CN110201232B - 一种自组装预血管化干细胞膜片的制备方法 - Google Patents
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Abstract
本发明公开了一种利用间充质干细胞的旁分泌作用在体外自组装形成预血管化干细胞膜片的制备方法，系使用人间充质干细胞(hMSC)在体外成膜片诱导6天，初步形成MSC膜片，之后将内皮细胞(EC)以饱和密度直接接种在其表面，并更换培养液为共培养条件培养基继续培养十天，完成预血管化EC‑MSC复合膜片的构建。与传统的添加生长因子促进膜片血管化的方法相比，本发明之血管化膜片形成的血管网更符合生理形态、血管成熟度高，制备成本低，且在植入体内后可快速与宿主血管形成吻合，无渗漏。该技术可在短时间内重建缺损组织的血管系统，有助于大面积组织缺损的修复与再生，为血管化组织工程提供了新的思路，具有广阔的应用价值。
Description
技术领域
本发明属于组织工程技术领域，尤其涉及一种自组装预血管化干细胞膜片的制备方法。
背景技术
细胞膜片技术是在体外连续培养高密度接种的细胞，使其复层生长，形成一张富含细胞和细胞外基质的膜状结构，并采用非酶解的方式对其进行收获，具有独特的保存细胞-细胞外基质与细胞间信号分子、细胞利用率高、可塑性好等优点。该技术已经成为现代再生医学的一个重要组成部分，并显示出巨大的临床应用前景。当使用多层细胞膜片对组织进行修复时，弥散作用仅能保证表层的细胞存活，而位于内部深层的细胞往往因营养缺乏而发生坏死，从而不能形成良好的组织再生效果。因此，构建含有微血管网络结构的细胞膜片是实现血管化组织工程以达到良好组织再生的关键。
目前，日本学者Sasagawa和Asakawa等设计了单层细胞叠加装置，分别将内皮细胞与成肌细胞或成纤维细胞层层叠加，并通过含因子的内皮培养液诱导形成预血管化的多层成肌细膜片或成胞纤维细胞膜片。国内学者张等将人血管内皮细胞接种于低氧诱导的人间充质干细胞膜片上，用含因子的内皮培养基EGM2诱导形成预血管化的干细胞膜片。以上研究均依赖在培养液里添加外源性促血管生长因子来实现预血管化膜片的构建。干细胞是一类具有自我更新和多向分化潜能的细胞，不仅如此它还具有自分泌和旁分泌多种细胞因子和免疫调节的作用。研究发现，干细胞能表达、合成、分泌生长因子、细胞因子、调节肽等多种生物活性因子，发挥抗凋亡、抗炎和促进血管生成等效应，进而促进缺血后心脏、肾脏和中枢神经器官功能的恢复。已经有大量研究表明MSC能通过旁分泌机制有效修复缺血损伤的血管：MSC可分泌趋化因子，如单核细胞化学趋化蛋白-1(MCP-1)、干细胞衍生因子-1(SDF-1)、巨噬细胞集落刺激因子(M-CSF)、肿瘤坏死因子(TNF-α)及白细胞介素8(IL-8)等细胞因子促进造血过程。在缺氧条件下，MSCs能上调一系列促血管生长因子及抗凋亡生长因子的表达，其中包括血管生成素-1/2(Ang-1/2)、血管内皮生长因子(VEGF)、胎盘生长因子(PLGF)、成纤维细胞生长因子(FGFs)、血小板源生长因子(PDGF)、表皮生长因子(EGF)、转化生长因子-β(TGF-β)、胰岛素样生长因子(IGF-1)和肝细胞生长因子(HGF)等。以上研究提示：干细胞潜在的旁分泌/自分泌功能可能参与调节正常血管的发生过程，包括内皮细胞增殖、分化、迁移、管腔形成等多个环节，进而调节血管生成与重塑。
发明内容
本发明将人间充质干细胞(hMSC)作为种子细胞构建MSC膜片，将血管内皮细胞(EC)与之共培养，利用MSC分泌的多种生物活性因子促进EC的分化、迁移、管腔形成，从而在体外无外源性生长因子的情况下自组装形成含有微血管网络结构的EC-MSC复合膜片。
一种自组装预血管化干细胞膜片的制备方法，按照如下步骤制成：
步骤1.MSC膜片的制备：取原代培养的人间充质干细胞扩增至第4代至第5代，调整细胞密度为2.6-3.6×104个/平方厘米接种在培养板中，第二天更换膜片诱导培养液，隔日换液，在37℃、饱和湿度、5％CO2条件下连续培养6天，初步构建MSC膜片；
步骤2.EC-MSC膜片共培养模型的建立：取原代培养的人内皮细胞扩增至第4代至第5代，调整细胞密度为2.6-4.7×104个/平方厘米接种到初步构建的MSC膜片表面，使内皮细胞和间充质干细胞膜片直接接触并整合，建立EC-MSC膜片共培养模型；
步骤3.预血管化EC-MSC复合膜片的制备：更换共培养EC-MSC膜片的培养液为共培养条件培养基，在37℃、饱和湿度、5％CO2条件下连续共培养10-14天，隔日换液，当在培养皿底部出现乳白色膜样物质，即完成预血管化EC-MSC复合膜片的构建。
所述步骤1中原代培养的人间充质干细胞为人骨髓间充质干细胞、脐带间充质干细胞、脂肪间充质干细胞，每种干细胞对应原代培养的方法不再赘述。
所述步骤2使用的人内皮细胞可为原代培养的人脐动、静脉内皮细胞，肺动、静脉内皮细胞，或者人其他组织来源的内皮细胞，每种内皮细胞对应原代培养的方法不再赘述。
所述步骤1中膜片诱导培养液包含10％胎牛血清(v/v)、0.2-0.4mg/mL谷氨酰胺、50-100μg/ml维生素C，余量为α-MEM培养液。
所述步骤3中共培养条件培养基包含体积百分比为66.7％-33.3％的无因子的内皮基础培养液和体积百分比为33.3％-66.7％(v/v)的低血清膜片诱导培养液。
所述低血清膜片诱导培养液为包含5％胎牛血清(v/v)、0.2-0.4mg/mL谷氨酰胺、50-100μg/ml维生素C，余量为α-MEM培养液。
所述无因子的内皮基础培养液为包含5％胎牛血清(v/v)的商用EBM培养液(美国Sciencell公司生产，货号1001-b)。
优选的，所述共培养条件培养基包含体积百分比为66.7％的无因子的内皮基础培养液，和体积百分比为33.3％的低血清膜片诱导培养液。
优选的，所述共培养条件培养基包含体积百分比为33.3％的无因子的内皮基础培养液，和体积百分比为66.7％的低血清膜片诱导培养液。
优选的，所述共培养条件培养基包含体积百分比为50％的无因子的内皮基础培养液，和体积百分比为50％的低血清膜片诱导培养液。
与现有技术相比，本发明具有如下优异效果：经CD31免疫荧光染色证实，无外源性生长因子的3#、4#、5#培养基配比可促使EC在膜片表面自组装形成含有微血管网络结构的EC-MSC复合膜片，具有制备简便、无外源性因子添加、无支架材料，生物相容性好等优点；进一步对各组膜片进行蛋白芯片和western-blot分析显示3#、4#、5#组培养基配比所生成的膜片血管网更符合生理形态，结构和功能更稳定，在植入体内后可快速与宿主血管形成吻合，实现移植组织的快速血管化。因此有望在短时间内重建大面积缺损组织的血管系统，加速组织缺损的修复与再生，为血管化组织工程提供了新的思路，具有广阔的应用价值。
附图说明
图1是采用本发明方法制备血管化EC-MSC复合膜片的流程图；
图1-A是P1代的MSC的显微照片(40倍)；图1-B是P1代的EC的显微照片(40倍)；图1-C为初步构建的MSC膜片的显微照片(40倍)；图1-D为最终共培养形成的EC-MSC复合膜片的显微照片(40倍)；图1-E为EC-MSC复合膜片的大体照片。
图2为不同共培养时间，不同共培养条件所形成的EC-BMSC膜片血管网的免疫荧光观察图(标尺＝200μm)。
图3为各组EC-BMSC膜片共培养体系上清的蛋白芯片分析图。
图4为各组EC-BMSC复合膜片血管生成相关蛋白的western-blot分析图。
图5膜片与支架材料复合图。
图6为各组膜片血管吻合的体式观察图(标尺＝1mm)。
具体实施方式
下面结合附图和实施例对本发明作详细描述，但本发明的实施不仅限于此。
实施例1血管化EC-BMSC膜片的构建
1.hBMSC的分离、培养和鉴定
采用全骨髓贴壁法分离培养hBMSC：取新鲜无菌骨髓，加入等量PBS混匀，800转/分钟离心5分钟，吸弃上清，清洗三次后加入含15％胎牛血清和0.4mg/mL谷氨酰胺的α-MEM培养基，接种于75cm2培养瓶中。3天后首次半量换液，之后第二天更换全新的培养液去除未黏附的细胞，继续培养贴壁细胞。每2天换液一次，待细胞生长至覆盖培养瓶约80％时，0.25％胰酶消化，传代培养。选择P4-P5代的细胞用于后续操作。
2.hUVEC的分离培养
取无菌游离脐带，分离脐静脉，用PBS缓冲液洗净静脉内血液，向静脉内注入300U/ml胶原酶，扎紧两端，置于无菌容器内，放入CO2培养箱内(37℃，5％CO2)孵育40min。PBS冲洗血管，收集细胞，离心后用培养液洗2次。之后用ECM培养基重悬细胞，并转移至25cm2的培养瓶中，于37℃,5％CO2环境下培养。24小时后更换培养液去除未贴壁的细胞,每2天换液一次至细胞铺满瓶底，0.25％胰酶消化细胞，按1:3比例传代培养。选择P4-5代的细胞进行后续实验。
3.EC-BMSC膜片共培养模型的建立
如图1所示：将BMSC以3×105个/孔的饱和密度接种到六孔板中，第二天将板内的基础培养液更换为膜片诱导培养液(SIM，含10％胎牛血清、0.4mg/mL谷氨酰胺和50μg/mlVc的商用α-MEM培养液)，连续培养6天，每两天换液一次。第8天时加入饱和密度(3×105个/孔)的hUVEC，使内皮细胞和hBMSC膜片直接接触并整合建立EC-BMSC膜片共培养模型。第9天,分别按照表1的配比制备共培养条件培养基，并更换培养液为共培养条件培养基，在常规培养条件下(37℃、饱和湿度、5％CO2)连续共培养10-14天，此时可在培养皿底部看到白色膜状物质，即完成预血管化膜片的构建。
表1共培养培养基成分配比(其中ECM为含因子的内皮培养液(美国Sciencell公司生产，货号1001)；EBM为无因子的内皮基础培养液，为含5％胎牛血清(v/v)的商用EBM培养液(美国Sciencell公司生产，货号1001-b)；LSIM为低血清的膜片诱导培养液包含5％胎牛血清(v/v)、0.4mg/mL谷氨酰胺、50μg/ml Vc，余量为α-MEM培养液)。
表1
4.不同共培养条件形成的EC-BMSC复合膜片血管网的荧光显微观察
分别在共培养第3、7和10天，将各组膜片用PBS轻轻冲洗2遍，4％多聚甲醛固定15min，PBS冲洗2遍；0.1％Triton X-100室温通透15min，PBS洗3遍；每孔加入1％的BSA室温封闭1h，甩干，每孔加入鼠抗人CD31一抗(1∶1000)500μL，37℃孵育2h，PBS清洗3次终止反应；后避光加Cy3标记的鼠二抗500μL/每孔，37℃孵育40min，PBS洗3次；加新鲜配制DAPI避光孵育10min，PBS清洗3次终止反应；然后在荧光体式显微镜下观察拍照。观察不同配比的条件培养基对共培养EC-BMSC膜片血管网形成的影响。
结果如图2所示：3天时1#、2#、3#三组共培养系统中，内皮细胞多以细胞岛的形式分布在膜片表面，而4#、5#和6#这三组共培养系统中的内皮细胞岛已逐渐伸长形成管状结构。随着共培养时间的延长，除2#组外其余各组的管状结构均不断延长并逐步交织成网，十天时含因子的1#组形成大量无序、无规则交织的细小血管网络；3#和6#组内皮岛均延伸形成脉管结构，但网络联接率较低；4#、5#组生成大量规则的血管网，且其形成的血管直径和网络面积明显高于1#组；2#组膜片表面仍然以内皮岛为主，形成的管状结构稀少。
实施例2血管化EC-BMSC复合膜片的体外、体内功能性评估
1.血管化EC-BMSC复合膜片的构建
血管化EC-BMSC复合膜片的构建同实施例1。
2.共培养细胞上清液的蛋白芯片分析
收集不同时间点(3天、7天、10天)共培养体系的细胞上清液，采用蛋白芯片分析共培养上清液中相关分泌性蛋白(表2)表达的差异，结果如图3所示：添加外源性生长因子的1#组其上清液高表达EGF、HGF、FGF、VEGF、Ang-1、Ang-2、MMP-9、IL-8。而外源性高含量的VEGF因子，一方面会使血管内皮细胞转化为大量的尖端细胞，造成膜片表面血管出芽过度，分支数目过度增加，不断启动血管新生；另一方面高含量的VEGF可上调ECM组中Ang2的表达水平，而高含量的Ang2与受体Tie2结合后会竞争性阻断Ang1/Tie2信号通路，抑制Tie2活化的内皮细胞吸引周细胞包围，抑制内皮细胞形成完整的血管壁，破坏血管结构的稳定。结合图2的结果可以得出ECM组形成的大多为新生的管壁结构不完整的初始毛细血管。而不添加外源性生长因子的3#、4#和5#组除高表达IL-6、TNF-α外，其余血管生成相关蛋白VEGF、Ang-1、Ang-2、MMP-9、MMP-2、FGF、SDF-1的表达均在适中水平。结合图2的结果，可以得出：不含外源性因子的3#、4#、5#组其自分泌的生长因子VEGF、Ang-1、Ang-2等与血管的新生更匹配，并促进血管壁成熟，形成的血管网更符合生理形态，有利于膜片血管网结构和功能的稳定。
表2干细胞可分泌的促血管生成的生物活性分子
3.各组膜片血管生成相关蛋白表达的Western blot分析
在各组膜片更换为共培养培养液的第10天终止培养，PBS清洗3次后进行总蛋白的提取，采用Western blot方法分析成血管相关蛋白(MT1-MMP，VEGFR2，VE-Cadherin，α-SMA)的表达。结果如图4所示：1#、2#组高表达与血管新生有关的金属基质蛋白酶类MT1-MMP；而4#、5#组高表达促进血管生成、稳定管壁结构的蛋白VEGFR、VE-Cadherin；且不含因子的3#、4#、5#、6#组高表达周细胞表面标志α-SMA。
4.EC-BMSC复合膜片的体内功能性评估
各组膜片成熟后揭起，分别包裹无菌医用聚氯乙烯软管(图5)。选取4-6周龄SCID免疫缺陷鼠9只，0.1％戊巴比妥钠腹腔(0.15ml/只)麻醉后,背部常规备皮、消毒、铺巾，在背部两侧分别剪开约1cm的切口，稍作分离后，将各组膜片分别植入裸鼠背部皮下，小针细线对位缝合皮肤组织，碘伏消毒，术后做好标记。术后10天颈部脱臼法处死裸鼠，并立即从切口处剪开取出移植的膜片，体视显微镜下观察并拍照。结果如图6所示：1#组膜片血管与宿主未形成良好吻合，局部血液渗漏明显；2#组和6#组表面仅观察到极少量的微血管；而在3#组、4#组和5#组表面均可观察到丰富的血管网，且血管网与宿主吻合程度较高，局部几乎未见血液渗漏。
以上公开的仅为本发明的具体实施例，但是，本发明并非局限于此，任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (7)
1.一种自组装预血管化干细胞膜片的制备方法，其特征在于：选择人间充质干细胞作为种子细胞初步构建MSC膜片；将人内皮细胞直接接种到MSC膜片表面，建立EC-MSC膜片共培养模型；使用共培养条件培养基连续培养10-14天完成预血管化EC-MSC复合膜片的构建；
具体步骤包括：
S1.MSC膜片的制备：取原代培养的人间充质干细胞扩增至第4代至第5代，调整细胞密度为2.6-3.6×104个/平方厘米接种在培养板中，第二天更换膜片诱导培养液，隔日换液，在37℃、饱和湿度、5％CO2条件下连续培养6天，初步构建MSC膜片；
S2.EC-MSC膜片共培养模型的建立：取原代培养的人内皮细胞扩增至第4代至第5代，调整细胞密度为2.6-4.7×104个/平方厘米接种到初步构建的MSC膜片表面，使内皮细胞和间充质干细胞膜片直接接触并整合，建立EC-MSC膜片共培养模型；
S3.预血管化EC-MSC复合膜片的制备：更换共培养EC-MSC膜片的培养液为共培养条件培养基，在37℃、饱和湿度、5％CO2条件下连续共培养10-14天，隔日换液，当在培养皿底部出现乳白色膜样物质，即完成预血管化EC-MSC复合膜片的构建；
步骤S3所述共培养条件培养基包含体积百分比为66.7％-33.3％的无因子的内皮基础培养液，和体积百分比为33.3％-66.7％的低血清膜片诱导培养液；
所述低血清膜片诱导培养液包含5％胎牛血清(v/v)、0.2-0.4mg/mL谷氨酰胺、50-100μg/ml维生素C，余量为α-MEM培养液；
所述无因子的内皮基础培养液包含5％胎牛血清(v/v)，余量为EBM培养液。
2.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，步骤S1中所述原代培养的人间充质干细胞为人骨髓间充质干细胞、脐带间充质干细胞或脂肪间充质干细胞。
3.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，步骤S2所述使用的人内皮细胞为原代培养的人脐动、静脉内皮细胞，或者肺动、静脉内皮细胞。
4.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，步骤S1所述膜片诱导培养液包含10％胎牛血清(v/v)、0.2-0.4mg/mL谷氨酰胺、50-100μg/ml维生素C，余量为α-MEM培养液。
5.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，所述共培养条件培养基包含体积百分比为66.7％的无因子的内皮基础培养液，和体积百分比为33.3％的低血清膜片诱导培养液。
6.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，所述共培养条件培养基包含体积百分比为33.3％的无因子的内皮基础培养液，和体积百分比为66.7％的低血清膜片诱导培养液。
7.根据权利要求1所述一种自组装预血管化干细胞膜片的制备方法，其特征在于，所述共培养条件培养基包含体积百分比为50％的无因子的内皮基础培养液，和体积百分比为50％的低血清膜片诱导培养液。
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