CN114940969A - 一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物 - Google Patents
一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物 Download PDFInfo
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Abstract
本发明提供了一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,包括间充质干细胞完全培养基,在每毫升间充质干细胞完全培养基中,还含有IFN‑γ8~12ng、TNF‑α9~12ng、GSK126 7~13µmol。本发明还提供了上述组合物在制备预防或治疗自身免疫性疾病或者炎性相关疾病的疾病的药物中的应用。本发明创新性地提出一种组合物因子在不影响细胞的形态和凋亡的同时,极大地增强了间充质干细胞的抗炎和免疫调控功能,且在以炎症反应为主要特征的免疫性疾病小鼠模型中具有显著的疗效。
Description
技术领域
本发明属于生物医药领域,涉及间充质干细胞,具体来说是一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物。
背景技术
间充质干细胞(mesenchymal stem cells,MSCs)呈现成纤维细胞形态,MSCs能够在体外扩增培养,在诱导的条件下分化为特定的细胞类型,如骨、软骨和脂肪细胞,此外,还包括内胚层的胰岛细胞和外胚层的神经细胞等。MSCs几乎存在于所有组织,如骨髓、脂肪、脐带、肌肉、胰腺、牙髓、羊水和脐带血等。
研究发现MSCs具有抗炎和免疫调控的功能,参与机体的免疫调节,因此其在组织修复、造血重建以及移植免疫中发挥重要的作用。近年来,MSCs已成为自身免疫疾病、炎症性疾病、脊髓损伤、中风、多种神经退行性疾病以及其他多种类型疾病的临床治疗的潜在治疗策略。已有的动物模型以及临床前试验结果表明,MSCs在安全性和有效性均展现良好的应用前景,目前将干细胞移植技术从实验研究转化到临床疾病治疗,仍然面临着很多困难和挑战。
MSCs因其组织来源、分离方法和培养环境的不同,导致MSCs具有很大的异质性,尤其是在抗炎和免疫调节方面的差异,会极大地影响临床前细胞质量控制以及潜在治疗效果的评估。如何提高MSCs具有的抗炎和免疫调控作用已成为目前研究中的难点。已有的增强间充质干细胞效能的方法如向细胞培养环境中加入激动剂、诱导试剂,虽能够提高干细胞效能,然而添加剂是否影响细胞的形态和凋亡尚不明确,且效果不够显著。
IFN-γ(γ干扰素)是可溶性二聚体细胞因子,是II型干扰素的唯一成员。它主要由自然杀伤细胞(NK)和自然杀伤T细胞(NKT)细胞分泌,在固有免疫中发挥作用;
TNF-α(肿瘤坏死因子-α)是一种涉及到系统性炎症的细胞因子,同时也是属于引起急相反应的众多细胞因子中的一员,其主要由巨噬细胞分泌。
GSK126是一种有效的,高选择性的,具有S-腺苷甲硫氨酸竞争性的小分子EZH2甲基转移酶活性抑制剂,对EZH2基因的过表达有明显的调控作用。
在炎症环境中,静息态的MSCs可以被微环境中的炎症因子诱导为具有抑制炎症的表型,明显提高MSCs的炎症抑制作用。微环境中的其他细胞因子也能够对MSCs的免疫调节能力产生协同或者拮抗作用。如何极大地提高MSCs的效能一直是干细胞领域重要的研究课题。本发明专利创新性地提出一种组合物因子在不影响细胞的形态和凋亡的同时,极大地增强了间充质干细胞的抗炎和免疫调控功能,且在以炎症反应为主要特征的免疫性疾病小鼠模型中具有显著的疗效。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,所述的这种用于提高间充质干细胞抗炎和免疫抑制功能的组合物要解决现有技术中的MSCs具有很大的异质性,在抗炎和免疫调节方面具有差异的技术问题。
本发明提供了一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,包括间充质干细胞完全培养基,在每毫升间充质干细胞完全培养基中,还含有IFN-γ8~12ng、TNF-α9~12ng、GSK126 7~13μmol。
进一步的,所述的组合物由间充质干细胞完全培养基、IFN-γ、TNF-α、GSK126组成,所述的间充质干细胞完全培养基、IFN-γ、TNF-α、GSK126的物料比为1ml:8~12ng:9~12ng:7~13μmol。
进一步的,所述的间充质干细胞完全培养基由高糖DMEM培养基、血清替代物、肝素钠组成,所述的高糖DMEM培养基、血清替代物、肝素钠的物料比为500ml:25ml:168ul。
具体的,所述的DMEM是一种含各种氨基酸和葡萄糖的培养基,是在MEM培养基的基础上研制的,高糖DMEM培养基是指葡萄糖的含量高于4500mg/L的DMEM培养基。高糖型DMEM有利于细胞停泊于一个位置生长,适于生长较快、附着较困难肿瘤细胞等。(DMEM培养基、高糖DMEM培养基为已有技术,在此不再赘述)
进一步的,所述间充质干细胞来源于人脂肪、骨髓、牙髓、脐带、胎盘或脐带血。
进一步的,所述间充质干细胞的制备方法包括:
1)用含有抗生素的缓冲液洗涤脐带组织,转移至干净的培养皿中,剖离去除脐带的2根脐动脉和1根脐静脉,取华氏胶部分,洗涤后,剪成约1mm3的组织块,加入质量百分比浓度为0.2%胶原酶;
2)将组织块转移至15ml离心管中,放置于37℃水浴锅中消化1小时;
3)完全培养基终止消化后,过70μm单细胞筛,去除未消化的组织块,1200rpm,离心5分钟;
4)完全培养基洗2次,计数,按照一定的细胞密度铺板,放入37℃、在体积百分比浓度为5%CO2的培养箱;第三天,弃去培养基中的未贴壁细胞,细胞长至汇合度~80%左右时,消化后,按1:3传代。
进一步的,所述间充质干细胞诱导前的接种方法包括:将1*10^5细胞接种于6孔板中或者1*10^6细胞接种于10cm细胞培养皿,所述间充质干细胞培养时间为18-24小时。
本发明还提供了上述组合物在制备预防或治疗自身免疫性疾病或者炎性相关疾病的疾病的药物中的应用。
进一步的,所述的自身免疫性疾病或者炎性相关疾病包括但不限于肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)、帕金森病(Parkinson’s disease,PD)或者脊髓损伤(spinal cord injury)。
本发明和已有技术相比,其技术进步是显著的。本发明创新性地提出一种组合物因子在不影响细胞的形态和凋亡的同时,极大地增强了间充质干细胞的抗炎和免疫调控功能,且在以炎症反应为主要特征的免疫性疾病小鼠模型中具有显著的疗效。
附图说明:
图1:GSK126、两因子组合物(2IF)以及三因子组合物(GSK126+2IF)处理hUMSCs的细胞形态。
图2:GSK126、两因子组合物(2IF)以及三因子组合物(GSK126+2IF)处理并不影响hUMSCs细胞凋亡。A-B:流式细胞检测hUMSCs细胞凋亡;C:四个处理组间的活细胞、早期凋亡以及死亡细胞比例均无明显差异(n=6)。
图3:相比两因子组合物,三因子组合物(GSK126+2IF)极大地提高了抗炎因子在hUMSCs中的表达水平。A:IDO1;B:IDO2;C:PGE2;D:IL6;E:CXCL9;F:CXCL11(*P<0.05,**P<0.01,***P<0.001,n=4)。
图4:不同处理条件下hUMSCs对PBMCs增殖的抑制效果(深绿色为三因子组合物(GSK126+2IF)处理组;淡绿色为两因子组合物(2IF)处理组;橙色为GSK126处理组;蓝色为空白组;红色为未与MSCs共培养的PBMCs。)相比两因子组合物,三因子组合物(GSK126+2IF)极大地提高了hUMSCs的免疫抑制功能。
具体实施方式:
实施例1组合物因子预处理脐带间充质干细胞
(1)将脐带样本收集到装有含抗生素的缓冲液中,冰上运输至细胞间;
(2)用含有抗生素的缓冲液洗涤脐带组织,转移至干净的培养皿中,剖离去除脐带的2根脐动脉和1根脐静脉,取华氏胶部分,洗涤后,剪成约1mm3的组织块,加入0.2%胶原酶;
(3)将组织块转移至15ml离心管中,放置于37℃水浴锅中消化1小时;
(4)在超净工作台中,配制间充质干细胞完全培养基:向500ml高糖DMEM培养基中加入占高糖DMEM培养基体积比例为5%的血清替代物和占(高糖DMEM培养基+血清替代物)总体积比例为0.032%的肝素钠溶液,混合均匀,静置待用;(高糖DMEM购自Gibco公司,型号为11965092;血清替代物购自Helios公司,型号为HPCPLCRL50;肝素钠溶液购自国药集团容生制药有限公司,效价为12500u)
(5)完全培养基终止消化后,过70μm单细胞筛,去除未消化的组织块,1200rpm,离心5分钟;
(6)完全培养基洗2次,计数,按照一定的细胞密度铺板,放入37℃、5%CO2培养箱。第三天,弃去培养基中的未贴壁细胞,细胞长至汇合度~80%左右时,消化后,按1:3传代。
(7)当细胞长至汇合度为90-100%时,胰酶消化细胞,计数,将1*10^5细胞接种于6孔板中或者1*10^6细胞接种于10cm细胞培养皿,培养18-24小时;
(8)弃掉原有培养基,更换新鲜完全培养基,设置不同分组:GSK126、两因子组合物(IFN-γ+TNF-α)、三因子组合物(GSK126+2IF),加入相应剂量的组合物因子(IFN-γ8~12ng、TNF-α9~12ng、GSK126 7~13μmol),继续培养24小时;
(9)弃掉含有小因子组合物的培养基,PBS缓冲液洗涤3次,继续培养48小时;
(10)胰酶消化细胞,计数,部分细胞用于凋亡检测,部分用于抽提细胞总RNA进行QPCR检测抗炎作用,部分细胞用于免疫抑制作用检测。
通过炎症因子组合物因子诱导24小时后,通过显微镜观察细胞形态。如图1所示,不同处理条件的细胞形态无明显差异,表明GSK126、两因子组合物以及三因子组合物处理不影响hUMSCs的细胞形态。
实施例2脐带间充质干细胞凋亡检测
(1)将细胞培养上清收集到合适的离心管,PBS洗涤细胞,胰酶消化细胞,将细胞轻轻吹打下来,转移至离心管内,1200rpm,离心3分钟,弃上清,收集细胞,PBS重悬细胞并计数;
(2)取适量重悬的细胞,离心,弃上清,加入500μl buffer重悬细胞;
(3)加入Annexin V-FITC和碘化丙啶染色液,轻轻混匀;
(4)室温避光,孵育15分钟,立即上机检测。
通过Annexin V/PI检测四个不同处理组的细胞凋亡情况,结果如图2所示,不同处理组之间的细胞凋亡情况并无明显差异。实验结果说明GSK126、两因子组合物以及三因子组合物处理hUMSCs并不会影响到细胞凋亡。
实施例3脐带间充质干细胞抗炎作用检测
1.细胞RNA抽提
(1)将组合物因子预处理24小时后的细胞从培养皿上消化下来,PBS洗涤细胞,离心弃上清,加入1ml Trizol,上下吹打混匀后将其转移至新的1.5ml EP管,室温裂解5分钟;
(3)向EP管中加入0.2ml氯仿,上下剧烈震荡15秒;
(4)室温静置5分钟;
(5)12000g,4℃离心15分钟(离心后,溶液分为上层含RNA的透明液体层、中间层含DNA和蛋白质、下层为苯酚氯仿);
(6)转移上层含RNA的溶液至新的EP管,加入等体积的异丙醇,上下颠倒混匀后,-20℃放置1-2小时;
(7)12000g,4℃离心10分钟,管底可以看到白色RNA沉淀,弃去上清;
(8)加入1ml 70%乙醇洗涤沉淀,7500rpm,4℃离心5分钟,重复2次,充分洗去RNA中的杂质;
(9)尽量去除乙醇,室温晾干沉淀,观察沉淀渐渐变透明即可;
(10)根据沉淀量,加入适量的RNAase-free水溶解RNA沉淀,冰上溶解10分钟,测浓度。
2.去除总RNA中的基因组DNA
(1)我们选择TURBO DNase Treatment kit(Ambion)来去除RNA中可能含有的DNA;
(2)首先,我们取适当总量的RNA,加入0.1倍体积的TURBO DNase buffer和1μlTURBO DNase,37℃孵育30分钟;
(3)孵育结束后,加入0.1倍体积DNase Inactivation Reagent,室温孵育5分钟;
(4)12000rpm,离心2分钟,转移上层水相到新的EP管,用于cDNA反转录,或者冻存于-80℃备用。
3.RNA反转为cDNA
(1)根据测量的RNA浓度,每个样本取0.5μg的RNA用于反转录;
(2)采用如下的反应体系:
试剂 | 使用量 | 终浓度 |
5×PrimeScript RT Master Mix(Perfect Real Time) | 2μl | 1× |
Total RNA | * | |
RNase Free dH<sub>2</sub>O | up to 10μl |
充分混匀后,短暂离心,放到PCR仪上,运行程序;
(3)反转录程序如下:
37℃,15minutes;85℃,5seconds;4℃,∞;
(4)反转录的cDNA立即用于q-PCR或者储存于-20℃。
4.实时定量PCR
(1)将所有引物稀释到10μM,备用,取适量cDNA样品1∶20加水稀释,作为模版;
(2)采用SYBR Green mix(takara)进行实时定量PCR,配制Q-PCR反应体系如下:10μl体系:
引物序列如下:
(3)加样后,2000rpm,离心1分钟,放到QPCR仪上,运行程序:50℃,2minutes hold;95℃,30seconds hold;95℃,15seconds,60℃,40seconds,40cycles;(加入溶解曲线分析引物特异性);
(4)根据内参,对q-PCR结果进行半定量分析。
与两因子诱导组相比,三因子诱导组细胞株#071701、#081602 IDO1 mRNA表达水平高70%,细胞株#041001、#010802 IDO1 mRNA表达水平高40%~50%;三因子诱导组细胞株#071501 PGE2 mRNA表达水平高于两因子诱导组50%,细胞株#010802高出300%;三因子诱导组细胞株#071501、#081602、#041001、#010802 CXCL9 mRNA表达水平高于两因子诱导组50%~75%;三因子诱导组细胞株#071501、#041001、#010802 IDO2 mRNA表达水平高于两因子诱导组40%~50%,细胞株#081602高出350%;三因子诱导组细胞株#041001、#010802 IL6 mRNA表达水平高于两因子诱导组25%~75%;三因子诱导组细胞株#071501、#081602、CXCL11 mRNA表达水平高于两因子诱导组80%~100%,三因子诱导组细胞株#041001、#010802 CXCL11 mRNA表达水平高于两因子诱导组180%~200%。由此可知,三因子组合物可以极大地提高hUMSCs所表达的免疫调控相关细胞因子的表达,从而高效提高hUMSCs的免疫调节作用。
实施例4脐带间充质干细胞免疫抑制作用检测
(1)铺板:当hUMSCs长至汇合度达到90-100%时,消化细胞,计数,按照3*10^4细胞/孔的细胞密度接种于24孔细胞培养板;
(2)预处理:细胞贴壁后,加入相应浓度的炎症因子及小分子组合,刺激细胞12小时;
(3)预处理hUMSCs的同时,加入anti-CD3和anti-CD28到PBMCs中,激活PBMCs;
(4)组合物因子预处理细胞24小时后,弃掉原有培养基,PBS洗2次,更换新鲜完全培养基,500μl/孔,放回培养箱中;
(5)采用CFDA SE标记PBMCs,将PBMCs收集到15ml离心管中,1500rpm,离心5分钟,弃去上清,PBS洗1次,用1ml CFDA SE细胞标记液重悬细胞沉淀(1~5*10^6细胞/ml);
(6)加入适当体积的CFDA SE储存液,轻轻混匀;
(7)放置于37℃水浴锅中孵育10分钟;
(8)加入10ml完全培养基,离心去上清,再洗一次,放置于37℃水浴锅中孵育5分钟,离心去上清;
(9)适当体积的完全培养基重悬细胞沉淀,细胞计数,按照hUMSCs:PBMCs=1:10的比例,将PBMCs接种于下层铺有hUMSCs的24孔板的上层transwell中,放回培养箱中继续培养5天;
(10)收集transwell中的PBMCs,离心去上清,PBS洗一次,用200μl PBS重悬细胞沉淀后,将细胞转移至流式管中,通过流式细胞仪检测PBMCs的增殖情况。
hUMSCs的免疫调节作用,重点表现为对免疫细胞增殖的抑制。通过密度梯度离心分离健康人外周血单个核细胞(PBMCs),经anti-CD3/CD28激活后,与hUMSCs以transwell法共培养1:10(MSC:PBMC),研究不同处理组hUMSCs对PBMCs的抑制作用。共培养5天后,以流式细胞术检测PBMCs的增殖情况。结果如图4所示,两因子组合物(2IF)刺激的MSCs提高了hUMSC对PBMCs增殖的免疫抑制作用,GSK126单独刺激hUMSCs对PBMCs的增殖抑制作用较低,且低于两因子刺激组,然而GSK126联合两因子组合刺激则能够极大地提高hUMSCs的免疫抑制力,且高于两因子组合物的刺激效果。该结果表明,相较于两因子组合物,三因子组合物能够极其高效地提高hUMSCs的免疫抑制力。
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Claims (9)
1.一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,包括间充质干细胞完全培养基,在每毫升间充质干细胞完全培养基中,还含有IFN-γ8~12ng、 TNF-α9~12ng、GSK126 7~13µmol。
2.根据权利要求1所述的一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,由间充质干细胞完全培养基、IFN-γ、 TNF-α、GSK126组成,所述的间充质干细胞完全培养基、IFN-γ、 TNF-α、GSK126的物料比为1ml:8~12ng:9~12ng:7~13µmol。
3.根据权利要求1或2所述的一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,所述的间充质干细胞完全培养基由高糖DMEM培养基、血清替代物、肝素钠组成,所述的高糖DMEM培养基、血清替代物、肝素钠的物料比为500ml:25ml:168ul。
4.根据权利要求3所述的一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,所述的高糖DMEM培养基是指葡萄糖的含量高于4500mg/L的DMEM培养基。
5.根据权利要求1所述的一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,所述间充质干细胞来源于人脂肪、骨髓、牙髓、脐带、胎盘或脐带血。
6.权利要求1所述一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,所述间充质干细胞的制备方法包括:
1)用含有抗生素的缓冲液洗涤脐带组织,转移至干净的培养皿中,剖离去除脐带的 2根脐动脉和 1 根脐静脉,取华氏胶部分,洗涤后,剪成约 1mm3的组织块,加入质量百分比浓度为 0.2%胶原酶;
2)将组织块转移至 15ml 离心管中,放置于 37°C 水浴锅中消化 1 小时;
3)完全培养基终止消化后,过 70µm 单细胞筛,去除未消化的组织块,1200rpm, 离心5 分钟;
4)完全培养基洗 2 次,计数,按照一定的细胞密度铺板,放入 37°C、在体积百分比浓度为5% CO2的培养箱;第三天,弃去培养基中的未贴壁细胞,细胞长至汇合度~80%左右时,消化后,按 1:3 传代。
7.根据权利要求1所述的一种用于提高间充质干细胞抗炎和免疫抑制功能的组合物,其特征在于,所述间充质干细胞诱导前的接种方法包括:将 1*10^5 细胞接种于 6 孔板中或者 1*10^6 细胞接种于 10cm 细胞培养皿,所述间充质干细胞培养时间为 18-24 小时。
8.权利要求1所述组合物在制备预防或治疗自身免疫性疾病或者炎性相关疾病的药物中的应用。
9.根据权利要求8所述的应用,所述的自身免疫性疾病或者炎性相关疾病包括但不限于肌萎缩侧索硬化、帕金森病或者脊髓损伤。
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