CN110777113A - 一种用于日本血吸虫感染治疗的间质干细胞处理方法 - Google Patents
一种用于日本血吸虫感染治疗的间质干细胞处理方法 Download PDFInfo
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Abstract
本发明公开了一种用于日本血吸虫感染治疗的间质干细胞处理方法,在MSC的培养基中添加IFN‑γ和LPS,37℃,培养24h,IFN‑γ的终浓度为5~100ng/ml,LPS的终浓度为10~100ng/ml,去除刺激因子,充分洗涤后用于治疗。其优势主要表现在以下方面:1、同目前主要是针对虫体的治疗相比,优化诱导后MSC可用于已感染患者肝脏纤维化治疗,且无明显毒副作用;2、同未诱导MSC相比,优化诱导后MSC可通过明显促进Th1反应,显著减轻血吸虫感染所致肝脏纤维化,获得更好疗效。
Description
技术领域
本发明涉及寄生虫感染治疗技术领域,更具体地,涉及一种用于日本血吸虫感染治疗的间质干细胞处理方法。
背景技术
血吸虫病是由血吸虫感染引起的严重影响人类健康以及社会经济发展的人兽共患寄生虫病,寄生于人体的血吸虫主要有日本血吸虫(Schistosomajaponicum)、曼氏血吸虫(S.mansoni)、埃及血吸虫(S.haematobium)、间插血吸虫(S.intercalatum)、湄公血吸虫(S.mekongi)和马来血吸虫(S.malayensis)。根据世界卫生组织(WHO)统计,全球共有74个国家的流行地区约有2亿人仍受到血吸虫病的影响和威胁。据统计,截至2016年底,我国仍有约5.4万血吸虫病患者,局部地区的防治形势依然严峻。在我国,引起血吸虫病的主要病原体是日本血吸虫,由于目前尚不能完全控制其传播,且晚期血吸虫病所致肝脏纤维化尚无有效方法,因此探讨研究血吸虫病所致肝纤维化的治疗方案依然是待攻克的卫生难题。
血吸虫虫卵在肝脏沉积引起的慢性肝损伤的伤口愈合反应是血吸虫病肝纤维化过程的实质,由此导致肝星状细胞(HSC)激活分泌大量胞外基质(ECM)促进肝纤维化。在感染初期,宿主以Th1反应为主(以IFN-γ,IL-12和NO升高为特征),从而抵御在宿主体内迁移的尾蚴和在肠系膜静脉中未性成熟的成虫,促进急性期生存。但是过度的Th1反应又可致感染鼠体重急剧下降、急性期死亡率明显升高;感染6周后,随着虫卵在组织中沉积,宿主转变为Th2反应为主(以IL-4和IL-13升高为标志),Th2反应为主的免疫反应可抑制过强的炎症反应,但另一方面,也促进肝纤维化形成,导致门脉高压等严重并发症。而慢性期肝脏纤维化临床尚无有效方法。
近发现间质干细胞(mesenchymal stem cell,MSC)可能通过旁分泌作用促进肝细胞再生或抑制肝星形细胞活化增殖而抑制纤维化,因而试用于血吸虫肝纤维化治疗,有一定疗效,但报道较少,效果尚不稳定。实际上MSC最明显的特点还表现在其免疫调节作用上,抑制淋巴细胞增殖或抑制Th1,Th2或Th17分化,具体到不同疾病的炎症环境,其免疫调节作用也不尽一致,从而疗效也不同。那么对于具有鲜明的免疫调节紊乱特征的血吸虫感染患者,MSC究竟发挥怎样的免疫调节,如何才能通过有效的免疫调节作用获得稳定疗效目前尚无系统研究。
因此尚无具体利用MSC治疗日本血吸虫感染的具体方案,制约了日本血吸虫感染的治疗。
发明内容
本发明的目的是为了克服现有技术尚无具体利用MSC治疗日本血吸虫感染的具体方案,制约了日本血吸虫感染的治疗的不足,提供一种用于日本血吸虫感染治疗的间质干细胞处理方法。
本发明的第一个目的是提供了一种处理MSC细胞的方法。
本发明的第二个目的是提供了一种所述方法处理得到的MSC细胞。
本发明的第三个目的是提供了一种所述MSC细胞在制备治疗血吸虫感染病中的制剂中的应用。
本发明的第四个目的是提供了一种所述MSC细胞在制备治疗由于Th2型免疫反应过度和/或紊乱引起的疾病的制剂中的应用。
为了实现上述目的,本发明是通过以下技术方案予以实现的:
首先分离小鼠骨髓MSC,采用不同剂量细胞因子和LPS作用一定时间后,初步比较其免疫调节差异,确定合适剂量和作用时间,将诱导后的MSC通过尾静脉注射血吸虫感染模型,分别比较未治疗感染组、未诱导MSC治疗组以及诱导后MSC治疗组的Th1/Th2分化情况、肝脏纤维化程度、胸腺以及脾脏的结构恢复以及血吸虫负荷指标,从而确定出优化的MSC预处理方案。按本方案处理的MSC较未治疗组显示明显促进Th1分化、改善肝脏纤维化、减轻虫卵负荷;而未诱导MSC治疗组则未见显著疗效,表明该优化MSC预处理方案用于血吸虫肝脏纤维化治疗可获得较好效果。
因此本发明要求保护一种处理MSC细胞的方法,其特征在于,MSC的培养基中添加IFN-γ和LPS,37℃,培养4h~24h,IFN-γ的终浓度为5ng/ml,LPS的终浓度为100ng/ml,去除刺激因子,充分洗涤后。
优选地,MSC的培养基中添加IFN-γ和LPS,37℃,培养24h,IFN-γ的终浓度为5ng/ml,LPS的终浓度为100ng/ml,去除刺激因子,充分洗涤后。
所述方法处理得到的MSC细胞,也属于本发明的保护范围。
本发明还要求保护所述MSC细胞在制备治疗血吸虫感染病中的制剂中的应用。
优选地,血吸虫感染病为血吸虫感染引起的肝纤维化、宿主的Th1型免疫反应、胸腺结构异常、和/或脾脏结构异常中的一种或几种。
本发明还要求保护所述MSC细胞在制备治疗由于Th2型免疫反应过度和/或紊乱引起的疾病的制剂中的应用。
优选地,Th2型免疫反应过度和/或紊乱引起的疾病如过敏性哮喘、过敏性鼻炎、变态反应性支气管肺曲霉菌病、肺囊性纤维化等
所述MSC来源包括小鼠或人骨髓、脂肪、肌肉、脐带血、以及脐带来源间质干细胞,MSC的界定除了符合国际细胞和基因治疗协会关于MSC的最低标准也符合国家卫生健康委员会(卫健委)、国家食药监总局的相关法规。
与现有技术相比,本发明具有如下有益效果:
为目前血吸虫感染主要还是针对虫体治疗而对诱发的肝脏纤维化效果不佳的治疗困境提供了可行方法。此外本方法由于可显著诱导体内Th1反应而对Th2有一定抑制作用,且无明显毒副作用,因此也适于Th2反应为主的免疫紊乱疾病,如过敏性肺炎、哮喘等,因此具有很好的应用前景和推广价值。其优势主要表现在以下方面
1、同目前主要是针对虫体的治疗相比,优化诱导后MSC可用于已感染患者肝脏纤维化治疗,且无明显毒副作用;
2、同未诱导MSC相比,优化诱导后MSC可通过明显促进Th1反应,显著减轻血吸虫感染所致肝脏纤维化,获得更好疗效。
附图说明
图1为流式检测MSC,显示CD11b,CD45均为阴性;CD44,CD106,Sca-1阳性,符合MSC的表型标准。
图2为诱导后的MSC的iNOS的表达和CD4+T细胞的的增值效率。
图3为采用IFN-γ和脂多糖诱导后MSC用于血吸虫感染治疗可显著减轻肝脏纤维化(A),减轻肉芽肿面积(B),降低羟脯胺酸含量(C)。
图4为诱导后MSC可促进血吸虫感染鼠血清表达Th1细胞因子IFN-γ,并抑制Th2细胞因子IL-5。
图5为诱导MSC可促进血吸虫感染鼠胸腺和脾脏结构恢复。
图6为诱导后MSC可减少血吸虫感染鼠的肝脏虫卵沉积。
图7为诱导后的MSC可显著促进IFN-γ表达并抑制IL-5表达,即促进Th1反应并削弱Th2反应。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1 MSC的分离培养
一、实验方法
取5-6W左右小鼠,颈椎脱臼处死,放于体积分数为75%的乙醇中浸泡5min,无菌状态下取出双侧股骨,在冰上仔细去除股骨上附带组织,用注射器吸取含100mL/L FCS的L-DMEM培养基,冲出骨髓腔中细胞,通过200目不锈钢网,收集细胞,红细胞裂解液溶解红细胞2min,离心去上清,用含100m L/L FCS的L-DMEM培养基重悬并调整细胞浓度,3ⅹ104接种于24孔板,置于37℃、体积分数为5%的CO2培养箱中,7d后第1次换液,此后每3d换液1次,待细胞有细胞克隆形成时,消化收集细胞克隆,转移至培养瓶,每3d换液1次,待细胞70%~80%融合时,消化传代,继续培养,细胞传至第5~6代细胞增殖速度稳定后,进行表型鉴定。
二、实验结果
表型鉴定结果见图1。流式检测显示MSC的CD11b、CD45均为阴性;CD44、CD106、Sca-1阳性,符合MSC的表型标准。
实施例2 MSC的处理
一、实验方法
将实施例1培养的MSC中加入IFN-γ(5ng/ml)和LPS(100ng/ml),37℃,作用24h,1500rpm,离心10min,弃培养液,用PBS重悬,离心去除上清,重复洗涤一次,收集细胞备用。
收集诱导后的MSC细胞,提取mRNA,real-time PCR定量检测关键免疫调节因子iNOS的表达,,并进一步通过淋巴细胞增殖抑制实验。
二、实验结果
较低浓度IFN-γ与较低浓度的LPS协同诱导后,可促进MSC iNOS的表达,并增强对淋巴细胞增殖的抑制作用,显示其免疫调节作用增强,如图2所示。
实施例3诱导后的MSC用于血吸虫感染肝脏纤维化治疗
一、治疗方法
1、取血吸虫尾蚴,经皮感染小鼠,建立血吸虫感染模型;
2、MSC治疗
将血吸虫感染模型随机分为三组(每组)血吸虫尾蚴16±2条,不进行任何处理作为空白对照组,另外两组分别于感染2,4周分别给予尾静脉注射实施例2制备的MSC;未经诱导的MSC作为对照组,1×105/每鼠;
3、感染后8周分别取材分析。
1)分别取三组血吸虫尾蚴的肝脏进行HE和masson染色以及羟脯胺酸定量,分析MSC对肝脏肉芽肿和纤维化影响。
2)分别取三组血吸虫尾蚴的血清检测Th1代表细胞因子IFN-γ,TNF,Th2代表细胞因子IL-5水平,综合分析MSC治疗对Th细胞分化的影响;同时取胸腺和脾脏病理。
3)分别取三组血吸虫尾蚴的一定量肝脏组织,计数虫卵含量;从门脉系统和肠系膜取成虫,分析MSC治疗对寄生虫负荷的影响
二、实验结果:
1、采用IFN-γ和脂多糖诱导后MSC用于血吸虫感染治疗可显著减轻肝脏纤维化,减轻肉芽肿面积,降低羟脯胺酸含量(见图3)。
2、优化诱导后MSC可促进血吸虫感染鼠血清IFN-γ显著升高;也促进胸腺和脾脏结构恢复,如图4和图5所示。
3、优化诱导后MSC可减轻肝脏虫卵负荷,如图6所示。
综上所述,实施例2制备的诱导后MSC治疗血吸虫感染,可促进血吸虫感染宿主的Th1反应以及胸腺和脾脏结构恢复,减轻虫卵负荷并显著改善血吸虫所致纤维化。
实施例3优化诱导MSC可显著用于Th2反应过度免疫紊乱疾病治疗
一、实验方法
体外实验,在体外将实施例2制备的诱导后MSC与脾脏淋巴细胞共同加入培养板,并加入抗小鼠CD3/CD28磁珠标记抗体(20ul/106cell),37℃培养48h,收集上清,ELISA检测IFN-γ和IL-5含量。
二、实验结果
如图7所示,实施例2制备的诱导后的MSC可显著促进IFN-γ表达并抑制IL-5表达,即促进Th1反应并削弱Th2反应。并且,实施例2制备的诱导后的MSC无明显相关毒副作用,适用于Th2反应过度紊乱为特征的免疫异常疾病,如过敏性哮喘、过敏性鼻炎等治疗,经过优化方案诱导后可静脉应用。
Claims (7)
1.一种处理MSC细胞的方法,其特征在于,MSC的培养基中添加IFN-γ和LPS,37℃,培养24h,IFN-γ的终浓度为5~100ng/ml,LPS的终浓度为10~100ng/ml,去除刺激因子,充分洗涤后。
2.根据权利要求1所述的方法,其特征在于,MSC的培养基中添加IFN-γ和LPS,37℃,培养24h,IFN-γ的终浓度为5ng/ml,LPS的终浓度为100ng/ml,去除刺激因子,充分洗涤后应用。
3.权利要求1或2所述方法处理得到的MSC细胞。
4.权利要求3所述MSC细胞在制备治疗血吸虫感染病中的制剂中的应用。
5.根据权利要求4所述的应用,其特征在于,血吸虫感染病为血吸虫感染引起的肝纤维化、宿主的Th1型免疫反应、胸腺结构异常、和/或脾脏结构异常中的一种或几种。
6.权利要求3所述MSC细胞在制备治疗由于Th2型免疫反应过度和/或紊乱引起的疾病的制剂中的应用。
7.根据权利要求6所述的应用,其特征在于,Th2型免疫反应过度和/或紊乱引起的疾病为过敏性哮喘、过敏性肺炎、变态反应性支气管肺曲霉菌病、或肺囊性纤维化中的一种或几种。
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