CN111437289A - 甘露糖在增强间充质干细胞免疫调节能力方面的应用 - Google Patents
甘露糖在增强间充质干细胞免疫调节能力方面的应用 Download PDFInfo
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Abstract
本发明提供了甘露糖在增强间充质干细胞免疫调节能力方面的应用，本发明利用甘露糖预处理间充质干细胞，显著增强了间充质干细胞的免疫调节能力，提高了间充干细胞在治疗自身免疫性疾病方面的疗效。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及甘露糖在增强间充质干细胞免疫调节能力方面的应用。
背景技术
间充质干细胞(mesenchymal stem cells, MSCs) 是来源于发育早期中胚层的一类重要的成体干细胞,具有多种组织修复能力。MSCs 最初是由骨髓基质中分离获得的非造血多能干细胞，现已证实MSCs 也存在于脂肪组织、脐带等多种组织内。根据国际细胞治疗协会（ISCT）下属间充质和组织干细胞委员会所提出的定义人MSCs最低标准：a )在标准培养条件下，MSCs必须具有对塑料底物的黏附性；b )CD105、CD73、CD90呈阳性，CD45、CD34、CD14或CD11b、CD79a或CD19和HLA-DR呈阴性；c )在体外标准分化条件下，MSCs能分化为成骨细胞、脂肪细胞和软骨细胞。
MSCs 除具有支持体外造血、促进体内造血重建、组织损伤修复外，还具有独特的免疫调节作用，而其调节作用又以T细胞为主，在混合淋巴细胞反应中，通过周期阻滞抑制T细胞增殖，但不引起T 细胞凋亡增加、活化受抑。同时MSCs 还能降低反应体系中的CD8T细胞和Thl细胞，由Th1 极化状态向Th2 漂移，升高Th2细胞，其分泌的IL-4,IL-10 等细胞因子含量明显增加，从而抑制炎症反应，在T细胞介导的自身免疫性疾病中发挥治疗作用。
目前，间充质干细胞在免疫调节领域的应用仍存在一些问题亟需解决。首先，由于间充质干细胞来源不同、细胞分离扩增的方法存在差异，间充质干细胞的质量也存在很大差异，不同批次的间充质干细胞的调节免疫效果差异较大。另外，由于细胞治疗本身的特点，间充质干细胞在治疗自身免疫性疾病方面的效果仍存在不确定性。
发明内容
针对现有技术存在的问题，本发明提供甘露糖在增强间充质干细胞免疫调节能力方面的应用。
本发明的目的是通过以下技术方案来实现的：
一方面，提供甘露糖在增强间充质干细胞免疫调节能力方面的应用。
优选地，所述应用过程具体为：取传代至P4代的间充质干细胞，接种于含有10% 胎牛血清和10-50mM甘露糖的α-MEM培养基中，于37℃，浓度为5 %的CO2培养箱中培养24-48h。
优选地，所述间充质干细胞选自骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞中的至少一种。
另一方面，提供甘露糖在制备治疗自身免疫性疾病的药物中的应用，包含以下步骤：
（1）取传代至P4代的间充质干细胞，接种于含有10% 胎牛血清和10-50mM甘露糖的α-MEM培养基中，于37℃，浓度为5 %的CO2培养箱中培养24-48h；
（2）将使用甘露糖处理后的间充质干细胞消化计数后，均匀悬于生理盐水或PBS缓冲液，至密度为1×106个/mL-5×106个/mL，获得所述治疗自身免疫性疾病的药物。
优选地，所述药物的组分包含间充质干细胞，所述间充质干细胞为经过甘露糖处理的间充质干细胞。
优选地，所述间充质干细胞选自骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞中的至少一种。
优选地，所述自身免疫性疾病为类风湿关节炎或系统性红斑狼疮。
相对于现有技术，本发明的有益效果在于：本发明利用甘露糖预处理间充质干细胞，显著增强了间充质干细胞的免疫调节能力，提高了间充干细胞在治疗自身免疫性疾病方面的疗效。
附图说明
图1A-图1H显示了甘露糖预处理后的间充质干细胞的免疫表型检测结果；
图2A显示了甘露糖预处理后的间充质干细胞的成脂分化能力检测结果；
图2B显示了甘露糖预处理后的间充质干细胞的成骨分化能力检测结果；
图3显示了实验组、对照组、空白对照组中间充质干细胞对T细胞增殖的影响；
图4A为实验组Th1细胞的表达检测图；
图4B为对照组Th1细胞的表达检测图；
图4C为空白对照组Th1细胞的表达检测图；
图5A为实验组Th2细胞的表达检测图；
图5B为对照组Th2细胞的表达检测图；
图5C为空白对照组Th3细胞的表达检测图；
图6A 显示了实验组和对照组中的间充质干细胞分泌TGF-β的能力检测结果；
图6B 间充质干细胞分泌HLA-G5的能力检测结果；
图6C 间充质干细胞分泌HGF的能力检测结果；
图6D 间充质干细胞分泌PGE-2的能力检测结果。
具体实施例
以下结合附图对本发明的技术方案通过具体的实施例进行进一步说明。
实施例1：间充质干细胞的分离培养
一、脐带间充质干细胞的制备
将脐带组织从采集瓶取出，置于酒精中清洗 2 次转入无菌 PBS 去除结缔组织、淤血等，之后充分清洗，去除血污。将脐带转入大号培养皿中，剪断成 3-4cm 长度之后沿纵向进行分离，是脐带组织管状变为片状。将片状脐带组织进行进一步的分离，切割为 2-3mm3大小的组织块，将组织块逐一种植到预先以含10 %胎牛血清的α-MEM培养基包被的培养皿中，置于37 ℃，体积分数为5 % CO2饱和湿度培养箱中，每24小试补加0.5 ml培养基，72小时后全量换液，每周换液 2 次，细胞生长融合只 80 % 左右时，用0.20%胰蛋白酶消化传代。
二、脂肪间充质干细胞的制备
获取脂肪组织，以 PBS 溶液冲洗 3 次，去除血管和结缔组织，剪碎，加入3 倍体积的I型胶原酶，37℃消化2 h，以200目滤网滤去未消化完全的大块脂肪组织，离心弃上清，以PBS重悬细胞，清洗 3 次，之后加入含10% FBS 的α-MEM重悬沉淀细胞，接种于平皿，于37℃、体积分数为5 %的CO2培养箱中培养，隔天换液，待细胞铺满瓶底 80% 时，用 0.25% 胰制剂消化传代。
实施例2：甘露糖预处理间充质干细胞
取实施例1中传代至P4代的间充质干细胞以105个/ml接种至培养皿中，使用含有10%FBS 和25mM甘露糖的α-MEM培养液进行培养，置于37 ℃，体积分数为5 % CO2饱和湿度培养箱中培养24小时，获得甘露糖预处理后的间充质干细胞。
实施例3：甘露糖预处理后的间充质干细胞免疫表型和分化能力的检测
一、间充质干细胞免疫表型的检测
取实施例2中甘露糖预处理后的间充质干细胞，用流式细胞术检测阳性抗体( CD90 ,CD105，CD73 )和阴性( CD45 ,CD34 ,CD11b ,CD19和HLA-DR )表达情况，见图1A-图1H。
二、成脂分化能力检测
取实施例2中甘露糖预处理后的间充质干细胞，常规培养，并每2d换一次培养液；取对数生长期的间充质干细胞，在约80％融合时加入脂肪诱导培养基,2周后油红O染色鉴定，显微镜下观察并拍照，见图2A。
三、成骨分化能力检测
取实施例2中预处理后的间充质干细胞，常规培养，并每2天换一次培养液；取对数生长期的人脐带间充质干细胞，在约80％融合时加入成骨诱导液，并每2天全量换液一次，21天后进行茜素红染色鉴定骨节形成，显微镜下观察并拍照，见图2B；
结果显示，实施例2培养得到的间充质干细胞符合干细胞标准。
实施例4：甘露糖预处理后的间充质干细胞的免疫调节能力检测
一、对T细胞增殖的影响
分离外周血淋巴细胞，磁珠分选CD3+T细胞。分为三组，一组为实验组将甘露糖预处理的间充质干细胞与T细胞共培养，一组为对照组将间充质干细胞于T细胞共培养，一组为空白对照组单独的T细胞，三组均加入PHA（2ug/mL）刺激。120h，3H-TdR渗入法检测T细胞增殖情况。
结果发现实验组的间充质干细胞和对照组的间充质干细胞后均可以抑制T细胞的增殖，但是实验组的间充质干细胞抑制作用更显著，见图3。
二、对T细胞分化的影响
分离外周血淋巴细胞，磁珠分选CD3+T细胞。分为三组，一组为实验组将甘露糖预处理的间充质干细胞与T细胞共培养，一组为对照组将间充质干细胞于T细胞共培养，一组为空白对照组单独的T细胞。72h后流失细胞数检测Th1和Th2细胞的表达。
结果发现实验组的间充质干细胞和对照组的间充质干细胞均可以抑制Th1，促进Th2的表达，但是实验组的间充质干细胞的作用增加明显，结果见图4A-图5C。图4A为实验组Th1细胞的表达检测图；图4B为对照组Th1细胞的表达检测图；图4C为空白对照组Th1细胞的表达检测图；图5A为实验组Th2细胞的表达检测图；图5B为对照组Th2细胞的表达检测图；图5C为空白对照组Th3细胞的表达检测图。
三、间充质干细胞抗炎因子的表达
取实施例1中传代至P4代的间充质干细胞接种至培养皿，分为两组，一组为实验组甘露糖预处理组，在培养基中加入25mM甘露糖，一组为对照组间充质干细胞组，不添加其他成分，培养24h后，收集细胞上清， 通过ELISA 法检测细胞上清中TGF-β1、HLA-G5、HGF及PGE-2等因子变化。
结果发现，甘露糖处理后的间充质干细胞其表达TGF-β、HLA-G5 、HGF以及PGE-2的能力增强，见图6A-图6D。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何在本申请揭露的技术范围内的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。
Claims (7)
1.甘露糖在增强间充质干细胞免疫调节能力方面的应用。
2.根据权利要求1所述的应用，其特征在于，所述应用过程具体为：取传代至P4代的间充质干细胞，接种于含有10% 胎牛血清和10-50mM甘露糖的α-MEM培养基中，于37℃，浓度为5 %的CO2培养箱中培养24-48h。
3.根据权利要求2所述的应用，其特征在于，所述间充质干细胞选自骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞中的至少一种。
4.甘露糖在制备治疗自身免疫性疾病的药物中的应用，其特征在于，包含以下步骤：
（1）取传代至P4代的间充质干细胞，接种于含有10% 胎牛血清和10-50mM甘露糖的α-MEM培养基中，于37℃，浓度为5 %的CO2培养箱中培养24-48h；
（2）将使用甘露糖处理后的间充质干细胞消化计数后，均匀悬于生理盐水或PBS缓冲液，至密度为1×106个/mL-5×106个/mL，获得所述治疗自身免疫性疾病的药物。
5.根据权利要求4所述的应用，其特征在于，所述药物的组分包含间充质干细胞，所述间充质干细胞为经过甘露糖处理的间充质干细胞。
6.根据权利要求4所述的应用，其特征在于，所述间充质干细胞选自骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞中的至少一种。
7.根据权利要求4所述的应用，其特征在于，所述自身免疫性疾病为类风湿关节炎或系统性红斑狼疮。
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