神经系统(central nervoussystem,CNS)的慢性炎症性自身免疫病,主要病理特点为CNS内多发的髓鞘脱失,轴突变性和神经元损伤。MS好发于20-40岁女性,其反复发作、逐渐进展导致神经功能的进行性损害,造成患者躯体残疾,认知功能下降,严重影响患者的生活质量。自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是MS的经典动物模型,与人类MS的临床特点及病理特征相似,被广泛应用于MS的基础研究。MS的病因及发病机制虽然尚未明确,但细胞免疫及体液免疫应答的异常被认为在其中发挥重要作用。目前,MS缓解期的治疗以疾病对症治疗(disease modifying therapies,DMTs)为主,然而现有的DMTs药物仅对部分患者有效,且可能产生严重的副作用。因此,积极探寻MS新的免疫调节方法具有重要科研价值。
发明内容
本发明的目的在于提供一种2′-脱氧肌苷在治疗自身免疫性脑脊髓炎中的应用。
为实现上述目的及其他相关目的,本发明提供的技术方案是:2′-脱氧肌苷在治疗自身免疫性脑脊髓炎中的应用。
优选的技术方案为:2′-脱氧肌苷的结构式如下:
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种治疗自身免疫性脑脊髓炎的药物组合物:包括2′-脱氧肌苷和药学可接受的材料。
由于上述技术方案运用,本发明与现有技术相比具有的优点是:
本发明证明了2′-脱氧肌苷(DI)抑制淋巴细胞增殖和炎症相关因子的表达。
附图说明
图1为EAE小鼠模型的建立流程图。
图2脊髓、脑单个核细胞的分离方法示意图。
图3EAE小鼠体重和临床评分图。
图4小鼠脊髓浸润淋巴细胞比较。
图5小鼠脊髓中淋巴细胞亚群比较。
图6小鼠脊髓淋巴细胞活化和增殖比较。
图7小鼠脊髓Th1/Th17/Treg基因表达变化(Q-PCR)。
图8小鼠脑部浸润淋巴细胞比较。
图9小鼠脑部淋巴细胞活化和增殖比较。
图10脾脏淋巴细胞细胞亚群比较。
图11脾脏淋巴细胞活化比较。
图12小鼠脾脏淋巴细胞基因表达变化(Q-PCR)。
图13小鼠外周血淋巴细胞和单核细胞比较。
图14小鼠外周血T淋巴细胞比较。
图15小鼠外周血CD4+/CD8+/Treg淋巴细胞比较。
图16DI对小鼠脾细胞体外增殖影响的大体图片。
图17DI对小鼠脾细胞体外增殖影响的荧光检测图。
图18DI对小鼠脾细胞细胞因子分泌的影响(QPCR/ck)。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本实施例所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1-18。须知,本说明书所附图式所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整。提供以下实施例以便更好地理解本发明,而非限制本发明。以下实施例中的实验方法如无特殊说明,均为常规方法。下述实施例中所用的实验材料如无特殊说明,均为常规生化试剂商店购买所得。
以下实施例所述试剂或材料若无说明,均为市售。
实施例1:2′-脱氧肌苷在治疗自身免疫性脑脊髓炎中的应用
一、EAE模型的建立步骤如下(如图1所示):
(1)抗原注射:按照为0.1ml:10g的体积比腹腔注射水合氯醛,麻醉小鼠。待小鼠麻醉后,背部剃毛,皮下注射200ul MOG35-55混合液,分四个部位注射,每个部位50ul。
(2)PTX注射:腹腔注射200ng PTX,48h后再次腹腔注射200ng PTX。
(3)体重及评分记录:以最后一次注射PTX作为第0天,从第1天开始对小鼠进行密切观察,记录每天体重,并按照下表对小鼠临床症状进行评分。
造模的小鼠都算模型小鼠(0-5)。
(4)小鼠分组:动物随机分为三组,每组6只,分别为对照组、模型组、2′-脱氧肌苷(以下简称DI)给药组。对照组为正常小鼠,未诱导EAE;模型组和DI给药组均为EAE模型小鼠,其中DI给药组于造模后第1天开始给予DI灌胃,1mg/kg,每天两次,对照组和模型组给予等量无菌水。第20天左右处死动物。
如图3所示,EAE模型小鼠经DI治疗后,体重减轻程度低(如图3的A),临床症状评分低(如图3的B),组内小鼠累计评分低((如图3的C)。
二、石蜡切片制作:
(1)固定:新鲜完整脊髓在PBS中迅速清洗,去除血污,放入4%多聚甲醛中固定。
(2)脱水:固定结束后流水冲洗三次,依次由70%乙醇、80%乙醇、95%乙醇I、95%乙醇II、无水乙醇I、无水乙醇II各浸泡1h,进行脱水。
(3)透明:依次由无水乙醇、二甲苯I、二甲苯II各浸泡10min透明。
(4)浸蜡:60℃融化的石蜡中浸泡1h。
(5)包埋:截取脊髓腰部膨大段放在包埋模具上,摆好位置,滴加石蜡,待石蜡表面凝固后放入冷冻台,过夜。蜡块可4℃长期储存。
(6)切片:包埋好的脊髓放入切片机进行切片,厚度为3um。
三、苏木素-伊红(HE)染色:
(1)脱蜡和水化:60℃烘箱烘片30min,依次放入一下溶液进行处理:二甲苯I10min;二甲苯II 10min;无水乙醇3min;95%乙醇3min;85%乙醇3min;75%乙醇3min;蒸馏水2min。
(2)苏木素染色:苏木素染色5min,蒸馏水冲洗;分化液分化10s,蒸馏水浸洗两次,每次5min。
(3)伊红染色:伊红染色1min。
(4)脱水、透明和封片:依次将切片作如下处理:75%乙醇2s;85%乙醇2s;95%乙醇2s;无水乙醇I 2s;无水乙醇II 1min;二甲苯I1min;二甲苯II 1min;中性树胶封片。
(5)镜检:按照下表评估脊髓炎症浸润程度。
分值 | 症状 |
0 | 无炎症细胞浸润 |
1 | 炎症细胞极少,仅浸润血管及被膜周围 |
2 | 每视野1-10炎症细胞浸润 |
3 | 每视野10-100炎症细胞浸润 |
4 | 每视野100-1000炎症细胞浸润 |
如图4所示,EAE小鼠DI治疗后脊髓病理改变轻,脊髓和大脑中浸润的炎性细胞数目少。模型组小鼠一组用DI治疗,一组用PBS。也可以直接说模型组。
四、小鼠CNS单个核细胞分离培养:
(1)脊髓与大脑分离:剪刀剪开小鼠背部皮肤,取出脊柱,注射器吸取5mlRPMI1640从尾端进针,用力将脊髓从髓腔内推出,与分离的小鼠大脑放入六孔板中。
(2)单个核细胞提取:
a.用剪刀将脊髓和脑子剪成小段,加入1.5mlRPMI1640、100mg/ml胶原酶Ⅳ和100mg/mlDNAaseⅠ,37℃消化45min。
b.加入2%FBS RPMI1640终止消化,充分研磨后经100目尼龙网过滤,500g离心5min;细胞沉淀以7ml RPMI1640悬浮,加入3ml 100% Percoll,迅速颠倒混匀,得到30%Percoll。
c.无菌吸管吸取2ml 70% Percoll,由下向上将30% Percoll顶起,20℃,500g离心30min,升5降0。
d.离心结束吸弃上层脂质层,吸取单个核细胞层转移至新的15ml离心管中,RPMI1640洗两遍,300ul完全培养基悬浮。
(3)淋巴细胞刺激:大脑和脊髓淋巴细胞刺激方法同脾脏。
五、流式细胞分析:
(1)流式细胞仪补偿调节
a.取10只流式管,每管加入100ulPBS,同时在各管中各加一滴混匀后的BD阴性对照补偿微球和抗大鼠/仓鼠Igκ补偿微球,混匀。
b.向管内分别加入1ul来源小鼠/仓鼠的抗鼠FITC、PE、APC、APC cy7、AF700、Percpcy5.5、BV421、BV605、BV500、PE CY7标记的抗体,并做好标记,4℃避光孵育15min。
c.加2mlPBS终止结合,200g离心10min,小心吸弃上清,微球沉淀以500ulPBS重悬混匀。
d.在流式细胞仪上分别运行每个试管,捕获完成后仪器自动调节补偿,调节后即可进行后续标本检测。
(2)Treg亚群检测
a.分别取100ul外周血、脾脏、脊髓、大脑细胞悬液于流式管中,2.5ml PBS洗涤,充分去除胎牛血清。
b.向管内加入含ghost(1:1000)的PBS,300ul/管,吹打混匀,4℃避光染色20min,离心。
c.向管内加入含有FITC-CD3抗体(1:100)、AF700-CD4抗体(1:100)以及Percpcy5.5-CD8抗体(1:100)的PBS,100ul/管,吹打混匀,4℃避光孵育15min,离心。
d.向管内加入1ml新配制的1xFix/Perm工作液,吹打混匀,4℃固定40min。
e.加入2ml 1x Perm/Wash工作液终止反应,离心弃上清,加入含有APC-Foxp3抗体(1:100)和BV421-Ki67抗体(1:100)的1x Perm/Wash工作液,100ul/管,混匀,4℃孵育40min。
f.再次加入2ml1x Perm/Wash工作液终止染色,离心,弃上清,细胞沉淀以200ulPBS悬浮,上机检测。
(3)TH1/Th2/TH17亚群分析
a.收取经刺激的脾脏、脊髓及大脑淋巴细胞,离心,PBS洗涤,去除胎牛血清。
b.除胞内分子替换为BV605-IL4抗体(1:100)、PE-IL17A抗体(1:100)和PE CY7-IFNγ(1:100)抗体外,其余染色步骤同上述Treg亚群。
如图5所示,EAE小鼠治疗后,脊髓中CD3+T细胞,CD4+T细胞和Th17细胞数目下降;CD8+T细胞,Th2细胞和Treg细胞数目上升。
如图6所示,EAE小鼠治疗后,脊髓中增殖和活化的T细胞数目减少。
六、实时荧光定量PCR:
(1)样本制备
分别收集剩余脊髓细胞,PBS洗涤两次后Trizol裂解,提取RNA进行实时荧光定量PCR。
(2)细胞RNA提取
a.向新鲜细胞裂解液中加入200μL三氯甲烷,颠倒混匀,室温静置10min。
b.4℃,12000g离心15min。
c.小心转移上层水相层于新除酶EP管中,加入等体积异丙醇,颠倒混匀,-20℃静置15min。
d.4℃,12000g离心10min。
e.移液器吸弃管内液体,保留管底白色RNA沉淀,并加入1ml 75%无水乙醇进行洗涤(75%无水乙醇以DEPC水进行配置)。
f.4℃,12000g离心5min
g.弃上清,沉淀以20μL DEPC水溶解,检测并记录RNA浓度及纯度。
(3)第一链cDNA的合成
按照HiScript III 1st Strand cDNA Synthesis Kit逆转录试剂盒标准操作流程对RNA进行逆转录。
(4)实时荧光定量PCR
按Taq Pro Universal SYBR qPCR Master Mix荧光定量试剂盒标准操作流程进行实时荧光定量实验。
a.反应体系配置
b.QPCR反应
如图7所示,EAE小鼠治疗后,脊髓中淋巴细胞表达更高水平的Foxp3,低水平低Il-17和ifn-r,与流式检测的结果一致。
如图8所示,EAE小鼠治疗后,大脑中CD3+T细胞,CD4+T细胞和Th17细胞数目下降;Treg细胞数目上升。
如图9所示,EAE小鼠治疗后,大脑中增殖和活化的T细胞数目减少。
七、小鼠脾脏淋巴细胞分离培养
(1)脾脏分离:取血后的小鼠断髓法处死,放入75%酒精中浸泡1min,于超净工作台中取出脾脏,放入无菌培养皿中,并于天平上称重记录。
(2)淋巴细胞提取:
a.在脾脏上放置一片无菌100目尼龙网,加入无血清RPMI 1640培养基,用10ml注射器内芯进行研磨,研磨后的悬液经100目尼龙网过滤,去除大块组织。
b.200g离心5min,收集上清存储于-20℃用于测定细胞因子,细胞沉淀以7ml RPMI1640培养基悬浮,吹打混匀。
c.无菌吸管吸取2.5ml 60%percoll,由下向上将混匀的细胞悬液顶起,20℃,500g离心30min,升5降0。
d.离心结束后吸取中间白膜层细胞,RPMI 1640洗两遍,收集细胞沉淀完全培养基悬浮,调整浓度至1*106/ml。
流式检测同脊髓细胞。
如图10所示,EAE小鼠治疗后,脾脏中CD3+T细胞数目下降;Treg细胞数目上升。
如图11所示,EAE小鼠治疗后,脾脏中活化的T细胞数目减少。
如图12所示,EAE小鼠治疗后,脾脏中淋巴细胞高表达抑制相关指标Foxp3、LAG-3和IL-4;低表达炎症相关指标CXCR3和IL-17。
八、小鼠外周血分离
(1)小鼠血液采集:抓取小鼠,水合氯醛麻醉;确定小鼠心脏位置,以1ml无菌注射器缓慢进针,针管内有回血时停止进针,缓慢抽取血液,抽取的血液分为两份,一份注入真空采血管内,进行免疫细胞测定,一份注入配置的400μl EDTA-Na2抗凝剂内,用于流式细胞检测。
(2)外周血免疫细胞检测:采用血常规仪器测定各组小鼠外周血中淋巴细胞比例和绝对计数。
(3)流式细胞检测:EDTA-Na2抗凝外周血以2ml红细胞裂解液裂解2min,离心,弃上清,PBS洗一次,再次离心,以PBS调整细胞浓度至2*106/ml。
流式检测同脊髓细胞。
如图13、14所示,EAE小鼠外周血淋巴细胞减少。
如图15所示,EAE小鼠治疗后,外周血中单核细胞数目增加,淋巴细胞数量减少,以CD4+T细胞减少为主。
九、MS患者外周血淋巴细胞的分离
(1)提前半小时取出Ficoll分离液,颠倒混匀,恢复室温。
(2)采集新鲜15ml MS患者无菌外周血于肝素抗凝管中,立即分离PBMC。
(3)于超净工作台中将采集的外周血转移至50ml离心管,加入等体积PBS进行稀释。另取一只50ml离心管,加入15ml Ficoll分离液,再用无菌吸管沿管壁缓缓加入上述血液稀释液,至45ml刻度处,切忌分离液液面波动。
(4)室温(18-20℃)500g离心30min,升5降0。根据血液中各组成成分密度的不同,离心后,50ml管内由上而下依次为淡黄色血浆层,单个核细胞白膜层,透明分离液层及最下方的红细胞层。
(5)移液管轻轻吸去上层血浆层,将白膜层转移至新的15ml离心管中,轻轻吹打混匀,同时加入10ml RPMI1640洗涤两次,每次200g离心5min。
(6)离心结束后弃上清,加入完全培养基4ml,轻轻吹打混匀。将细胞转移至6孔板中,每孔2ml,37℃培养箱孵育2h,使巨噬细胞贴壁。
(7)2h后移液枪吸取培养孔内悬浮细胞,即为外周血淋巴细胞。200g离心5min,弃上清,完全培养基调整细胞浓度为1*106/ml,备用。
十、淋巴细胞细胞增殖能力测定
(1)CFTR染色:取上述淋巴细胞悬液,离心弃上清,PBS悬浮,加入CTFR染料至终浓度为1μM,37℃避光染色15min,完全培养基终止染色,200g离心5min;离心的细胞沉淀以完全培养基悬浮,加入2μg/ml anti-CD28抗体,同时调整细胞浓度为1*106/ml。
(2)刺激细胞增殖:移液枪吸弃OKT3包被板内悬液,并以无菌PBS重复洗涤两次,去除所的未结合抗体。洗板结束后,每孔加入100μl淋巴细胞悬液,同时加入不同浓度等体积DI,使DI最终浓度依次为0mM、0.25mM、0.5mM、1mM。37℃孵箱培养48h。
(3)流式检测:收集孔内细胞至流式管中,1000rpm离心5min,PBS悬浮细胞,流式细胞仪检测细胞增殖情况。
十一、细胞因子测定
(1)细胞因子收集:除淋巴细胞染色外其余操作同2.3,收集48h上清,进行细胞因子测定。
(2)流式CBA法测定细胞因子:
a.标准品制备:复溶细胞因子标准品冻干微球,依据1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256、1:512在流式内进行倍比稀释,并设置空白管。
b.捕获微球制备:依据样本数及标准品数计算各种捕获微球体积需要量,涡旋混合捕获微球,离心后在血清增强缓冲液中重悬备用;
c.检测抗体制备:计算所需要PE检测抗体体积,并进行稀释
d.标本反应:分别吸取50μL捕获微球、PE检测试剂、样本/标准品于相应流式管中,室温避光孵育3h。
e.检测与分析:1mL洗涤缓冲液终止结合,离心吸弃上清液后再以300μL洗涤缓冲液重悬微球,上机检测PE荧光强度。以FCAP Array软件绘制标准曲线,计算样本浓度。
QPCR方法同脊髓细胞。
如图18所示,DI抑制淋巴细胞增殖和炎症相关因子的表达。
以上所述者仅为用以解释本发明之较佳实施例,并非企图具以对本发明做任何形式上之限制,是以,凡有在相同之发明精神下所作有关本发明之任何修饰或变更,皆仍应包括在本发明意图保护之范畴。
Claims (3)
1.2'-脱氧肌苷在治疗自身免疫性脑脊髓炎中的应用。
2.根据权利要求1所述的应用,其特征在于:2'-脱氧肌苷的结构式如下:
3.一种治疗自身免疫性脑脊髓炎的药物组合物,其特征在于:包括2'-脱氧肌苷和药学可接受的材料。
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