CN117660538A - 一种具有增强免疫抑制能力和迁移能力的间充质干细胞及其在gvhd上的应用 - Google Patents
一种具有增强免疫抑制能力和迁移能力的间充质干细胞及其在gvhd上的应用 Download PDFInfo
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- CN117660538A CN117660538A CN202311681768.5A CN202311681768A CN117660538A CN 117660538 A CN117660538 A CN 117660538A CN 202311681768 A CN202311681768 A CN 202311681768A CN 117660538 A CN117660538 A CN 117660538A
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Abstract
本发明属于生物医学检测技术领域,尤其涉及一种增强免疫抑制能力和迁移能力的间充质干细胞及其在GVHD上的应用。本发明通过TGF‑β提高MSC的抗炎能力,通过CXCR3增强MSC向疾病受损部位的迁移;且进一步采用含雷帕霉素的培养基培养,可进一步增强MSC的免疫抑制作用,可以阻断IL‑2启动的T细胞增殖而选择性抑制T细胞,增加骨髓移植后的抗移植排斥反应。本发明还提供了一种表达增强型人脐带间充质干细胞用于治疗GVHD的应用。本发明发现表达TGF‑β以及CXCR3且经过雷帕霉素预处理的人脐带间充质干细胞通过增强抗炎能力、免疫抑制能力以及增强MSC在体内的迁移能力,显著提高了干细胞对GVHD的治疗效果。
Description
技术领域
本发明属于生物医学检测技术领域,尤其涉及一种增强免疫抑制能力和迁移能力的间充质干细胞及其在GVHD上的应用。
技术背景
间充质干细胞具有天然的免疫耐受特性,不激活异体免疫系统产生免疫排斥反应,且MSC分泌的各种细胞因子如HGF、PGE2,在免疫调节过程中具有抑制淋巴细胞的作用,在移植物抗宿主病中表现出比较好的治疗效果。MSC还具有趋向发炎部位的能力,在发炎部位它们可以调节炎症性反应并有助于修复受损组织。
但是MSC的迁移能力有限,随着血液循环绝大部分倍截留在肺部组织,并不能有效到达全身的炎症部位。而且,MSC的免疫调节能力也是有限的,导致MSC的治疗效果受限。雷帕霉素是一种新型强效免疫抑制剂,临床多用于器官移植的排斥反应和自身免疫疾病的治疗,它的免疫抑制活性是环孢素的数十倍,副作用小,用量低,效果显著。现有技术中,尚未有采用雷帕霉素对MSC进行预处理的相关记载。
发明内容
针对现有技术中存在的问题,为了提高MSC的免疫抑制能力、抗移植排斥能力以及向炎症部位的迁移能力,以及为治疗GVHD提供一种更有效的治疗方式,本发明提供了一种具有增强免疫抑制能力和迁移能力的间充质干细胞mTORC1-Ad-MSC。
本发明还提供了上述具有增强免疫抑制能力和迁移能力的间充质干细胞在GVHD中的应用。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种重组载体,所述重组载体携带人TGF-β基因和趋化因子受体3型CXCR3的序列。
进一步的,所述趋化因子受体3型CXCR3的编码序列如SEQ ID NO.2所示;所述TGF-β基因的编码序列如SEQ ID NO.1所示。
本发明提供的重组载体,具体包含启动子(PGK)、人TGF-β基因和趋化因子受体3型CXCR3的序列、增加转基因表达的调控元件(WPRE)、自催化肽的序列(E2A)。
进一步的,所述自催化肽位于TGF-β和CXCR3序列之间。
本发明还提供了一种重组慢病毒,通过权利要求1-4任一项所述的重组载体与慢病毒辅助包装质粒公转染宿主细胞获得。
本发明进一步的提供了一种转染有上述重组慢病毒的间充质干细胞。
进一步的,所述间充质干细胞来源于自骨髓、胎盘、脐带、羊膜、脂肪组织、脐带血。
本发明还提供了一种上述间充质干细胞在作为治疗GVHD药物中的应用。
进一步的,所述间充质干细胞使用时采用雷帕霉素预处理;优选的,所述雷帕霉素预处理的浓度为30-80nM。
本发明通过TGF-β提高MSC的抗炎能力,通过CXCR3增强MSC向疾病受损部位的迁移;另外,本发明还使用含雷帕霉素的培养基培养,雷帕霉素预处理可进一步增强MSC的免疫抑制作用,可以阻断IL-2启动的T细胞增殖而选择性抑制T细胞,增加骨髓移植后的抗移植排斥反应。
本发明的有益效果为:
(1)本发明提供细胞为人脐带间充质干细胞,表达TGF-β以及CXCR3,且经过雷帕霉素预处理。制备后的增强型mTORC1-Ad-MSC细胞表现出良好的免疫抑制能力、抗炎能力、迁移能力以及抗免疫排斥能力。
(2)本发明还提供了一种表达增强型人脐带间充质干细胞用于治疗GVHD的应用。本发明发现表达TGF-β以及CXCR3且经过雷帕霉素预处理的人脐带间充质干细胞通过增强抗炎能力、免疫抑制能力以及增强MSC在体内的迁移能力,显著提高了干细胞对GVHD的治疗效果。
附图说明
图1为细胞形态图;
图2为流式细胞术检测Ad-MSC表达CXCR3的流式图;
图3为mTORC1-Ad-MSC培养上清中TGF-β表达量的结果;
图4为mTORC1-Ad-MSC免疫表型检测的结果;
图5为mTORC1-Ad-MSC迁移能力的结果;
图6为mTORC1-Ad-MSC的免疫抑制能力的结果;
图7为两种MSC治疗GVHD模型的体重变化、WBC、GVHD评分以及生存率结果;
图8为两种MSC在GVHD小鼠体内各组织迁移能力的比较结果。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
本发明提供的具有增强免疫一直能力和迁移能力的MSC,其中:
第一方面,本发明涉及的MSC来源于自骨髓、胎盘、脐带、羊膜、脂肪组织、脐带血等的MSC。
第二方面,本发明提供的增强型MSC表达TGF-β、CXCR3;优选的,所述TGF-β包括如SEQ ID NO:1所示的氨基酸序列;优选的,所述CXCR3包括如SEQ ID NO:2所示的氨基酸序列;
第三方面,在本发明的背景下构建了编码TGF-β和CXCR3的慢病毒载体。该表达载体用于转导MSC,从而稳定共表达TGF-β、CXCR3。
第四方面,本发明涉及的慢病毒载体的包含启动子(PGK)、人基因TGF-β(SEQ IDNO: 1)和CXCR3(SEQ ID NO:2)的序列、增加转基因表达的调控元件(WPRE)、自催化肽的序列(E2A);自催化肽位于TGF-β和CXCR3序列之间,作用是易于两个基因更好的表达。
第五方面,增强型MSC在治疗GVHD疾病模型前,给与雷帕霉素预处理,雷帕霉素预处理的浓度为30-80nM。
本发明还提供了一种表达TGF-β和CXCR3的人脐带间充质干细胞用于治疗GVHD的应用。本发明发现表达TGF-β和CXCR3的mTORC1-Ad-MSC人脐带间充质干细胞显著提高了干细胞对GVHD的治疗效果。
实施例1
(一)脐带来源的MSC的制备和扩增
通过供者筛查,筛选到供者年龄,妊娠情况,健康状况满足供者筛查标准的产妇,签署供者知情同意书后进行脐带采集。将脐带组织经过洗涤后,减去两端结扎部分,将脐带组织上的静脉和动脉血管剥离干净,剪成肉糜状,接种于T175培养瓶中,加MSC完全培养基后,置于37℃,5%CO2的培养箱中进行培养; 待培养瓶中长出较多克隆,通过显微镜观察细胞形态及汇合度,MSC呈梭型贴壁生长;当汇合度≥80%,使用4ml /瓶TrypLE(TM) Select进行消化传代。传代按8000/cm2浓度接种于T175培养瓶,于37℃,5%CO2的培养箱中继续培养,待汇合度≥80%,用4ml /瓶TrypLE(TM) Select 进行消化,依次类推,传至P5代进行以下各项实验。
(二)TGF-β/CXCR3慢病毒载体构建
通过用AgeI和SacII限制性酶同时酶切pC/RRL-PGK-FANCA-WPRE质粒,在5’端和3’端去除FANCA基因,以获得慢病毒骨架和PGK启动子的片段。获取TGF-β、CXCR3的编码序列信息,设计引物并由第三方公司进行引物合成;引物包括5’端AgeI和SacII限制性位点以及TGF-β、CXCR3序列,进行PCR扩增,PCR产物再用AgeI和SacII酶切,NucleoSpin凝胶和PCRClean‑up试剂盒进行纯化;将酶切纯化后的慢病毒骨架和目的片段与T4 DNA连接酶连接,将连接的产物转化入Stable3细菌,以获得pC/RRL-PKG‑CXCR4‑TGF-β-WPRE质粒。
表1 各基因的编码序列信息及其对应的引物信息
将总量12×106个HEK293T细胞在前一天铺板在T175瓶中。待细胞汇合度达80%时进行转染。转染前1小时将培养基更换为含有10%HyClone和1%青霉素/链霉素的新鲜DMEM‑Glutamax的培养基;制备含有基因转移质粒、病毒基因组和包装构建体的三种质粒的等分子混合物:30μg pC/RRL-PKG‑CXCR4‑TGF-β-Wpre质粒、15μg携带异源VSVg包膜的pMD2.VSVg包装质粒(BioVector NTCC Inc,Catalog #pMD2.G-VSVG慢病毒包装质粒载体)和30μg携带gag‑pol‑rev病毒基因的pCMVdR8.74包装质粒(addgene,Catalog #22036)进行转染。以4.0mL超纯H2O的最终体积来制备这些质粒混合物,并小心地加入450μl 2.5M CaCl2。室温下孵育5min之后,逐滴地加入3 .8mL 2×Hank's缓冲盐水(HBS)缓冲液。将该溶液加入HEK293T细胞中,5小时后,更换为新鲜培养基,转染后48小时收集上清液。上清液用0.22μm孔径的过滤器过滤,于4℃ 20000rpm离心2小时进行浓缩。然后向病毒沉淀中加入DMEM,混匀4℃孵育1小时,离心后弃掉细胞碎片,‑80℃保存。MSC贴壁后加入制备好的慢病毒浓缩液进行转导,在转导过程期间加入polybrene,提高转导效率,转染后的MSC命名为Ad‑MSC。
(三)CXCR3和TGF-β蛋白共表达
用PE-anti-human CXCR3抗体对Ad-MSC染色30min后,通过流式细胞术检测CXCR3在Ad-MSC细胞表面的表达。使用ELISA方法检测人TGF-β检测细胞的Ad-MSC上清液中分泌的TGF-β。如图2所示,流式检测转导后的Ad‑MSC高表达CXCR3,而WT-MSC不表达CXCR3,表明CXCR3基因成功转导进MSC细胞;ELISA检测的TGF-β数据结果见图3,Ad‑MSC分泌的TGF-β远高于WT-MSC;以上数据表明重组MSC构建成功。
(四)雷帕霉素预处理Ad-MSC及表型检测
将雷帕霉素(ab120224)用DMSO配置成50mM的储液。用含50nM雷帕霉素的完全培养基培养Ad-MSC 48小时,将此细胞命名为mTORC1-Ad-MSC。mTORC1-Ad-MSC的细胞形态如图1,为长梭形贴壁细胞,与WT-MSC无差异。培养结束后,用TrypLE(TM) Select 将细胞消化成单细胞悬液;取mTORC1-Ad-MSC细胞悬液,300g,5min弃上清。加入600μlPBS重悬混匀后,均匀分为6管,按照表2中的组合加入相应的抗体,按照2μl/1×106细胞加入相应抗体,漩涡震荡仪上混匀。
表2
避光放15min后,每管加1mlPBS混匀,300g,5min弃上清。每管加入200-400μlPBS重悬混匀后,转移细胞悬液至5ml圆底流式分析管中,流式检测以上表面抗体的表达。检测结果如图4所示,符合MSC表型标准:CD44+/CD73+/CD90+/CD105+≥95%;CD34+/HLA-DR+/CD19+/CD11b+/CD45+≤2%,说明雷帕霉素处理MSC,不会影响MSC的表型表达。
效果验证
(一)细胞迁移实验
含50nM雷帕霉素的完全培养基培养Ad-MSC 48小时。吸弃培养基,用MMC处理4h,按4×105/孔铺入Transwell上室,放有transwell小室的下层6孔板中加入3ml MSC完全培养基,培养48h后,取出小室,用棉棒轻轻擦去上室细胞,用PBS轻轻冲洗小室膜的下表面后,将小室置于4%多聚甲醛溶液中固定30min。固定后小室,晾干,置于2%结晶紫染色液中染色5min,轻轻冲洗,至流出液无紫色。将小室晾干后于倒置显微镜下拍照。选取5个视野计数,统计迁移细胞数目。如图5所示,雷帕霉素预处理的mTORC1-Ad-MSC的细胞数目明显增多,表明mTORC1-Ad-MSC的迁移能力增强。
(二)体外免疫抑制实验
含50nM雷帕霉素的完全培养基培养mTORC1-Ad-MSC 48小时;吸弃培养基,用MMC处理4h,按2×10^5/孔铺6孔板板,待汇合率达80%,进行消化。
(1)MSC和雷帕霉素预处理的mTORC1-Ad-MSC分别与PBMC按照1:5的比例进行共培养铺板,培养18h后,加PMA(50ng/ml)和离子霉素(1ug/ml)用于激活细胞,产生细胞因子;同时加BFA(10ug/ml),阻断细胞因子向胞外分泌;三种刺激因子孵育6-8h后收集PBMC;将细胞用FITC Anti-Human CD3、PE Anti-Human CD8室温孵育30min,PBS洗涤后将用1x Fix/Permworking solution,4℃破膜50min,1x Fix/Wash working solution洗涤细胞,然后将细胞分为两份,一份用APC Mouse anti-Human IFN-γ染色,另一份用APC Mouse anti-HumanIL-17A破膜染色。流式流式细胞检测仪检测Th1/Th17的表达。
(2)WT-MSC和雷帕霉素预处理的mTORC1-Ad-MSC分别与PBMC按照1:5的比例进行共培养铺板,共培养5天后收集细胞,用FITC anti-human CD4、PE-CD25室温孵育30min,按上述方式对细胞进行破膜,破膜后Alexa Fluor 647 Mouse anti-Human FoxP3 染色,4℃染50min;用流式细胞检测仪检测Treg细胞。
(3)MSC和雷帕霉素预处理的Ad-MSC分别与PBMC按照1:5的比例进行共培养铺板,加5ug/ml 的PHA以刺激PBMC增殖;共培养3D后,收集各组细胞上清液,采用酶联免疫吸附法检测TNF-α的表达。
(4)WT-MSC和雷帕霉素预处理的mTORC1-Ad-MSC分别与PBMC按照1:5的比例进行共培养铺板,PBMC铺板前进行以下处理:使用5uM的CFSE进行染色标记PBMC细胞,当细胞进行分裂增殖时,CFSE标记荧光被平均分配到两个子细胞中;因此可用于标记细胞检测细胞增殖;WT-MSC/mTORC1-Ad-MSC与PBMC按照1:5的比例进行共培养铺板,加1ug/mL的PHA以刺激PBMC增殖;PBMC与WT-MSC/mTORC1-Ad-MSC共培养3天后收集PBMC,用APC-CD3标记淋巴细胞。检测结果如图6所示,与WT-MSC相比,雷帕霉素预处理的mTORC1-Ad-MSC与PBMC共培养后对Th1,Th17具有更强的抑制作用,对Treg具有更强的促增殖作用(图6A);mTORC1-Ad-MSC对PBMC具有更强的增殖抑制作用(图6B),对PBMC分泌的TNF-α的抑制作用也增强了(图6C)。因此,经雷帕霉素预处理的mTORC1-Ad-MSC在免疫抑制方面具有很大的提升,更适用于治疗一些免疫相关以及炎症相关的疾病。
(三)mTORC1-Ad-MSC在GVHD中的应用
对受体鼠BALB/C小鼠(8-10周,雄性)进行X射线辐照,辐照剂量为8Gy,剂量率为1.0Gy/min。对供体鼠C57BL/6J小鼠(5-6周,雄性)进行骨髓进和脾脏细胞的获取,将得到的小鼠骨髓细胞/脾细胞悬液,用100um滤网过滤,滤液离心1500rpm,5min,上清;分别向骨髓细胞/脾细胞悬液中加入2倍体积的红细胞裂解液,轻轻混匀,静置5min,溶解红细胞。室温离心1500rpm,5min,弃上清。用PBS洗涤两遍细胞,并将细胞浓度调整为1×108/ml,按照1:1的比例将脾脏细胞和骨髓细胞进行混匀,4℃放置备用。待辐照后6h,按0.2ml/只的剂量混合细胞进行骨髓细胞和脾淋巴细胞移植。移植后1D、3D、5D分别用WT-MSC以及雷帕霉素处理后mTORC1-Ad-MSC进行间充质干细胞的治疗,剂量为1×106/只。将治疗组小鼠分为两组,一组用于评分及生存率的长期观察,另一组用于检测重组MSC体内迁移能力的检测。
长期观察:给药治疗后,每天观察小鼠生存情况,每两天进行体重称重和GVHD评分,每3天进行眼眶取血测WBC。GVHD严重程度评分标准:GVHD的严重程度进行临床评估,评估方法采用Cooke等创建的评分系统,共涉及5项临床参数,具体如下:体重缺失(1分,体重缺失为10-25%;2分,体重缺失>25%);姿势(1分,仅在休息弯腰驼背;2分,严重弯腰驼背妨碍运动);活力(1分,>45%时间处于静止状态;2分,不刺激不活动),毛发整洁度(1分,轻度至中度毛发蓬乱;2分,全身毛发蓬乱);皮肤完整性(1分,轻微毛发缺损;2分,明显的毛发缺损,裸露皮肤)。
每隔2天对小鼠进行称重,评分,每天观察小鼠的生存状态,实验结果如图7所示,治疗祖小鼠体重在治疗10天左右开始回升,且mTORC1-Ad-MSC治疗组体重回升的更快;WBC检测数据各组小鼠在移植后前4天都持续性降低,其中WT-MSC和mTORC1-Ad-MSC治疗组小鼠移植后6天开始回升,而GVHD组一直维持在很低的水平(0-0.2×109/L);GVHD组小鼠在第6-8天已经体重严重缺失,严重严重弯腰驼背,几乎不怎么运动,全身炸毛和腹泻,治疗组评分明显比GVHD组要低,而且mTORC1-Ad-MSC治疗组的GVHD评分也比WT-MSC组低;生存率方面,GVHD组小鼠在5天开始死亡,第11天全部死亡,死亡率100%;移植组小鼠截止第20天的存活率为25%;WT-MSC治疗组的生存率为40%;mTORC1-Ad-MSC治疗组的生存率为60%,大大提高了小鼠的生存率。
体内迁移能力的检测:将WT-MSC以及mTORC1-Ad-MSC治疗后D6的小鼠进行解剖,收集小鼠肝、肺、肠、胃、皮肤组织,将组织在组织研磨仪上研磨均匀。标准曲线的制备:裂解MSC细胞提取DNA,以不同量的DNA为模板、以CD105为引物进行PCR;绘制标准曲线:标曲DNA溶液对应的CT值和对应的间充质干细胞细胞量的对数制作标准曲线。取0.3mg组织匀浆提取组织DNA,以CD105为引物,进行QPCR,试剂盒采用的是天根SuperReal PreMix Plus(SYBR Green);采用25ul体系,反应体系为:2×SuperReal PreMix Plus 12.5ul,正向10um引物0.75ul,反向10um引物0.75ul,DNA模板:100ng~0.04ng/孔,50×Rox Reference Dye0.5ul;补RNase-free ddH2O至25ul。反应程序为:预变性95℃,15min;变性95℃,10sec;退火62℃,32sec;延伸:72℃,30sec;40个循环。如图8结果所示,WT-MSC在输注到GVHD小鼠体内后,WT-MSC主要要肺部截留,迁移到肝、肠、胃、皮肤中的WT-MSC很少;而mTORC1-Ad-MSC由于整合了CXCR3基因,显著增强mTORC1-Ad-MSC的迁移能力,迁移到病变部位肠、胃、皮肤中的MSC显著增多;且显著增加了体内MSC的存活。
Claims (10)
1.一种重组载体,其特征在于,所述重组载体携带人TGF-β基因和趋化因子受体3型CXCR3的序列。
2.根据权利要求1所述的重组载体,其特征在于,所述趋化因子受体3型CXCR3的编码序列如SEQ ID NO.2所示;所述TGF-β基因的编码序列如SEQ ID NO.1所示。
3.根据权利要求1或2所述的重组载体,其特征在于,具体包含启动子(PGK)、人TGF-β基因和趋化因子受体3型CXCR3的序列、增加转基因表达的调控元件(WPRE)、自催化肽的序列(E2A)。
4.根据权利要求3所述的重组载体,其特征在于,所述自催化肽位于TGF-β和CXCR3序列之间。
5.一种重组慢病毒,其特征在于,通过权利要求1-4任一项所述的重组载体与慢病毒辅助包装质粒公转染宿主细胞获得。
6.一种转染有权利要求5所述的重组慢病毒的间充质干细胞。
7.根据权利要求6所述的间充质干细胞,其特征在于,所述间充质干细胞来源于自骨髓、胎盘、脐带、羊膜、脂肪组织、脐带血。
8.一种如权利要求6或7所述的间充质干细胞在作为治疗GVHD药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述间充质干细胞使用时采用雷帕霉素预处理。
10.根据权利要求9所述的应用,其特征在于,所述雷帕霉素预处理的浓度为30-80nM。
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