CN115261313A - 一种人胎盘间充质干细胞来源的外泌体制备方法及应用 - Google Patents
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Abstract
本发明公开了一种人胎盘间充质干细胞来源的外泌体制备方法及应用,制备方法为:步骤一,人胎盘来源间充质干细胞的原代培养;1,材料获取;2,分离培养;3,换液和传代;4,冻存与复苏;5,细胞表面抗原检测;步骤二,人胎盘间充质干细胞来源外泌体(PMSCs‑exosomes)的提取;1,无外泌体FBS制备:120000g过夜超速离心FBS;2,PMSCs条件培养基的制备;3,外泌体的分离。制备的人胎盘间充质干细胞来源的外泌体可以应用于制备治疗慢性疼痛(炎性疼痛,神经病理性疼痛以及化疗药物所致疼痛)的药物。
Description
技术领域
本发明公开了一种人胎盘间充质干细胞来源的外泌体制备方法及应用,涉及生物与新医药技术领域。
背景技术
间充质干细胞(MSC)是一种可以自我更新并分化成各种细胞谱系的细胞。在过去的几十年中,已经发现来自各种组织的MSCs可以通过其分化和/或分泌功能产生治疗效果。与其他组织相比,人胎盘是一种理想的MSCs储存库,可以通过非侵入性方式获得MSCs,并且很少引起伦理问题。此前有报道称,人胎盘来源的间充质干细胞(hPMSC)移植可以恢复卵巢功能,保护脑缺血再灌注损伤后的脑功能,减轻脊髓损伤等。然而,MSCs移植治疗受到细胞保留率和肿瘤发生潜在风险的限制。更重要的是,许多研究发现其治疗效果主要归因于间充质干细胞的分泌功能,如细胞因子、生长因子和外泌体。
外泌体是细胞外囊泡(EVs)的一种,可通过促进细胞间各种RNA、DNA、细胞因子和生长因子的转移,作为细胞间通讯的一种手段。自2004年在人胎盘中发现hPMSCs并证实其多向分化潜能以来,近年来以hPMSCs及其外泌体为基础的疾病治疗研究逐渐增多。除了hPMSCs可以直接对疾病产生治疗作用外, hPMSCs衍生的外泌体也具有治疗潜力。
慢性疼痛的发生往往由多种因素所介导,包括炎症性疼痛,神经病理性疼痛,癌症相关性疼痛等。炎症是免疫系统消除有害刺激,恢复内环境平衡的重要机制。然而,有害刺激和炎症的持续存在是许多慢性炎症性疾病的基础,包括糖尿病、心血管疾病、关节炎等。在这种情况下,炎症不再是身体的保护性反应,并可能引起以痛觉过敏、异常性疼痛和特发性疼痛为特征的持续性慢性疼痛,并释放多种促炎介质,包括细胞因子、趋化因子、神经生长因子等,导致外周伤害感受器的敏化。神经病理性疼痛(Neuropathic pain, NeuP)是由物理性的机械损伤、代谢或营养性神经改变、病毒感染、药物或放疗的神经毒性、缺血性神经损害、神经递质功能障碍等多种因素引发的慢性疼痛,表现为自发性疼痛、痛觉过敏、异常疼痛和感觉异常等临床特征。2011年国际疼痛研究协会(International Associationfor the Study of Pain,IASP)将神经病理性疼痛(Neuropathic pain,NeuP)定义更新为“由躯体感觉神经系统的损伤或疾病而直接造成的疼痛”,各国流行病学调查显示NeuP发病率在6-10%之间,且发病率呈逐年增加趋势。癌症相关性疼痛包括化疗药物诱导的神经痛,通常是由长春新碱或紫杉醇等化疗药物所介导。慢性疼痛的发病机制复杂,主要包括离子通道功能变化、神经炎症、外周和(或)中枢敏化、上行和下行通路功能失调、脊髓胶质细胞的活化等。目前临床治疗慢性疼痛尚无较好方案,数据表明至少有50%的患者未能获得有效的疼痛缓解,严重影响了患者的生活质量,增加了个人和社会经济负担。综上所述,探寻对于各种慢性疼痛更有效的治疗药物和方案具有重要的临床和社会意义。
据目前的相关报道,MSCs衍生的外泌体在慢性疼痛模型中表现出有希望的治疗潜力。但是,hPMSCs 来源的外泌体是否能缓解包括炎症性疼痛,神经病理性疼痛,癌症相关性疼痛等在内的慢性疼痛及其可能机制尚未见报道。
发明内容
本发明旨在提供一种人胎盘间充质干细胞来源的外泌体制备方法,探讨hPMSCs-exosomes对不同类型慢性疼痛(炎性疼痛,神经病理性疼痛,化疗药物所致疼痛)的治疗作用,实现本发明专利的核心用途:鞘内注射hPMSCs-exosomes能够通过其内容物的免疫调节和抗炎作用产生缓解/治疗不同类型慢性疼痛的作用,具体包括:1)明确hPMSCs-exosomes缓解/治疗不同类型慢性疼痛的作用;2)明确hPMSCs-exosomes 缓解/治疗不同类型慢性疼痛所导致的炎症反应,为后续开发生物治疗药物奠定基础。
与普通药物治疗相比,人胎盘间充质干细胞衍生的外泌体具有以下优点:1)镇痛效果明显且持久;2) 易于获取与制备;3)无明显不良反应;4)蛋白质、活性因子和生物活性物质含量高、活性强;5)更灵活,更易于生物改造。
而我们的胎盘间充质干细胞衍生的外泌体和其他间充质干细胞外泌体相比,可以通过非侵入的方式获取,更简易。此外,也面临更少的伦理问题。与单独miRNA治疗疼痛相比,通过外泌体中包含多种活性物质,这些物质如蛋白质、非编码RNA如miRNA均可能产生缓解疼痛、抗炎等作用;其次以外泌体为载体给药,可以显著提高生物吸收率,减少不良反应,提高给药效率。
具体技术方案:
1.1研究方法和实验方案
1.1.1人胎盘来源间充质干细胞的原代培养
(1)材料获取:获得医院伦理委员会批准,与健康产妇及其家属签署知情同意书后,于胎盘娩出后立即取5mL胎盘后置于冰盒内,避免组织与冰袋的直接接触,1h内转移至实验室,准备实验。
(2)分离培养
1)剪取胎儿侧绒毛膜组织5mL,置于10cm培养皿,皿中装有2/3体积含有1%双抗的PBS。
2)除去取得胎盘组织的羊膜部分,用组织剪将较大组织剪成小块,并用手术镊夹持清洗,避免溢出,如此反复3遍,至漂洗液清亮,弃除漂洗液。
3)将漂洗好的绒毛膜组织剪细至5mm3左右,转移绒毛膜组织至50mL体积的离心管中。
4)向各离心管中加入II型胶原酶消化液,各加入等体积II型胶原酶(酶消化终浓度0.1%),置于摇床内37℃、250rpm震荡,约60min后终止消化。(置于37℃孵箱/水浴箱内,每5min轻轻震荡离心管,使其消化充分)
5)加完全培养基至50mL,300rpm、5min离心,过70μm细胞筛收集上清。
6)分为20mL/管,加完全培养基至50mL,2000rpm、10min离心,弃上清。
7)10mL完全培养基重悬细胞,将两管合并混匀。加完全培养基至50mL,接种至5皿10cm直径培养皿。置于37℃,5%CO2培养箱中培养。
(3)换液和传代:每3天换液一次。细胞生长至80%时,每皿加入1.0-2.0mL的0.25%胰酶(含EDTA) 进行消化传代。传代时,消化三至四分钟,加含有血清的细胞培养液终止消化,将细胞悬液转移至50mL 离心管中,并补加完全培养基至50mL,1200rpm,离心5min,弃含消化液的上清,加入细胞完全培养液吹打混匀,计数,按照约1.5×106/皿接种于直径10cm的细胞培养皿中,置于37℃,5%CO2培养箱中培养。
(4)冻存与复苏
本研究采用冷冻保护液:5%二甲基亚砜(dimethylsulfoxide,DMSO)+50%标准胎牛血清+45% DMEM/F12。
复苏培养:解冻时从液氮罐中取出冷冻管,将其放入37℃水浴锅迅速融化约1~2min(或放入37℃、 5%CO2、饱和湿度的培养箱中使其融化)。将冻存管中液体加入有培养基的离心管中,1500rpm离心5min,去掉上清,接种到新的培养基皿中,于37℃、5%CO2、饱和湿度条件下培养,第2天更换培养液。
(5)细胞表面抗原检测
根据ISCT(国际细胞治疗协会)间充质干细胞委员会制定的标准,流式细胞仪分析鉴定MSC应细胞表达黏附分子和基质细胞标记CD73、CD90、CD105,整合蛋白家族CD29,以及透明质酸盐受体CD44,低表达造血细胞标记CD11b、CD34、CD45和HLA-DR。
1.1.2人胎盘间充质干细胞来源外泌体(PMSCs-exosomes)的提取与鉴定
(1)PMSCs-exosomes的提取:本研究采用国际通用的超高速离心法进行提取,结合已发表的 PMSCs-exosomes文献提取方法:
1)无外泌体FBS制备:120000g过夜超速离心FBS
2)PMSCs条件培养基(PMSCs-condition medium,PMSCs-CM)的制备:培养传代至P3的PMSCs 于10cm培养皿中,待细胞增殖融合至80%,按照1:5进行传代;P4细胞融合至对数生长期时,弃培养基,无外泌体PBS漂洗两次,加入无FBS的DMEM/F12培养基6mL/皿,24h后收取培养基于离心管中,该培养基即为PMSCs-CM,并计数5个培养皿中的细胞总数。
3)外泌体的分离:
①预冷低温离心机,超速离心机至4℃,300g,4℃离心PMSCs-CM 10min,除细胞碎片,将离心后上清液移植超速离心管中,用PBS补齐;
②2000g,4℃离心20min,进一步去除细胞及碎片,0.22μm滤器过滤离心后上清,去除大于200 nm的粒子;
③过滤后上清于另一超速离心管中,120000g,4℃离心90min;
④弃上清,无菌滤纸吸净管壁液体,管底即为PMSCs-exosomes,加入相应液体,-80℃冰箱保存待用;
(2)PMSCs-exosomes的鉴定:主要包括电镜、Westernblot、粒径分析。
1)透射电镜鉴定PMSCs-exosomes形态的实验:
①50μL PBS溶解超速离心后exosomes
②取20-30μL exosomes悬液于载体铜网上,室温静置1min
③滤纸沿铜网外侧吸干液体
④铜网上滴加20ml/L磷钨酸溶液约30μL,室温负染1min
⑤滤纸吸干磷钨酸溶液,铜网放置白炽灯下烤干约10min
⑥透射电镜下观察并采集照片
2)Western blot鉴定PMSCs-exosomes表面标志物实验
①exosomes蛋白的提取:50μL蛋白裂解液(RIPA:PMSF=100μL:1μL)加入超速离心后获得的 exosomes中,涡旋裂解3次,每次5min,13000g,室温离心5min,吸取上清于新的EP管中,使用 BCA蛋白浓度测定试剂盒进行蛋白浓度测定后,取20-25μg exosomes的蛋白于上样缓冲液中,煮沸 5min,进行蛋白变性
②SDS-PAGE电泳:配置12%分离胶及5%浓缩胶,加入电泳液及样品后调节电泳条件为80V,30min,120V,2h进行电泳。
③转膜:切胶放于转膜缓冲液中,将0.22μm PVDF膜浸泡于甲醇中活化15s,铺“三明治”结构,即3层滤纸+膜+胶+3层滤纸,其中膜面积>胶>滤纸,注意赶走气泡,调节电压100V转膜100min。
④封闭和一抗孵育:将膜取出放于封闭液中,37℃摇床1h,一抗稀释液配制抗体CD9、CD63、 TSG101及ALIX,4℃摇床过夜。
⑤二抗孵育及显色曝光:一抗孵育过夜后取膜放于PBST中洗3次,每次5min,随后加入辣根酶标记山羊抗兔Ig G(1:10000),37℃摇床1h;接着取膜放于PBST中洗3次,每次5min,使用ECL 化学发光法进行曝光。
3)纳米追踪分析(Nano Tracking Analysis)检测外泌体密度分布
使用N4 Plus纳米颗粒粒度分析仪(Beckmen)检测。将冻存于-80℃的外泌体于4℃冰箱解冻;然后用PBS冲洗仪器连接管,抽取100μL外泌体溶液缓慢注射入连接管,仪器检测后可通过纳米微粒的布朗运动成像来检测其密度分布。
1.1.3不同类型疼痛模型的构建
1)炎症性小鼠疼痛:在用七氟醚(2%~5%)麻醉小鼠后,通过向左足底皮下注射CFA(20μL,Sigma, St.Louis,MO)在小鼠中建立炎症性疼痛。假手术组小鼠左爪皮下注射生理盐水20μL。
2)神经病理性疼痛小鼠模型:小鼠用七氟醚(2%~5%)麻醉后,分离大腿附近的坐骨神经,并暴露其分支:腓肠神经、腓总神经和胫神经。腓总神经和胫神经在三叉处用4-0细丝紧紧结扎,然后在丝结的远端切断,然后去除远端神经末端3~5mm。腓肠神经在手术过程中小心地保持其完好无损。假手术组小鼠只用同样的方法分离和暴露坐骨神经,不结扎和切断其分支。
3)癌症相关疼痛(化疗药导致的疼痛)小鼠模型:在用七氟醚(2%~5%)麻醉小鼠后,通过连续5 天向腹腔注射Paclitaxel(2mg/kg/day)在小鼠中建立紫杉醇所致周围神经性疼痛模型。假手术组小鼠腹腔注射同等体积生理盐水。
1.1.4行为学检测
机械缩足反射阈值(paw withdrawal mechanical threshold,PWMT)测定:小鼠在机械性痛觉测试架上适应3天,适应时禁食、禁水,室温在23±1℃,在上午9:00~下午17:00进行。适应3天后,用von Frey 进行小鼠爪底机械感受阈值测试,测量时以von Frey弯曲45°作为完全受力的标准,用up and down检测方法得到每只小鼠的PWMT,单位为g。基础缩爪阈值一般控制在2.0g左右。缩爪阈值越低说明机械性触诱发痛越严重。
1.1.5 PMSCs-exosomes治疗后缓解慢性疼痛的作用机制研究
(1)结合既往文献结论和前期结果,不同类型慢性疼痛给药方案如下:
炎症性疼痛:小鼠造模后6~8h鞘内注射外泌体,在第1,3,5,7天进行机械缩足反射阈值和热潜伏期的测量。
神经病理学疼痛:小鼠在建模后7天能够达到一个比较稳定的平台期,据此设计的给药方式和时间点如下,并在术后7、8、10、12、14、21、28、35天进行机械缩足反射阈值的测量。
癌症相关性疼痛:在紫杉醇连续5天腹腔注射后一天鞘内注射外泌体,并在10天内进行机械缩足反射阈值的测量。
(2)在鞘内给药后1天处死小鼠并取背根神经节,应用ELISA检测炎症因子的变化。
本发明的有益效果:1)通过明确hPMSCs-exosomes缓解/治疗慢性疼痛(包括炎症性疼痛,神经病理性疼痛,癌症相关疼痛)的作用,可以预见慢性疼痛的生物治疗方法;2)通过对胎盘间充质干细胞来源的外泌体通过抗炎缓解慢性疼痛的作用机制研究,明确了胎盘间充质干细胞来源的外泌体通过抗炎缓解慢性疼痛方面的应用。
附图说明
图1为实施例2中(1)小鼠在术后7、8、10、12、14、21、28、35天进行机械缩足反射阈值的测量;
图2为实施例2中(1)小鼠造模后6-8h鞘内注射外泌体,在第1,3,5,7天进行机械缩足反射阈值和热潜伏期的测量;
图3为实施例2中(1)紫杉醇连续5天腹腔注射,在注射第一天预先鞘内注射外泌体,并在10天内进行机械缩足反射阈值的测量;
图4为实施例2中PMSCs-exosomes显著缓解小鼠SNI神经病理性疼痛模型机械敏感阈值;
图5为实施例2中PMSCs-exosomes显著缓解小鼠CFA致炎症性疼痛模型机械敏感阈值和热痛潜伏期;
图6为实施例2中PMSCs-exosomes显著缓解紫杉醇所致小鼠周围神经痛模型机械敏感阈值;
图7为实施例2中PMSCs-exosomes显著降低SNI小鼠背根神经元的TNF-α和IL-1β水平,*表示与其他各组相比,P<0.05;
图8为实施例2中PMSCs-exosomes显著降低CFA所致炎性疼痛小鼠背根神经元的TNF-α和IL-1β水平,**表示与CFA组相比,P<0.01;
图9为实施例2中PMSCs-exosomes显著降低紫杉醇所致周围神经性疼痛小鼠背根神经元的TNF-α和 IL-1β水平,**表示与Pacl组相比,P<0.01。
具体实施方式
下面结合具体实施方式,对本发明的权利要求作进一步的详细说明,但不构成对本发明的任何限制,任何人在本发明权利要求保护范围之内所做的有限次的修改,仍然在本发明的权利要求保护范围之内。
试验材料的来源:获得医院伦理委员会批准,与健康产妇及其家属签署知情同意书后,于胎盘娩出后立即取5ml胎盘后置于冰盒内,避免组织与冰袋的直接接触,1h内转移至实验室,准备实验。
实施例1
1.1人胎盘来源间充质干细胞的原代培养
(1)材料获取:获得医院伦理委员会批准,与健康产妇及其家属签署知情同意书后,于胎盘娩出后立即取5ml胎盘后置于冰盒内,避免组织与冰袋的直接接触,1h内转移至实验室,准备实验。
(2)分离培养
1)剪取胎儿侧绒毛膜组织5mL,置于10cm培养皿,皿中装有2/3体积含有1%双抗的PBS。
2)除去取得胎盘组织的羊膜部分,用组织剪将较大组织剪成小块,并用手术镊夹持清洗,避免溢出,如此反复3遍,至漂洗液清亮,弃除漂洗液。
3)将漂洗好的绒毛膜组织剪细至5mm3左右,转移绒毛膜组织至50mL体积的离心管中。
4)向各离心管中加入II型胶原酶消化液,各加入等体积II型胶原酶(酶消化终浓度0.1%),置于摇床内37℃、250rpm震荡,约60min后终止消化。(置于37℃孵箱/水浴箱内,每5min轻轻震荡离心管,使其消化充分)
5)加完全培养基至50mL,300rpm、5min离心,过70μm细胞筛收集上清。
6)分为20mL/管,加完全培养基至50mL,2000rpm、10min离心,弃上清。
7)10mL完全培养基重悬细胞,将两管合并混匀。加完全培养基至50mL,接种至5皿10cm直径培养皿。置于37℃,5%CO2培养箱中培养。
(3)换液和传代:每3天换液一次。细胞生长至80%时,每皿加入1.0-2.0mL的0.25%胰酶(含EDTA) 进行消化传代。传代时,消化三至四分钟,加含有血清的细胞培养液终止消化,将细胞悬液转移至50mL 离心管中,并补加完全培养基至50mL,1200rpm,离心5min,弃含消化液的上清,加入细胞完全培养液吹打混匀,计数,按照约1.5×106/皿接种于直径10cm的细胞培养皿中,置于37℃,5%CO2培养箱中培养。
(4)冻存与复苏
本研究采用冷冻保护液:5%二甲基亚砜(dimethylsulfoxide,DMSO)+50%标准胎牛血清+45% DMEM/F12。
复苏培养:解冻时从液氮罐中取出冷冻管,将其放入37℃水浴锅迅速融化约1~2min(或放入37℃、 5%CO2、饱和湿度的培养箱中使其融化)。将冻存管中液体加入有培养基的离心管中,1500rpm离心5min,去掉上清,接种到新的培养基皿中,于37℃、5%CO2、饱和湿度条件下培养,第2天更换培养液。
(5)细胞表面抗原检测
根据ISCT(国际细胞治疗协会)间充质干细胞委员会制定的标准,流式细胞仪分析鉴定MSC细胞表达黏附分子和基质细胞标记CD73、CD90、CD105,整合蛋白家族CD29,以及透明质酸盐受体CD44,低表达造血细胞标记CD11b、CD34、CD45和HLA-DR。
1.1.2人胎盘间充质干细胞来源外泌体(PMSCs-exosomes)的提取与鉴定
(1)PMSCs-exosomes的提取:本研究采用国际通用的超高速离心法进行提取,结合已发表的 PMSCs-exosomes文献提取方法:
1)无外泌体FBS制备:120000g过夜超速离心FBS
2)PMSCs条件培养基(PMSCs-condition medium,PMSCs-CM)的制备:培养传代至P3的PMSCs 于10cm培养皿中,待细胞增殖融合至80%,按照1:5进行传代;P4细胞融合至对数生长期时,弃培养基,无外泌体PBS漂洗两次,加入无FBS的DMEM/F12培养基6mL/皿,24h后收取培养基于离心管中,该培养基即为PMSCs-CM,并计数5个培养皿中的细胞总数。
3)外泌体的分离:
①预冷低温离心机,超速离心机至4℃,300g,4℃离心PMSCs-CM 10min,除细胞碎片,将离心后上清液移植超速离心管中,用PBS补齐;
②2000g,4℃离心20min,进一步去除细胞及碎片,0.22μm滤器过滤离心后上清,去除大于200nm 的粒子;
③过滤后上清于另一超速离心管中,120000g,4℃离心90min;
④弃上清,无菌滤纸吸净管壁液体,管底即为PMSCs-exosomes,加入相应液体,-80℃冰箱保存待用;
(2)PMSCs-exosomes的鉴定:主要包括电镜、Western blot、粒径分析。
1)透射电镜鉴定PMSCs-exosomes形态的实验:
①50μL PBS溶解超速离心后exosomes
②取20-30μL exosomes悬液于载体铜网上,室温静置1min
③滤纸沿铜网外侧吸干液体
④铜网上滴加20ml/L磷钨酸溶液约30μL,室温负染1min
⑤滤纸吸干磷钨酸溶液,铜网放置白炽灯下烤干约10min
⑥透射电镜下观察并采集照片
2)Western blot鉴定PMSCs-exosomes表面标志物实验
①exosomes蛋白的提取:50μL蛋白裂解液(RIPA:PMSF=100μL:1μL)加入超速离心后获得的 exosomes中,涡旋裂解3次每次5min,13000g,室温离心5min,吸取上清于新的EP管中,使用BCA 蛋白浓度测定试剂盒进行蛋白浓度测定后,取20-25μg exosomes的蛋白于上样缓冲液中,煮沸5min,进行蛋白变性。
②SDS-PAGE电泳:配置12%分离胶及5%浓缩胶,加入电泳液及样品后调节电泳条件为80V, 30min,120V,2h进行电泳。
③转膜:切胶放于转膜缓冲液中,将0.22μm PVDF膜浸泡于甲醇中活化15s,铺“三明治”结构,即3层滤纸+膜+胶+3层滤纸,其中膜面积>胶>滤纸,注意赶走气泡,调节电压100V转膜100min。
④封闭和一抗孵育:将膜取出放于封闭液中,37℃摇床1h,一抗稀释液配制抗体CD9、CD63、 TSG101及ALIX,4℃摇床过夜。
⑤二抗孵育及显色曝光:一抗孵育过夜后取膜放于PBST中洗3次,每次5min,随后加入辣根酶标记山羊抗兔IgG(1:10000),37℃摇床1h;接着取膜放于PBST中洗3次,每次5min,使用ECL 化学发光法进行曝光。
3)纳米追踪分析(Nano Tracking Analysis)检测外泌体密度分布
使用N4 Plus纳米颗粒粒度分析仪(Beckmen)检测。将冻存于-80℃的外泌体于4℃冰箱解冻;然后用PBS冲洗仪器连接管,抽取100μL外泌体溶液缓慢注射入连接管,仪器检测后可通过纳米微粒的布朗运动成像来检测其密度分布。
实施例2
本发明的人胎盘间充质干细胞来源外泌体在治疗不同类型慢性疼痛的应用研究
(1)不同类型慢性疼痛给药方案如下:
神经病理性疼痛:小鼠在建模后7天能够达到一个比较稳定的平台期,据此设计的给药方式和时间点如下,并在术后7、8、10、12、14、21、28、35天进行机械缩足反射阈值的测量(参见附图1)。
炎症性疼痛:小鼠造模后6-8h鞘内注射外泌体,在第1,3,5,7天进行机械缩足反射阈值和热潜伏期的测量(参见附图2)。
癌症相关性疼痛:紫杉醇连续5天腹腔注射,在注射第一天预先鞘内注射外泌体,并在10天内进行机械缩足反射阈值的测量(参见附图3)。
(2)CFA造模小鼠和SNI小鼠在鞘内给药后1天处死小鼠并取背根神经节,紫杉醇所致神经性疼痛小鼠在第8天处死并取背根神经节,并应用ELISA检测炎症因子的变化。
PMSCs-exosomes鞘内注射能够缓解不同类型慢性疼痛模型小鼠的伤害性感觉
1)神经病理性疼痛
我们采用部分神经损伤(SNI)模型来模拟神经损伤引起的神经性疼痛。然后,我们评估了不同时间点模型小鼠的机械刺激缩足阈值(图4)并观察到小鼠损伤侧后爪的阈值明显降低,且在6-7天达到最小阈值。在SNI术后的第7天,给予小鼠0.5μg/μl浓度的hPMSCs衍生外泌体单次鞘内注射,剂量为10μL。与既往的研究不同,我们没有观察到给予外泌体早期对神经病理性疼痛的缓解作用(注射后6小时内)。然而,我们观察到与SNI和盐水组相比,鞘内注射外泌体24小时后机械阈值显著增加。更令人振奋的是,来自hPMSCs的外泌体在单次鞘内注射后可产生持久的镇痛作用,与SNI组相比直到35天仍有显著差异。
PMSCs-exosomes显著缓解小鼠SNI神经病理性疼痛模型机械敏感阈值(参见附图4)
Baseline:术前1天基础阈值;SNI 7d:术后七天机械敏感阈值;*,与其他各组相比,P<0.05,**,与其他各组相比,P<0.01。
2)炎症性疼痛
我们采用CFA脚掌皮下注射来模拟炎症引起的慢性疼痛。在用七氟醚(2%-5%)麻醉小鼠后,通过向左足底皮下注射CFA(20μL,Sigma,St.Louis,MO)在小鼠中建立炎症性疼痛。假手术组小鼠左爪皮下注射生理盐水20μL。然后,我们评估了不同时间点CFA炎性疼痛小鼠的机械痛阈值和热潜伏期(图4),我们看到在鞘内注射0.5μg/μl浓度外泌体后24h,炎性疼痛小鼠的机械痛阈和热潜伏期较Sham组都没有出现明显的下降,于炎性痛组相比则有显著提高,而且这种效果持续至少第七天。
PMSCs-exosomes显著缓解小鼠CFA致炎症性疼痛模型机械敏感阈值和热痛潜伏期(参见附图5)
Baseline:术前1天基础阈值;D1,3,5,7为分别给予溶媒和外泌体鞘内注射后1,3,5,7天后的机械敏感阈值。**,与其他各组相比,P<0.01。
3)癌症相关性疼痛(化疗药导致的疼痛)
我们对小鼠以2mg/kg/day的剂量连续5天腹腔注射紫杉醇以引起小鼠周围神经病理性疼痛。然后,我们评估了不同时间点模型小鼠的机械刺激缩足阈值(图5)并观察到小鼠在连续腹腔注射紫杉醇后,后爪机械阈值明显降低,且在第8天达到最小阈值。我们在第1天给予小鼠0.5μg/μl浓度的hPMSCs衍生外泌体单次鞘内注射,剂量为10μL。我们观察到鞘内注射外泌体组和Pacl组相比,鞘内注射外泌体后小鼠的机械阈值在后续的第2,5,8,10天机械阈值显著增加,具有良好的疼痛缓解作用。
PMSCs-exosomes显著缓解紫杉醇所致小鼠周围神经痛模型机械敏感阈值(参见附图6)
Baseline(BL):术前1天基础阈值;*,与其他各组相比,P<0.05,**,与其他各组相比,P<0.01。
4)PMSCs-exosomes鞘内注射降低不同类型慢性疼痛模型小鼠脊髓的炎症因子水平
为了明确外泌体是否有减轻神经炎症的作用,我们对炎症性疼痛小鼠,神经病理性疼痛小鼠,紫杉醇导致的神经性疼痛小鼠在PMSCs-exosomes注射后取背根神经节进行Elisa检测炎症因子表达情况,可见炎症因子TNF-α和IL-1β都出现了明显下调(参见附图7-9)。
Claims (2)
1.一种人胎盘间充质干细胞来源的外泌体制备方法,其特征在于,按照下述方法制备,具体步骤为:
步骤一:人胎盘来源间充质干细胞的原代培养
(1)材料获取:获得医院伦理委员会批准,与健康产妇及其家属签署知情同意书后,于胎盘娩出后立即取5ml胎盘后置于冰盒内,避免组织与冰袋的直接接触,1h内转移至实验室,准备实验;
(2)分离培养:剪取胎儿侧绒毛膜组织,置于培养皿,皿中装有2/3体积含有1%双抗的PBS;除去组织的羊膜部分,将组织剪成小块,反复清洗,至漂洗液清亮;将漂洗好的绒毛膜组织剪细至5mm3左右,转移绒毛膜组织至50mL体积的离心管中;向各离心管中加入II型胶原酶消化液,消化约50~70min终止;加完全培养基至50mL,300rpm、5min离心,过70μm细胞筛收集上清;分为20mL/管,加完全培养基至50mL,2000rpm、10min离心,弃上清;10mL完全培养基重悬细胞,将两管合并混匀;加完全培养基至50mL,接种至5皿直径10cm的培养皿;置于37℃,5%CO2培养箱中培养;
(3)换液和传代:每3天换液一次;细胞生长至80%时,每皿加入1.0~2.0mL的0.25%胰酶进行消化传代;
传代时,消化3~4min,加含有血清的细胞培养液终止消化,将细胞悬液转移至50mL离心管中,并补加完全培养基至50mL,1200rpm,离心5min,弃含消化液的上清,加入细胞完全培养液吹打混匀,计数,按照约1.5×106/皿接种于细胞培养皿中,置于37℃,5%CO2培养箱中培养;
(4)冻存与复苏:采用冷冻保护液:5%二甲基亚砜+50%标准胎牛血清+45%DMEM/F12;复苏培养:解冻时从液氮罐中取出冷冻管,将其放入37℃水浴锅迅速融化约1~2min或放入37℃、5%CO2、饱和湿度的培养箱中使其融化;将冻存管中液体加入有培养基的离心管中,1500rpm离心5min,去掉上清,接种到新的培养基皿中,于37℃、5%CO2、饱和湿度条件下培养,第2天更换培养液;
(5)细胞表面抗原检测:流式细胞仪分析鉴定MSC应细胞表达黏附分子和基质细胞标记CD73、CD90、CD105,整合蛋白家族CD29,以及透明质酸盐受体CD44,低表达造血细胞标记CD11b、CD34、CD45和HLA-DR;
步骤二:人胎盘间充质干细胞来源外泌体(PMSCs-exosomes)的提取
(1)无外泌体FBS制备:120000g过夜超速离心FBS;
(2)PMSCs条件培养基的制备:培养传代至P3的PMSCs于10cm培养皿中,待细胞增殖融合至80%,按照1:5进行传代;P4细胞融合至对数生长期时,弃培养基,无外泌体PBS漂洗两次,加入无FBS的DMEM/F12培养基6mL/皿,24h后收取培养基于离心管中,该培养基即为PMSCs-CM,并计数5个培养皿中的细胞总数;
(3)外泌体的分离:预冷低温离心机,超速离心机至4℃,300g,4℃离心PMSCs-CM10min,除细胞碎片,将离心后上清液移植超速离心管中,用PBS补齐;2000g,4℃离心20min,进一步去除细胞及碎片,0.22μm滤器过滤离心后上清,去除大于200nm的粒子;过滤后上清于另一超速离心管中,120000g,4℃离心90min;弃上清,无菌滤纸吸净管壁液体,管底即为PMSCs-exosomes,加入相应液体,-80℃冰箱保存待用。
2.一种人胎盘间充质干细胞来源的外泌体在制备具有治疗慢性疼痛药物中的应用,其特征在于,所述慢性疼痛为炎性疼痛,神经病理性疼痛,化疗药物所致疼痛一种或多种。
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