CN117431208B - 一种trim15高表达细胞外囊泡的制备及应用 - Google Patents
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Abstract
本发明公开了一种TRIM15高表达细胞外囊泡的制备及应用,在5% CO2和2%低氧分压条件下培养间充质干细胞至P30代,收集间充质干细胞培养时使用的条件培养基,通过分离纯化制备获得TRIM15高表达细胞外囊泡;本发明提供的细胞外囊泡在治疗过敏性气道炎症中能够抑制肺部炎性细胞浸润,改善肺部气道高反应,并促使CD4+CD25+Foxp3+调节性T细胞增殖,抑制促炎因子(IL‑4和IL‑17)的表达,促进抗炎因子(IL‑10和TGF‑β1)的表达,有效控制Th0向Th2/Th17的极化;体内体外产生良好的免疫调节反应;同时,本发明提供的低氧分压条件下培养的间充质干细胞来源的细胞外囊泡可制备成生物制剂,用于生产过敏性疾病的药物组合物。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种TRIM15高表达细胞外囊泡的制备及应用。
背景技术
过敏性疾病是人类常见的一种免疫性疾病,这类疾病是由过敏原引发的免疫变态反应,敏感个体接触到过敏原后,导致辅助性T淋巴细胞(Helper T cells,Th)向Th2/Th17极化倾斜,其分泌的促炎因子包括白细胞介素-4(Interleukin 4,IL-4)、IL-5和IL-13等水平增高,这些促炎因子通过诱导嗜酸性、中性粒细胞浸润和免疫球蛋白IgE合成发挥致病作用。
最常见的并且研究最充分的过敏性疾病是特应性的,特应性过敏性疾病具有共同的潜在病理生理机制。过敏性疾病由IgE介导,IgE介导的过敏反应包括三个时期:致敏期、激活期和效应期。对过敏原的健康免疫耐受机制是调节性T细胞(Regulatory Tlymphocytes,Treg)起着核心作用,Treg细胞失调会导致过敏性致敏和过敏性疾病;树突状细胞可以捕获抗原,然后迁移到引流淋巴结,在那里它们诱导幼稚T细胞分化为Treg细胞并促进它们归巢到肠道。Treg细胞可通过产生IL-10、转化生长因子-β(transforming growthfactor-β,TGF-β)和IL-35从而抑制Th2依赖性过敏性炎症和减少肥大细胞脱颗粒。
目前治疗过敏性疾病的药物主要为抗组胺药和激素类药物,临床上有较好效果,但长期服用可能具有一定的副作用。另外,脱敏治疗即过敏原特异治疗是一种对因治疗,可以阻断过敏性疾病自然进程;脱敏治疗能够改善传统的激素类化学药物只在疾病发作时对症治疗的问题,但具有治标不治本的局限性。并且,脱敏治疗的目的是让机体产生耐受,这与树突状细胞密切相关。
来源于人自身的间充质干细胞(mesenchymal stem cell,MSC)的细胞外囊泡(extracellular vesicles,EVs)体积小,相对稳定,易透过屏障并具有免疫调节作用,使其成为近年来研究的热点;并且,自身干细胞来源的细胞外囊泡与树突状细胞有着良好的相容性。
发明内容
本发明旨在提供一种人三结构域蛋白15(TRIM15)高表达细胞外囊泡的制备及应用,改善传统的激素类化学药物只在疾病发作时对症治疗,具有治标不治本的局限性,提供一种与树状突细胞具有良好相容性的可用于脱敏治疗的细胞外囊泡;本发明提供的TRIM15高表达细胞外囊泡是指相对于常氧条件下,通过本发明提供的低氧条件下制备得到的细胞外囊泡中的TRIM15蛋白表达量更高。
一方面,本发明提供一种TRIM15高表达细胞外囊泡的制备方法,包括以下步骤:
在5% CO2和2%低氧分压的三气培养箱中进行间充质干细胞的传代培养;
收集所述传代培养至P30代使用的条件培养基;所述条件培养基为包括重组人血白蛋白、血清素、重组人转铁蛋白、重组人胰岛素、乙醇胺、亚硒酸钠、β巯基乙醇、非必需氨基酸、丙氨酰谷氨酰胺、脂质浓缩液、L-抗坏血酸-2-磷酸、孕酮、重组人表皮生长因子、重组人碱性成纤维生长因子、重组人血小板衍生生长因子、骨形态生成蛋白-2、生长分化因子-9、转化生长因子-β1的无血清干细胞培养基;
将所述条件培养基通过分离、纯化,制备获得TRIM15高表达的细胞外囊泡。
可选地,将所述条件培养基通过分离纯化制备获得TRIM15高表达的细胞外囊泡,所述分离纯化包括,超高速离心法、超滤法、空间排阻色谱法、免疫亲和法。
可选地,所述超高速离心法的离心力为100000×g。
第二方面,本发明提供一种所述细胞外囊泡在制备过敏性疾病的药物组合物中的应用。
可选地,所述药物组合物激活了调节性T细胞的增殖,有效控制Th0向Th2/Th17的极化。
可选地,所述药物组合物抑制了促炎因子IL-4和IL-17的表达,促进了抗炎因子IL-10和TGF-β1的表达。
可选地,所述药物组合物用于抑制肺部炎症浸润,降低炎性细胞数量。
可选地,所述药物组合物用于降低肺气道阻力,提高肺顺应性。
本发明具备的有益效果包括:
(1)本发明提供的无血清培养物,制备方法简单、便捷,添加全面,可用于大规模生产;
(2)本发明提供的制备细胞外囊泡的方法,选择机体内干细胞生长的微环境和产生耐受的机制,得到更适合间充质干细胞生长并使细胞外囊泡具有更好的免疫调控功能的培养体系,获得更优秀的干细胞及其来源的细胞外囊泡;并且通过低氧培养制备的细胞外囊泡的TRIM15的表达量显著高于常氧培养的细胞外囊泡;
(3)本发明提供的细胞外囊泡,通过表达TRIM15激活了调节性T细胞的增殖,抑制了促炎因子IL-4和IL-17的表达,促进了抗炎因子IL-10和TGF-β1的表达,有效控制Th0向Th2/Th17的极化,能够达到高效抗过敏作用。
附图说明
图1为实施例2中低氧分压条件下培养的P10、P20、P30代间充质干细胞形态观察结果图;
图2为本发明低氧和常氧制备的P30代间充质干细胞通过CCK-8检测细胞增殖活力图;
图3为本发明常氧条件下制备的P30代间充质干细胞流式检测结果图;
图4为本发明低氧条件下制备的P30代间充质干细胞流式检测结果图;
图5为本发明低氧和常氧制备的P30代间充质干细胞通过β-半乳糖苷酶染色检测衰老水平结果图;
图6为本发明低氧和常氧制备的P30代间充质干细胞干性基因表达水平图;
图7为蛋白印迹检测间充质干细胞来源细胞外囊泡表达标志蛋白水平图;
图8为实施例3制备的间充质干细胞来源的细胞外囊泡TRIM15基因表达;
图9为实施例3制备的间充质干细胞来源的细胞外囊泡对调节性T细胞增殖的流式结果图;
图10为实施例3制备的间充质干细胞来源的细胞外囊泡治疗AAI小鼠后肺泡灌洗液中免疫细胞分析结果图;
图11为实施例3制备的间充质干细胞来源的细胞外囊泡治疗AAI小鼠后肺组织H&E病理染色与结果统计图;
图12为实施例3制备的间充质干细胞来源的细胞外囊泡治疗AAI小鼠后气道高反应结果图;
图13为实施例3制备的间充质干细胞来源的细胞外囊泡治疗AAI小鼠后细胞因子分泌水平结果图。
具体实施方式
本发明将结合说明书附图,通过以下实施例作进一步说明。
一方面,本发明实施例优先提供了一种无血清培养物的制备方法。
一些实施例中,无血清培养物中包括骨形态生成蛋白2、生长分化因子9、转化生长因子β1。
一些实施例中,将制备的无血清培养物添加至MEM alpha(美国Gibco)无血清培养基中制得本发明实施例中所用的条件培养基。
第二方面,本发明提供了一种间充质干细胞在低氧条件下,使用条件培养基的培养方法。
一些实施例中,间充质干细胞需传代培养至P30代。
具体的,P30代间充质干细胞具有良好的增殖活力。
具体的,P30代细胞保持干细胞良好的细胞形态、细胞表型,并且可分化为相应的成体细胞。
一些实施例中,本发明制备的间充质干细胞在动物体内存活时间更长,具有很好的干细胞特性。
第三方面,本发明实施例提供一种TRIM15高表达细胞外囊泡的制备方法,包括以下步骤:
在5% CO2和2%低氧分压的三气培养箱中进行间充质干细胞的传代培养;
收集所述传代培养至P30代使用的条件培养基;所述条件培养基为包括重组人血白蛋白、血清素、重组人转铁蛋白、重组人胰岛素、乙醇胺、亚硒酸钠、β巯基乙醇、非必需氨基酸、丙氨酰谷氨酰胺、脂质浓缩液、L-抗坏血酸-2-磷酸、孕酮、重组人表皮生长因子、重组人碱性成纤维生长因子、重组人血小板衍生生长因子、骨形态生成蛋白-2、生长分化因子-9、转化生长因子-β1的无血清干细胞培养基;
将所述条件培养基通过分离、纯化,制备获得TRIM15高表达的细胞外囊泡。
一些实施例中,将所述条件培养基通过分离纯化制备获得TRIM15高表达的细胞外囊泡,所述分离纯化包括,超高速离心法、超滤法、空间排阻色谱法、免疫亲和法。
具体的,所述超高速离心法的离心力为100000×g。
第四方面,本发明实施例提供一种所述细胞外囊泡在制备过敏性疾病的药物组合物中的应用。
一些实施例中,所述药物组合物激活了调节性T细胞的增殖,有效控制Th0向Th2/Th17的极化。
一些实施例中,所述药物组合物抑制了促炎因子IL-4和IL-17的表达,促进了抗炎因子IL-10和TGF-β1的表达。
一些实施例中,所述药物组合物用于抑制肺部炎症浸润,降低炎性细胞数量。
一些实施例中,所述药物组合物用于降低肺气道阻力,提高肺顺应性。
本发明实施例中所用试剂、试剂盒均为市售;间充质干细胞来源包括,人羊膜、胎盘、羊水、脐带、骨髓、脂肪、脐血、牙髓、外周血、尿液等。
实施例1
本发明实施例1提供一种培养干细胞的50×无血清培养物及条件培养基的制备方法,包括以下步骤:
分别称取各成分并在超纯水中溶解,需称取的物质及最终浓度和购买渠道如表1所示;
磁力搅拌下,4 ℃层析柜中溶解30 min,调节pH值为7.2-7.6,配制的溶液通过孔径为0.22 μm的真空过滤器抽滤除菌后,再使用孔径为0.1 μm的真空过滤器二次抽滤除菌;
除菌完成后,灌装至无菌西林瓶中,每瓶容量10 mL,压盖密封后于-86 ℃超低温冰箱保存;
灌装完毕的10 mL西林瓶可于冷冻干燥机中进行冷冻干燥,得到无血清培养物冻干粉,压盖密封后于4 ℃冰箱保存,可延长有效期;
将制备得到的50×无血清培养物稀释为1×无血清培养物,加入MEM alpha无血清培养基中,制得条件培养基;
表1 50×无血清培养物的组成及其浓度
。
实施例2
本发明实施例2提供一种低氧条件下使用条件培养基培养间充质干细胞的方法,包括以下步骤:
将人废弃脐带(与捐献者签署知情同意书)在75%酒精中浸泡3-5 min;
浸泡完成后,使用2 mL pH为7.4的1×PBS缓冲液(含2倍双抗),洗涤3次;
洗涤完成后,用眼科剪剪碎至1 mm3左右,吸取到50 mL离心管中,1000 rpm/min,离心10 min;
将离心所得细胞沉淀,即间充质干细胞,用pH为7.4的1×PBS缓冲液洗涤1次,加入条件培养基重悬,并通过台盼蓝染色进行细胞计数;
计数完成后,按1×106-107细胞/mL加入10 cm细胞培养皿中,于低氧条件下(37℃,5% CO2和2%低氧分压)三气培养箱中培养48 h,作为实验组;
同时,设置对照组,对照组为常氧条件下培养,氧分压为21%,其他条件与实验组一致;
培养完成后,对间充质干细胞进行换液,将初始培养液使用移液器吸弃,加入条件培养基;
换液完成后,放置于10 cm细胞培养皿中,各组培养条件下继续培养;每隔2 d用条件培养基进行换液1次;
间充质干细胞在细胞培养皿中生长融合达到80%以上,用5 mL移液管吸弃培养液,取5 mL pH为7.4的1×PBS缓冲液洗涤1次细胞培养皿;
洗涤完成后,加入0.5 mL质量体积比为0.25%的胰酶(含0.05% EDTA)(美国Gibco)于细胞培养皿中,于各组培养条件下消化2 min;
消化完成后,加入2 mL细胞培养液终止消化,并加入5 mL pH为7.4的1×PBS缓冲液,混匀后吸至15 mL离心管中;加入5 mL 1×PBS缓冲液洗涤1次培养皿,将洗涤液加入到上述15 mL离心管中;
将15 mL离心管,于1000 rpm/min,离心5 min,收集沉淀,所得沉淀即为第2代间充质干细胞,加入1 mL条件培养基对沉淀进行重悬,得到第2代间充质干细胞悬浮液,进行计数;
计数完成后,加入10 mL条件培养基于新的175 cm2的细胞培养瓶中继续在各组培养条件下进行培养,每隔3 d更换新配制的条件培养基;
待干细胞生长融合达到80%以上时,按上述经胰酶消化收集干细胞,连续传代培养到P10代、P20代和P30代,并收集培养至各代时所用条件培养基;
获得的各代沉淀,所得沉淀即为各代间充质干细胞,分别用pH为7.4的1×PBS缓冲液重悬,台盼蓝染色计数,即制得P10代、P20代和P30代间充质干细胞,置于冰箱4 ℃存放备用。
实施例3
本发明实施例3提供一种间充质干细胞来源的细胞外囊泡制备方法,包括以下步骤:
在实施例2进行传代培养间充质干细胞过程中,分别收集低氧和常氧条件下培养至P10代、P20代、P30代时所用条件培养基;采用超高速冷冻离心的方法制备细胞外囊泡:
将收集得到的条件培养基加入到50 mL离心管中,1000×g,离心10 min,离心完成后,转移上清液一至新的离心管中;
将上清液一在3000×g,离心15 min,离心完成后,转移上清液二至新的离心管中;
将上清液二在10000×g,离心30 min,离心完成后,将上清液三转移到专用离心管中;
将上清液三在4 ℃,SW32Ti转子,100000×g,离心1.5 h;
离心完成后,得到沉淀,所得沉淀即为细胞外囊泡,加入1 mL pH为7.4的1×PBS缓冲液重悬,储存在-86 ℃超低温冰箱备用;制得低氧条件下培养的间充质干细胞来源的细胞外囊泡(Hy-EVs)和常氧条件下培养的间充质干细胞来源的细胞外囊泡(No-EVs)。
性质检测
实施例2中传代培养的MSC性质检测
1.低氧培养条件下传代MSC形态观察:
取培养至P10、P20和P30代MSC,在倒置显微镜下观察各代MSC的形态,结果如图1所示;低倍镜下(4倍镜)观察,细胞均呈小突起、梭形、呈环状生长。
2.低氧条件和常氧条件下传代培养的MSC增殖活性分析:
低氧条件下培养得到的P30代MSC作为实验组以及通过常氧培养的P30代MSC作为空白组进行增殖活性分析:
取不同条件培养的P30代间充质干细胞接种到96孔板中,每孔接种细胞数量为1×103;接种完成后,每孔加入0.2 mL条件培养基,均设置3个重复;
分别在本发明对应的低氧和常氧培养条件下培养24 h、48 h、72 h、96 h和120 h;
分别在培养24 h、48 h、72 h、96 h和120 h时,通过CCK-8法检测细胞增殖活率,即通过酶标仪(美国Thermo公司)检测OD值,结果如图2所示,细胞活力的计算公式为:
细胞活力=[(实验孔-空白孔)/(对照孔-空白孔)]×100%得出细胞增殖活力。
3.低氧条件和常氧条件下传代培养的MSC的表型分析:
取实施例2中分别在低氧和常氧条件下制备的P30代MSC,所取细胞数量为3×106,低氧条件下的P30代MSC作为实验组,常氧条件下的作为对照组;
实验组和对照组各分4组:第1组为同型对照,分别添加到含有20 μL FITC标记鼠IgG1、20 μL PE标记鼠IgG1和20 μL PerCP标记鼠IgG1的混合液中;
第2组分别添加到含有20 μL FITC标记鼠抗人CD34单抗、20 μL PE标记鼠抗人CD90单抗和20 μL Percp标记鼠抗人HLA-DR单抗的混合液中;
第3组分别添加到含有20 μL FITC标记鼠抗人CD44单抗和20 μL PE标记鼠抗人CD73单抗的混合液中;
第4组分别添加到含有20 μL FITC标记鼠抗人CD45单抗和20 μL PE标记鼠抗人CD105单抗(全部流式抗体购自美国Biolegend公司)的混合液中;
将样本置于4 ℃冰箱染色30 min;染色完成后,加入1 mL pH为7.4的1×PBS缓冲液洗涤3次;
洗涤完成后,加入0.5 mL的1×PBS缓冲液重悬洗涤后的细胞,所得洗涤后的细胞用FCS Calibur流式细胞仪检测(美国BD公司),检测结果如图3和图4所示;
4.低氧条件和常氧条件下传代培养的MSC衰老水平分析:
取实施例2中低氧条件和常氧条件下培养得到的P30代MSC,接种于6孔板上,每孔接种的细胞数量为2×105;接种完成后,每孔加入3 mL条件培养基,分别在低氧和常氧条件下进行培养;
细胞生长融合到70-80%后,以10 μmol/L 4-羟基壬烯酸(4-Hydroxynonenal,4-HNE)处理48 h;
处理结束后用pH为7.4的1×PBS缓冲液清洗细胞1次,加入固定液固定细胞15min,然后用1×PBS缓冲液清洗细胞3次;
配置β-半乳糖苷酶反应液(碧云天生物公司),并在37 ℃生化培养箱中孵育12 h;
孵育完成后,取出细胞并在倒置显微镜下观察细胞染色情况,计数200个细胞,统计染色阳性细胞百分比;结果如图5所示(图5中的A为显微镜下MSC染色情况;图5中的B为β-半乳糖苷酶染色阳性细胞率统计图)。
5.实施例2中传代培养的MSC干性基因表达:
分别取终止消化的低氧和常氧条件下培养的P30代MSC,所取细胞的数量为1×106,加入pH为7.4的1×PBS缓冲溶液洗涤2次,得到细胞沉淀;
将细胞沉淀溶于1 mL Trizol(美国Invitrogen)中,参照试剂说明书提取总RNA,经Nanodrop 2000C浓度测定仪测定RNA浓度;
调整反转录的模板RNA为1 μg,参照Prime ScriptTM RT试剂盒(日本TAKARA)说明书进行反转录,得到P30代MSC的cDNA;
使用TB GreenTM Premix Ex Taq TM试剂盒(日本TAKARA)进行SOX2、OCT4、Nanog干性标志基因的定量PCR扩增;选择GAPDH作为内参基因,引物序列如SEQ ID NO.1-SEQ IDNO.8所示(均由上海生工合成);
PCR扩增的条件为:94 ℃,5 min,94 ℃,15 s,60 ℃,30 s,40个循环;得到的结果通过2-ΔΔt计算方法分析目的基因的相对表达水平,结果如图6所示。
实施例3中制备的MSC来源的EVs的性质检测
1.MSC来源的EVs表面结构及其在条件培养基中的含量检测:
表面结构检测:取实施例3中提取的EVs使用扫描电镜进行观察其表面结构:实施例3中制备的MSC来源的EVs的平均粒径为80.42 nm,在30 nm-150 nm之间,符合外泌体粒径标准;
含量检测:取实施例3提取的EVs采用BCA蛋白分析试剂盒(美国Abcam公司)进行蛋白定量,根据试剂盒产品使用说明书,使用酶标仪并拟定标准曲线进行计算其蛋白浓度并定量,检测结果如表2所示;
表2 间充质干细胞来源的细胞外囊泡在条件培养基中的含量
,
2.MSC来源的EVs的靶蛋白表达比率检测:
通过蛋白印迹检测EVs标志蛋白水平:
取实施例3中制备的EVs,通过Ready PrepTM蛋白萃取试剂盒(蛋白总量)(Bio-Rad,USA)提取总蛋白;
提取完成后,将总蛋白于4 ℃,12000×g,离心15 min;离心完成后,取上清液,使用BCA检测试剂盒(PierceTM),根据说明书测量上清液蛋白质浓度;
检测完成后,100 ℃下将上清液煮沸5 min后,得到蛋白质提取物;
在电流为20 mA条件下,取20 μg 蛋白质提取物在12% SDS-聚丙烯酰胺凝胶上进行电泳;
电泳完成后,在电流为90 mA的条件下将凝胶转移到聚偏氟乙烯膜上,转膜时间为90 min;
将膜放置在含有8 mL封闭缓冲液的小塑料盒中,于室温25 ℃下温和搅拌孵育2h;
将孵育完成的膜暴露于抗TRIM15(稀释倍数为1:500)、CD63(稀释倍数为1:1000)、和TSG101(稀释倍数为1:1000,美国Santa Cruz公司)中,在4 ℃下静置12 h;
静置完成后,使用TBS-T(Tris缓冲盐水,含有Tween 20)漂洗膜3次,每次15 min;
漂洗完成后,膜与辣根过氧化物酶偶联的第二抗体(华安生物),25 ℃下孵育1h,并用TBS-T洗涤15 min;
洗涤完成后,用增强化学发光剂(美国伯乐公司)观察蛋白质,蛋白质的相对量通过密度计测定并表示为吸光度单位;同时测定靶蛋白TRIM15的表达比率;每个实验重复3次,结果如图7所示(图7中的A为蛋白印迹结果;图7中的B为TRIM15蛋白表达水平统计图)。
3.MSC来源的EVs的TRIM15基因表达检测:
取实施例3中制备的Hy-EVs和No-EVs,分别加于1 mL Trizol中,进行TRIM15基因表达检测,后续步骤与检测实施例2中传代培养的间充质干细胞干性基因表达的条件、步骤一致;所用试剂盒均相同;对照组为P30代间充质干细胞;
TRIM15基因的定量PCR扩增使用的引物序列如SEQ ID NO.9和SEQ ID NO.10所示;结果如图8所示。
4.MSC来源EVs免疫调节作用的检测:
通过检测Treg细胞增殖的结果来分析EVs免疫调节作用:
在6孔板中加入含有10%胎牛血清(FBS,Gibco)的RPMI 1640培养基;
在含培养基的6孔板中加入外周血淋巴细胞(peripheral blood lymphocytes,PBL),外周血淋巴细胞来源于签署知情同意书的健康捐赠者;
在含有培养基和PBL的6孔板中加入No-EVs和Hy-EVs,每孔中EVs的最终浓度为20µg/mL;
将6孔板于5% CO2、37 ℃的培养箱中连续培养3 d;
培养完成后,吸取6孔板中的PBL,1200 rpm/min,离心5 min,收集细胞沉淀,用pH为7.4的1×PBS洗涤1次;
将洗涤后的细胞于1200 rpm/min,离心5 min;将离心得到的细胞沉淀用0.2 mL 1×PBS重悬;
取50 µL重悬细胞液加入到含有流式抗体的2 mL离心管中;
第1组为FITC-CD25、PE-小鼠IgG1 κ和PerCP-CD4;
第2组为FITC-CD25、PE-Foxp3和PerCP-CD4;
第3组为空白组;免疫细胞在黑暗中用第1组和第2组的流式抗体在4 ℃下染色30min;
检测Foxp3的细胞内表达情况,结果如图9所示(图9中的A为直观图;图9中的B为CD4+CD25+Foxp3+Treg细胞比例统计图)。
5.MSC来源的EVs治疗过敏性气道炎症的效果验证:
6周龄雌性Balb/c小鼠购自长沙SLAC实验动物公司;动物程序按照南昌大学转化医学研究所实验动物中心指南进行,并经南昌大学动物护理和使用委员会审查批准;
将小鼠随机分为以下5组,每组8只小鼠,包括:正常对照组、过敏性气道炎症模型(AAI模型组,PBS处理)、屋尘螨处理(HDM组)、No-EVs联合HDM处理(No-EVs组)、Hy-EVs联合HDM处理(Hy-EVs组);
从第0天起,用50 μg HDM(美国Greer labs公司)和5 mg氢氧化铝(Sigma)溶解在100 μL 1×PBS缓冲液中并通过腹膜内注射致敏小鼠,隔2周注射一次,共注射2次,正常对照组不进行注射;
No-EVs组和Hy-EVs组(200 µg/200 µL 1×PBS缓冲液)每隔1天通过尾静脉注射1次;HDM(250 µg/100 µL 1×PBS缓冲液)从第35 d到第40 d每隔1 d进行皮下注射,共3次;每次注射100 µL;
从第47天开始,每隔1天用HDM(25 µg/25 µL 1×PBS缓冲液)进行鼻内激发,共6次;在第58天处死所有小鼠后,进行后续检测分析,包括:
(1)小鼠支气管肺泡灌洗液血常规分析:
在最后一次HDM鼻内激发后收集各组的肺泡灌洗液(Bronchoalveolar lavagefluid,BALF);
将小鼠处死后,在上部水平结扎气管,并通过21号导管将1 mL冷1×PBS缓冲液轻轻推入肺部;
收集的BALF在4 ℃,3000 rpm/min,离心5 min;
离心完成后,弃上清液,将离心管中BALF中的细胞重悬于50 μL 1×PBS中,得到BALF细胞悬浮液;
通过对实验小鼠进行BALF细胞悬浮液血常规检查,分析免疫细胞计数情况;各组的细胞计数结果如图10所示(图10中的A为各组小鼠BALF中白细胞计数统计图;图10中的B为各组小鼠BALF中淋巴细胞计数统计图;图10中的C为各组小鼠BALF中中间细胞计数统计图;图10中的D为各组小鼠BALF粒细胞计数统计图);
(2)小鼠肺组织病理学分析:
完成各组BALF的收集后,取各组的肺组织样本,在4%福尔马林中性缓冲液中固定48 h,脱水后包埋在石蜡中进行固定;
固定完成后,用苏木精和伊红对石蜡包埋切片(切片厚度为4 μm,每组8只动物,每只动物制3个切片)进行染色,以评估肺部炎症水平;
使用立式显微镜(日本奥林帕斯,BX63)观察H&E染色,并对肺部炎症评分,炎症分级如下:0级(未观察到炎症细胞)、1级(偶尔观察到炎性细胞)、2级(粘膜被1-3层炎性细胞包围)、3级(粘膜或血管被4-5层炎性细胞包围)和4级(大多数粘膜或血管由5层以上的炎性细胞围绕);肺组织病理学如图11所示(图11中的A为各组H&E染色切片图;图11中的B为各组炎症评分统计图);
(3)小鼠气道高反应(Airway hyperresponsiveness,AHR)的测定:
通过FlexiVent系统(美国SCIREQ公司)测量各组实验小鼠AHR的两个参数:呼吸阻力(Rrs)和静态顺应性(Crs)反应小鼠AHR的程度和特点,包括以下步骤:
呼吸机设置为以150次/min的呼吸频率,产生潮气量为10 mL/kg;
将0、7.8125、15.625、31.25、62.5和125 mg/mL的乙酰甲胆碱的连续剂量雾化以激发气道,直到该剂量导致小鼠产生持续的气道阻力,该阻力大约是基线时的4-5倍;
呼吸阻力通过SnapShot的扰动来测量,分别包括Rrs和Crs;Rrs和Crs的检测结果如图12所示(图12中的A为乙酰甲胆碱剂量为31.25 mg/mL时Rrs的测量值统计图;图12中的B为乙酰甲胆碱剂量为62.5 mg/mL时Rrs的测量值统计图;图12中的C为乙酰甲胆碱剂量为31.25 mg/mL时Crs的测量值统计图;图12中的D为乙酰甲胆碱剂量为62.5 mg/mL时Crs的测量值统计图);
(4)小鼠细胞因子分泌的检测:
在处死各组的实验小鼠后,使用酶联免疫吸附测定法(ELISA)测量,按照ELISA试剂盒说明书检测血清中促炎因子IL-4、IL-17和抗炎因子IL-10、TGF-β1的表达水平;结果如图13所示(图13中的A为各组小鼠促炎因子IL-4的表达水平;图13中的B为各组小鼠促炎因子IL-17的表达水平;图13中的C为各组小鼠抗炎因子TGF-β1的表达水平;图13中的D为各组小鼠抗炎因子IL-10的表达水平)。
结果分析:
参见图2,本发明低氧条件下制备的P30代MSC和常氧制备的P30代MSC相比,随着培养时间增长,细胞增殖活性明显增强,两者之间具有显著的差异(* P<0.05,** P<0.01和*** P<0.001),表明本发明制备的P30代MSC仍具有更好的增殖活性。
参见图3和图4,本发明制备的MSC均高表达CD90、CD73、CD105和CD44,而低表达CD34、CD45和HLA-DR,表明在本发明提供的常氧和低氧条件下,在条件培养基中连续培养MSC到P30代,所得P30代MSC仍符合MSC表型标志物要求。
参见图5,通过β-半乳糖苷酶检测低氧和常氧条件下制备的MSC染色水平,4-HNE引起的氧化应激引起了阳性MSC数量逐渐增多(图5中的A和图5中的B);在常氧条件下,不同浓度4-HNE(0 μmol/L和10 μmol/L)处理后,MSC细胞染色阳性率分别为5.57±0.68%和44.84±3.27%;而低氧条件下,不同浓度4-HNE(0 μmol/L和10 μmol/L)引起的染色阳性率分别为3.90±0.68%和13.92±2.45%,其在10 μmol/L 4-HNE处理后,产生的MSC染色阳性比例显著低于常氧(P=0.001)(图5中的B);结果表明,本发明实施例2中提供的低氧条件下培养的方法可以改善氧化应激引起的MSC衰老或死亡的问题,减缓MSC衰老进程,使其保持更好的生长增殖状态。
参见图6,本发明实施例2中低氧条件下制备的P30代间充质干细胞均表达干性标志基因:SOX2、OCT4和Nanog,且高于常氧条件下制备的P30间充质干细胞,并具有显著的差异(** P<0.01,*** P<0.001和**** P<0.0001);因此,在本发明提供的低氧分压条件下,间充质干细胞培养至P30代仍具有保持很好的细胞干性,有助于其保持良好的分化潜能和体内存活能力。
参见表2,分别取低氧和常氧条件下不同代次MSC的条件培养基25 mL进行EVs的提取,所取不同条件培养得到的MSC细胞数均为1×107;从表中结果可以看到本发明低氧条件下制备的MSC分泌的EVs数量显著高于常氧条件下制备的MSC分泌的EVs(* P<0.05和** P<0.01);表明通过本发明的方法进行EVs的制备时能够得到更好的效果;同时表明,本发明提供的制备方法能够为规模化制备细胞外囊泡提供方法和思路。
参见图7,通过蛋白印迹检测,本发明实施例3中制备的低氧和常氧条件下培养的MSC来源的EVs均表达CD63和TSG101,表明超高速离心提取获取的沉淀为EVs,且表达外泌体标志蛋白;同时,两者均能够表达TRIM15蛋白,但Hy-EVs的TRIM15蛋白的表达量高于No-EVs,其与调节干扰素表达相关的免疫抑制作用密切相关,可能改变Th1/Th2和Th17极化的倾斜。
参见图8,本发明实施例3中制备的低氧和常氧培养MSC来源的EVs与对照组相比均表达TRIM15基因,且本发明在低氧分压条件培养的MSC来源的EVs与对照组相比,TRIM15基因表达水平显著增高。
参见图9,本发明实施例3中制备的的低氧和常氧条件下培养的MSC来源的EVs均促进Treg细胞增殖,低氧MSC来源的EVs促进Treg细胞增殖的比例高于常氧(* P<0.05);因此,本发明提供的低氧分压条件培养的MSC来源的EVs促进了Treg细胞增殖,可以产生明显的免疫调节作用。
参见图10,本发明低氧和常氧条件下培养MSC来源的EVs有效抑制了AAI模型组白细胞的增殖(图10中的A),且特别地抑制了粒细胞的增殖;Hy-EVs组显著地抑制了粒细胞增殖,与No-EVs组差异显著(* P<0.05)(图10中的D);本发明提供的在低氧分压条件培养MSC来源的EVs抑制了BALF中白细胞和粒细胞的增殖,而粒细胞参与激活过敏性气道炎症。
参见图11,可以观察到AAI模型组显示支气管周围和血管周围有大量炎症浸润;与AAI模型组和HDM处理组相比,No-EVs组和Hy-EVs组处理均明显减轻了过敏性炎症,炎症评分分别比HDM处理组下降38.46%和61.54%;此外,注射Hy-EVs处理组的小鼠的炎症评分下降了37.5%,远低于非EVs处理组的小鼠;因此证明,Hy-EVs可以更大程度地减轻慢性气道炎症反应。
参见图12,通过检测实验小鼠的AHR的呼吸阻力Rrs和静态顺应性Crs探究EVs对AAI小鼠模型AHR的作用;结果表明,随着乙酰甲胆碱浓度的增加,Rrs持续增加,其中在AAI模型组和HDM组处理中均明显增加;通过比较不同浓度乙酰甲胆碱(31.25和62.5 mg/mL)处理的结果,与HDM组相比,No-EVs组和Hy-EVs组均显著地降低了Rrs;不同浓度乙酰甲胆碱处理后,No-EVs组下降5.02和3.74倍,Hy-EVs组下降9.77和13.54倍;同时,Hy-EVs组显著提高AHR的Crs,在不同浓度乙酰甲胆碱条件下,Hy-EVs组分别提高8.75和3.21倍,与AAI模型组和HDM组相比有显著差异;此外,Hy-EVs组的Crs均高于No-EVs(图12中的C和图12中的D);证明MSC来源的EVs可以调节炎症反应并改善AHR,且本发明提供的低氧条件下制备的MSC来源的EVs的作用更明显。
参见图13,可以发现,MSC来源的EVs能够抑制过敏性气道炎症小鼠促炎因子IL-4和IL-17的分泌,与AAI模型组和HDM组相比,IL-4和IL-17分泌水平均显著下降,且Hy-EVs组的抑制效果更为明显(* P<0.05和** P<0.01);同时,EVs处理后促进了抗炎因子IL-10和TGF-β1的分泌,且Hy-EVs组的促进效果明显高于No-EVs组,统计学上均具有显著差异(* P<0.05);结果表明,本发明提供的低氧条件下制备的MSC来源的EVs具有抑制炎症因子分泌的作用,能够逆转Th0向Th1/Th17极化,从而促进Treg细胞增殖和抗炎因子的表达。
虽然在上文中详细说明了本发明的实施方式,但是对于本领域的技术人员来说显而易见的是,能够对这些实施方式进行各种修改和变化。但是,应理解,这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且,在此说明的本发明可有其它的实施方式,并且可通过多种方式实施或实现。
Claims (8)
1.一种TRIM15高表达细胞外囊泡的制备方法,其特征在于,包括以下步骤:
在5% CO2和2%低氧分压的三气培养箱中进行间充质干细胞的传代培养;
收集所述传代培养至P30代使用的条件培养基;所述条件培养基为包括5%重组人血白蛋白、50 mg/L血清素、2 g/L重组人转铁蛋白、1 g/L重组人胰岛素、2 g/L乙醇胺、2 g/L亚硒酸钠、2 mmol/L β巯基乙醇、50×的非必需氨基酸、20 mmol/L丙氨酰谷氨酰胺、50×的脂质浓缩液、2 mmol/L L-抗坏血酸-2-磷酸、10 μg/L孕酮、5 μg/L重组人表皮生长因子、5 μg/L重组人碱性成纤维生长因子、5 μg/L重组人血小板衍生生长因子、20 μg/L骨形态生成蛋白-2、10 μg/L生长分化因子-9、20 μg/L转化生长因子-β1的无血清干细胞培养基;将所述条件培养基通过分离、纯化,制备获得TRIM15高表达的细胞外囊泡。
2.根据权利要求1所述的方法,其特征在于,将所述条件培养基通过分离纯化制备获得TRIM15高表达的细胞外囊泡,所述分离纯化包括,超高速离心法、超滤法、空间排阻色谱法、免疫亲和法。
3.根据权利要求2所述的方法,其特征在于,所述超高速离心法的离心力为100000×g。
4.如权利要求1-3任一项所述制备方法在制备过敏性疾病的药物组合物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述药物组合物激活了调节性T细胞的增殖,有效控制Th0向Th2/Th17的极化。
6.根据权利要求4所述的应用,其特征在于,所述药物组合物用于抑制促炎因子IL-4和IL-17的表达,促进了抗炎因子IL-10和TGF-β1的表达。
7.根据权利要求4所述的应用,其特征在于,所述药物组合物用于抑制肺部炎症浸润,降低炎性细胞数量。
8.根据权利要求4所述的应用,其特征在于,所述药物组合物用于降低肺气道阻力,提高肺顺应性。
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