CN113943699B - 对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用 - Google Patents
对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用 Download PDFInfo
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Abstract
本发明提供了对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用。建立了一种模拟糖尿病患者体内的高糖环境诱导培养液，通过添加抗氧化剂GSH，降糖的利拉鲁肽和碱性成纤维细胞生长因子(bFGF)等物质，经过5～10天的体外高糖环境和抗氧化诱导，增加了间充质干细胞的在高糖环境下的细胞活性和功能，能够更有效的促进创面血管和神经的生成，达到移植治疗糖尿病创面的目的。研究证实，经本方法诱导培养的间充质干细胞可显著促进大鼠糖尿病创面皮肤创伤的愈合，具有更好的改善代谢、降低血糖、促进血管再生、促进神经细胞修复、伤口愈合的功能。为糖尿病创面的临床治疗选择提供了优良的种子细胞。
Description
技术领域
本发明属于干细胞培养技术领域，涉及对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用。
背景技术
糖尿病创面愈合不良是糖尿病的严重并发症之一，也一直是是临床治疗的难题。创面愈合是一个非常复杂的过程，涉及到修复细胞、炎症细胞、血管生成和末梢神经受损等多方面的相互协调，需要通过细胞因子、抗氧化作用、能量代谢等细胞微环境进行非常精细的调控。任何因素的失衡均会导致愈合延迟、难以愈合或创面不愈合。
糖尿病患者体内环境复杂，长期的高血糖、高血脂、酮体增多、活性氧增加等，使自身的MSCs动员迁移及损伤修复的能力减弱，创面常常经久不愈。
间充质干细胞(Mesnchymal Stem Cells，MSCs)是一类具有极强的自我更新和多项分化的潜能的细胞，可向骨、软骨、肌肉、韧带、神经等多个方向分化。MSCs是通过分泌的多种细胞因子发挥作用的，而目前的研究发现，在不同的环境刺激下，MSCs的分泌谱会随之发生改变。MSCs局部应用可调控炎症、增加血管化和促进上皮化，被认为是创面最有前景的治疗方法之一。
近年研究显示干细胞疗法对糖尿病创面具有一定的治疗效果。其中脐带间充质干细胞(Umbilical Cord Mesnchymal Stem Cells，UC-MSCs)因来源广泛，免疫原性低，用于移植治疗糖尿病及其并发症研究已经日成趋势。研究显示间充质干细胞能够移动和归巢到受伤和缺血组织，并分泌生长因子以促进血管生成和细胞外基质重塑；同时干细胞还能分化成许多细胞类型，如成纤维细胞、脂肪细胞、上皮细胞等，通过分化作用促进血管生成、神经细胞修复促进糖尿病创面愈合。
上述研究通常采用的是常规的干细胞培养技术，虽然收获一定量的干细胞用于创面的修复，但是由于糖尿病患者复杂的高糖高氧化损伤的身体条件，常常存在治疗效果不理想、修复功效缓慢的现象。
发明内容
针对上述技术问题，本发明提供了对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用。
为实现本发明的目的，本发明采用以下技术方案：
对抗高糖损伤的脐带间充质干细胞诱导液，包括：高糖DMEM培养基，所述高糖DMEM培养基中加有人源化血小板裂解物，GSH，利拉鲁肽，bFGF以及青霉素/链霉素双抗。
H-DMEM培养能够体外模拟糖尿病患者体内的高糖环境，将UC-MSCs在无血清的H-DMEM里培养，能够诱导MSCs耐受高糖环境，使MSCs移植后在糖尿病患者的高糖环境中更好的耐受存活。本诱导培养基选择H-DMEM作为基础培养基，增加UC-MSCs的输注效果。
作为优选，高糖DMEM培养基(H-DMEM)的葡萄糖浓度为4.5g/L。
人源化血小板裂解物，含有多种生长因子(血小板衍生生长因子，PDGF；血管内皮生长因子，VEGF；表皮生长因子，EGF等)和蛋白质，能够有效促进MSCs增殖，且无异源的动物成分，可替代胎牛血清为UC-MSCs提供营养支持，同时有效规避了胎牛血清可能带来的过敏和动物源性疾病风险。
作为优选，培养基中人源化血小板裂解物的浓度为5～10％。
GSH是细胞内含有巯基的抗氧化剂，通过直接参与抗氧化酶的酶促反应，起到抗氧化，抗凋亡作用。还能预防损伤，调整基因表达，调节细胞的代谢活性，参与信号转导***，保护细胞免受氧化损伤，并使内环境处于稳定的还原态。高血糖环境刺激下，导致细胞氧化应激反应增强及抗氧化能力减弱。培养基中添加GSH可以保护间充质干细胞免受氧化应激损伤，保持MSCs的活性和功能，增加干细胞的输注效果。
作为优选，培养基中GSH的浓度为1～20mmol/L。
利拉鲁肽是一种人胰高糖素样肽-1(GLP-1)类似物，用于治疗糖尿病。GLP-1是肠道L细胞分泌的一种肠促胰素，可以通过促进胰岛β细胞增殖，抑制其凋亡来增加β细胞数量。既往研究已经证实，在干细胞分化过程中，GLP-1可以充当诱导分化剂促进干细胞向胰岛素分泌细胞的成熟，提高分化效率。本诱导培养基中添加利拉鲁肽可促进UC-MSC向胰岛素分泌细胞分化。
作为优选，培养基中利拉鲁肽的使用浓度范围是1～10nmol/L。
碱性成纤维细胞生长因子(bFGF)能促进纤维细胞的有丝***，同时可以刺激血管生成、在体内能维持神经元、神经胶质细胞的存活。能促进细胞的黏附、增值与分化。本诱导培养基中添加bFGF可有效促进创面中神经和血管的修复，更有效促进创面愈合。
作为优选，培养基中bFGF的使用浓度范围是10～30ng/mL。
作为优选，培养基中青霉素/链霉素双抗为100U/mL青霉素，100mg/L链霉素。
本发明还提供了对抗高糖损伤的脐带间充质干细胞诱导方法，包括将UC-MSCs的传代细胞接种到培养瓶中，加入上述诱导液，进行诱导分化培养。
作为优选，诱导时间为5～10天。
本发明还提供了通过上述诱导方法得到的脐带间充质干细胞在用于糖尿病创面的药物中的应用。
本发明建立了一种模拟糖尿病患者体内的高糖环境诱导培养液，通过添加抗氧化剂GSH，降糖的利拉鲁肽和碱性成纤维细胞生长因子(bFGF)等物质，经过5～10天的体外高糖环境和抗氧化诱导，增加了间充质干细胞的在高糖环境下的细胞活性和功能，能够更有效的促进创面血管和神经的生成，达到移植治疗糖尿病创面的目的。
本发明的有益效果如下：
(一)针对现有的诱导培养技术存在细胞活性低，诱导转化方法复杂等缺陷，本发明设计了一种体外高糖和抗氧化诱导间充质干细胞的方法。
(二)本发明提供的诱导液用于体外诱导UC-MSCs耐受高糖环境，并且抵抗由于高糖诱导发生的氧化应激，能够减少细胞凋亡损伤。
(三)本发明建立的对抗高糖损伤诱导液诱导的IUC-MSCs能够加速大鼠糖尿病溃疡皮肤创伤的愈合，具有更好的改善代谢、降低血糖、促进血管再生、伤口愈合的功能。为糖尿病创面的临床治疗选择提供了优良的种子细胞。
(四)本发明建立的对抗高糖损伤诱导液诱导的IUC-MSCs相比常规UC-MSCs能分泌更高的HGF、FGF-9、NGF等细胞因子。
附图说明
图1为本发明实施例诱导分化过程中细胞形态学观察图，A.IUC-MSCs细胞；B.常规UC-MSCs细胞；C.单纯高糖培养液培养的UC-MSCs细胞。
图2为本发明实施例体外分泌细胞因子鉴定结果。
图3为本发明实施例三组大鼠创面愈合趋势折线图，A.PBS对照组；B.UC-MSCs组；C.IUC-MSCs组。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
1.UC-MSCs体外分离培养
(1)脐带来源间充质干细胞的分离和原代细胞培养
1)无菌条件下，将脐带(脐带组织采集自足月剖宫产的健康产妇，采集之前与产妇及其家属均签署知情同意书，方案经医院医学伦理会批准。)用含青霉素/链霉素双抗的生理盐水反复冲洗，洗去脐带残留血液，将脐带剪成3.0-4.0cm的小段，每段均沿脐静脉腔剪开，平铺后剔除静脉和动脉，取出血管之间、血管与外膜之间的胶状物—华通胶，用小剪刀将华通胶剪切成约1mm3小块，接种于T75培养瓶内，使脐带华通胶均匀分布瓶底。置于二氧化碳培养箱内培养，培养箱内温度为37℃，CO2浓度为5％，贴壁4～18hr后加入15ml DM-PL干细胞培养液。进行原代细胞的爬出。
DM-PL干细胞培养液的配制：DMEM/F12基础培养液加入体积分数为5％人源化血小板裂解物。
2)5d后首次全量换液，弃去未贴壁细胞，每3～4d换液一次。倒置相差显微镜观察贴壁细胞，待细胞游出后，待细胞长至75％～85％融合时，采用0.25％胰蛋白酶于37℃消化贴壁细胞不超过5分钟。加入完全培养液终止消化，收集细胞悬液置50ml离心管，离心，1200rpm，5min，弃去含胰酶上清液，生理盐水清洗两次，弃上清，得到原代人UC-MSCs。
(2)细胞传代培养
将获得的原代细胞置于新的培养液中进行传代培养，倒置显微镜观察贴壁细胞融合达75％～85％，胰酶消化，生理盐水清洗2次，得到P1细胞；加入新的培养液按照1:3～1:4的比例重新进行接种，进行传代培养，并使用同样方法重复进行P2和P3代人脐带间充质干细胞的培养。培养温度为37℃，环境空气为5％CO2。
2.UC-MSCs体外诱导培养
将P3代UC-MSCs接种到新的T75培养瓶中，加入高糖抗氧化损伤培养液，进行诱导分化培养。常规培养基培养的细胞和单纯高糖抗氧化损伤培养液培养的细胞为对照细胞。诱导时间为9天。
常规培养基成分为DMEM培养液中添加10％FBS；
高糖抗氧化损伤培养液成分为高糖DMEM培养基(H-DMEM，4.5g葡萄糖/L)中添加5％人源化血小板裂解物，10mmol/L GSH，5nmol/L利拉鲁肽，100U/mL青霉素，100mg/L链霉素，20ng/mL bFGF。
3.诱导分化过程中细胞形态学观察
本发明诱导获得的IUC-MSCs细胞(A图)可稳定贴壁生长，所获得的细胞生物学特征同常规培养方法(B图)得到的细胞特征相似，显微镜下细胞为典型的梭形漩涡状两者都呈现梭行，为正常干细胞形态，两者没有大的差别。
单独用高糖培养液(C图)培养的UC-MSCs细胞，细胞生长缓慢，部分细胞变圆，胞核较大，细胞界限清晰。后期基本停止生长，出现凋亡状态。
4.UC-MSCs体外分泌细胞因子鉴定
实验分为2组：①UC-MSCs：脐带间充质干细胞组；②IUC-MSCs，诱导后脐带间充质干细胞组。分别取两组细胞上清液进行干细胞生长因子(HGF)、成纤维细胞生长因子(FGF)、神经生长因子(NGF)的检测。如图2可见，经IUC-MSCs组间充质干细胞分泌的组织创伤修复相关细胞因子显著高于非诱导组。且具有统计学意义，***，P<0.001，**P<0.01。
5.UC-MSCs治疗糖尿病创面
(1)糖尿病创面试验模型建立
Wistar雄性大鼠(6-8周龄，160-180g重)适应性饲养1周后，腹腔注射链脲佐菌素(streptozotocin，STZ)制作大鼠糖尿病模型，注射3天后，监测大鼠的血糖、体重、饮水量和尿量，待出现血糖≥16.7mmol/L及多饮、多尿、多食且体重减轻等糖尿病症状后，用打孔器在大鼠背部制作6mm×6mm的圆形全层皮肤缺损创面，建立大鼠糖尿病创面模型。
(2)干细胞准备
将诱导培养好的UC-MSCs细胞终止培养，生理盐水冲洗2遍，0.25％胰蛋白消化，进行细胞活性检测。细胞活性90％以上的细胞进行计数，PBS清洗两遍，将不同数量细胞分别溶于1ml PBS溶液，混匀备用。每毫升细胞悬液所含细胞数量为0.5～1.5×107。
(3)干细胞移植治疗
1)造模成功后，大鼠随机分成3组：A组为糖尿病创面对照组(PBS对照组)；B组为常规脐带间充质干细胞组(UC-MSCs组)；C组为高糖抗氧化诱导培养脐带间充质干细胞组(IUC-MSCs组)。每组15只大鼠。
2)用微量进样器抽取细胞悬液UC-MSCs及IUC-MSCs后沿创面边缘皮下注射，每个创面选择8个注射点，每点注射10μl。对照组抽取PBS沿创面边缘皮下注射，每个创面选择8个注射点均匀注射，每点注射10μl。
3)对各组大鼠分别在创面形成术后当天(0天)、第3天、第7天、第11天、第14天的创面愈合情况跟踪检测。计算不同时间点的创面愈合率，公式如下：
愈合率＝(原始创面面积－剩余创面面积)/原始创面面积×100％
(4)实验结果
第14天IUC-MSCs组大鼠创面的愈合率时间为79％，与UC-MSCs组相比无差异。PBS组愈合率较低48％，与各组相比均有显著差异(P<0.05)。与PBS对照组相比，局部注射脐带间充质干细胞能明显加快皮肤溃疡面积的愈合。两组干细胞组相比，用高糖抗氧化损伤诱导过的UC-MSCs具有更好的促进糖尿病创面皮肤愈合效果，表明诱导的IUC-MSCs具有比普通间充质干细胞更好的改善代谢、降低血糖、促进血管再生、伤口愈合的功能。
Claims (5)
1.对抗高糖损伤的脐带间充质干细胞的诱导方法，其特征在于，所述方法包括将脐带间充质干细胞的传代细胞接种到培养瓶中，加入对抗高糖损伤的脐带间充质干细胞诱导液，进行诱导分化培养获得对抗高糖损伤的脐带间充质干细胞；所述对抗高糖损伤的脐带间充质干细胞诱导液为添加有5％人源化血小板裂解物，10mmol/L GSH，5nmol/L利拉鲁肽，100U/mL青霉素，100mg/L链霉素和20ng/mL bFGF的高糖DMEM培养基。
2.根据权利要求1所述的诱导方法，其中所述高糖DMEM培养基中葡萄糖浓度为4.5g/L。
3.根据权利要求1或2所述的诱导方法，其特征在于，所述诱导分化培养为5～10天。
4.根据权利要求1或2所述的诱导方法，其特征在于，所述诱导分化培养为9天。
5.使用根据权利要求1-4任一项所述诱导方法获得的对抗高糖损伤的脐带间充质干细胞在制备用于治疗糖尿病创面的药物中的应用。
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