CN114377208A - 微型活性真皮替代物及其构建方法 - Google Patents
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Abstract
本发明属于皮肤组织工程技术和医药生物技术领域，具体提供了一种微型活性真皮替代物，具有这样的特征：以活性微粒羊膜为微载体构建而成。本发明还提供了一种微型活性真皮替代物的构建方法，具有这样的特征，包括以下步骤：制备活性微粒羊膜；成纤维细胞的分离培养；旋转培养系统中同时加入培养的成纤维细胞以及活性微粒羊膜，旋转培养得到微型活性真皮替代物。
Description
技术领域
本发明属于皮肤组织工程技术和医药生物技术领域，具体涉及一种微型活性真皮替代物及其构建方法。
背景技术
含成纤维细胞的活性真皮替代物的构建与移植是皮肤组织工程研究中的重要内容，可帮助创面快速重构真皮，提高创面愈合速度并改善创面愈合质量，在临床应用时显示出良好的疗效。
目前传统活性真皮替代物经多次消化传代培养以扩增一定数量的成纤维细胞，然后接种于真皮支架上进行2-3周的培养构建活性真皮替代物。此两步法构建方式不仅耗时长，而且反复消化、长时间培养会对成纤维细胞造成损伤，影响其增殖；同时，移植到创面后成纤维细胞缺乏足够营养支持及创面炎症因子等对成纤维细胞的直接损伤，使得移植后细胞在创面存活率低。因此，构建新型真皮替代物，缩短细胞体外培养时间，维持细胞高增殖活性，改善创面微环境提高活性真皮替代物移植存活率，具有重大的临床意义。
人羊膜主要包括单层羊膜上皮细胞层及含极少量成纤维细胞的无血管基质层。羊膜上皮细胞具有干细胞特征，能向三个胚层细胞分化并能通过旁分泌多种因子改善创面微环境，进而促进创面修复；羊膜基质层成分与人体真皮成分类似，不仅含有天然的基底膜结构，且富含I-VII型胶原、糖胺聚糖、层黏连蛋白、整合素、弹性蛋白等多种细胞外基质成分，能作为真皮支架提供天然的土壤促进表皮细胞、成纤维细胞粘附增殖和分化。本发明人在前期研究中将大张新鲜羊膜经过自制微粒皮切割机直接切割为300-600μm微载体大小的活性微粒羊膜后，发现其能完好保存羊膜天然结构、羊膜上皮细胞活性及羊膜生物活性因子，应用到创面后通过调控创面炎症反应、促进新生血管化加速创面修复(Zheng Y,Ji S,WuH,Tian S,Zhang Y,Wang L,Fang H,Luo P,Wang X,Hu X,Xiao S and Xia Z.Topicaladministration of cryopreserved living micronized amnion accelerates woundhealing in diabetic mice by modulating local microenvironment.Biomaterials2017；113:56-67)。此研究成果也获得了中国发明专利，发明名称为“负载羊膜固有干细胞的冻存活性羊膜微粒及其条件培养基与应用”，专利号201510435208.0，授权公告号为CN105018417A。
文献报道300-1000微米大小的微载体结合旋转细胞培养系统能为细胞的培养提供较大表面积和三维立体空间，模拟细胞体内生长环境，快速大量扩增细胞的同时一步法同步构建含细胞的活性组织工程皮肤，从而极大缩短复合皮体外构建时间；同时，加入新的微载体就可通过微载体间的接触、碰撞完成细胞到新的微载体的转移，从而实现细胞的自动扩增，避免反复消化培养对细胞活性的损伤。然而，能否直接使用活性羊膜微粒作为微载体扩增成纤维细胞，一步法构建微型活性真皮替代物，目前未见报道。
发明内容
本发明是为了解决上述问题而进行的，目的在于提供一种微型活性真皮替代物及其构建方法。
本发明提供了一种微型活性真皮替代物，具有这样的特征：以活性微粒羊膜为微载体构建而成。
在本发明提供的微型活性真皮替代物中，还可以具有这样的特征：其中，活性微粒羊膜由新鲜羊膜经微粒皮切割机制备成300μm-600μm大小的活性羊膜微粒，该活性羊膜微粒包含完整的羊膜上皮细胞层及羊膜基质层。
在本发明提供的微型活性真皮替代物中，还可以具有这样的特征：其中，微型活性真皮替代物包括活性微粒羊膜以及成纤维细胞。
在本发明提供的微型活性真皮替代物中，还可以具有这样的特征：其中，成纤维细胞来自于包皮或皮肤。
本发明提供了一种微型活性真皮替代物的构建方法，具有这样的特征，包括以下步骤：制备活性微粒羊膜；成纤维细胞的分离培养；旋转培养系统中同时加入培养的成纤维细胞以及活性微粒羊膜，旋转培养得到微型活性真皮替代物。
在本发明提供的微型活性真皮替代物的构建方法中，还可以具有这样的特征：其中，旋转培养系统中同时加入的成纤维细胞以及活性微粒羊膜的体积质量比为20ml：600mg，成纤维细胞的浓度为1×105个/ml，活性微粒羊膜的质量为湿重。
在本发明提供的微型活性真皮替代物的构建方法中，还可以具有这样的特征：其中，旋转培养的旋转速度为20rpm。
在本发明提供的微型活性真皮替代物的构建方法中，还可以具有这样的特征，还包括：其中，向旋转培养得到微型活性真皮替代物中加入新的活性羊膜微粒，成纤维细胞向新的活性羊膜微粒的表面迁移，从而实现成纤维细胞的自动扩增及成纤维细胞-活性微粒羊膜复合皮的一步法同步构建。
发明的作用与效果
根据本申请所提供的微型活性真皮替代物及其构建方法，因为以活性微粒羊膜为微载体构建微型活性真皮替代物，活性微粒羊膜具有完好的羊膜组织结构及丰富的羊膜生物活性因子，能为成纤维细胞的扩增提供理想的土壤-营养作用，一步法实现成纤维细胞的快速扩增及活性真皮替代物的同步构建，缩短复合皮体外构建时间并避免反复消化培养对成纤维细胞造成的损伤。
此外，因为活性微粒羊膜具有主动调控创面微环境的天然优势，移植成纤维细胞-活性微粒羊膜复合皮于创面后不仅能直接调控创面微环境促进创面内源性修复，且能为成纤维细胞在创面的存活提供更理想的生长微环境，提高移植后细胞存活率。
另外，羊膜为胎盘废弃组织，以此构建活性真皮替代物，价格低廉，能最大程度实现羊膜组织“变废为宝”。
附图说明
图1为本发明的实施例中活性微粒羊膜结合旋转细胞培养系统扩增成纤维细胞的状态图，图1A为培养1天的相差显微镜下照片，图1B为培养1天的荧光显微镜下照片，图1C为培养4天的相差显微镜下照片，图1D为培养4天的荧光显微镜下照片；
图2为本发明的实施例中成纤维细胞在活性微粒羊膜结合旋转体系中培养与普通培养皿中培养的相对增殖活性比较图；
图3为本发明的实施例中微型活性真皮替代物中羊膜上皮细胞干性标志物OCT-4的表达图；
图4为本发明的实施例中微型活性真皮替代物中生长因子、炎症因子及趋化因子的表达。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，以下实施例结合附图对本发明微型活性真皮替代物及其构建方法作具体阐述。
<实施例>
本实施例提供了一种微型活性真皮替代物及其构建方法。
微型活性真皮替代物的构建方法包括以下步骤：
步骤1，制备活性微粒羊膜。
经医院伦理委员会批准，产妇知情同意，选择肝炎病毒抗体、梅毒抗体及HIV均为阴性的剖腹产妇的新鲜胎盘组织。在无菌条件下钝性分离绒毛膜，剥离去除纤维母细胞层和海绵层，抗生素清洗后经自制微粒皮切割机制备成300μm-600μm大小的活性羊膜微粒，其包含完整的羊膜上皮细胞层及羊膜基质层，能为后续成纤维细胞的扩增提供理想的土壤-营养作用，并且活性微粒羊膜具有主动调控创面微环境的天然优势，能为后续成纤维细胞在创面的存活提供更理想的生长微环境，提高移植后细胞存活率。
上述制备过程为现有技术，具体可参见本申请人已获得授权的专利，发明名称为“负载羊膜固有干细胞的冻存活性羊膜微粒及其条件培养基与应用”，专利号为2015104352080。
步骤2，成纤维细胞的分离培养。
患者同意并签署知情同意书，获取健康儿童包皮环切术后丢弃的包皮组织，反复PBS清洗后，剪成1cm左右大小的组织块，dispase酶4℃消化过夜后剥去表皮，真皮部分使用0.2％胶原蛋白酶消化约2-3h，悬液采用200目滤网过滤后收集离心，完全培养基重悬培养，使用第2-3代细胞进行下一步实验。
步骤3，旋转培养系统培养。
旋转培养系统(RCCS)中同时加入20ml成纤维细胞(浓度为1×105个/ml)及600mg湿重活性微粒羊膜，旋转速度20rpm，4天后羊膜微粒布满成纤维细胞。此时，布满成纤维细胞的羊膜微粒已经可以作为微型活性真皮替代物使用。
此外，在上述羊膜微粒布满成纤维细胞后，还可以向旋转培养系统(RCCS)中加入新的活性羊膜微粒，实现成纤维细胞向新的微粒表面迁移，完成成纤维细胞的自动扩增及成纤维细胞-活性微粒羊膜复合皮的一步法同步构建。这样可以获取更多的微型活性真皮替代物。
验证实验
1.实验方法
1.1 CCK-8法检测成纤维细胞增殖活性
为比较成纤维细胞增殖活性，以6孔板的普通二维培养作为对照，每孔中加入1ml密度为1×105个/ml的成纤维细胞；旋转培养系统中加入20ml密度为1×105个/ml的成纤维细胞及600mg湿重活性微粒羊膜，或单独20ml完全培养基中加入600mg湿重活性微粒羊膜，分别培养1天、3天、5天、7天、9天、11天后采用CCK-8试剂盒检测成纤维细胞增殖活性。检测时从旋转培养系统中取出1ml微型活性真皮替代物或1ml活性微粒羊膜置于6孔板中，每孔加入100μlCCK-8试剂，避光孵育培养3小时后使用酶标仪检测每孔在450nm吸光度下的OD值。旋转培养系统中成纤维细胞增殖值＝微型活性真皮替代物OD值-活性微粒羊膜OD值。检测对照组中普通二维培养时成纤维细胞增殖，每孔加入100μl CCK-8试剂，避光孵育培养3小时后使用酶标仪检测每孔在450nm吸光度下的OD值。
相对增殖活性(％)＝第n天OD值/第1天OD值×100％
1.2免疫荧光检测OCT-4的表达
采用免疫荧光的方法检测微型活性真皮替代物中羊膜上皮细胞干性标志物OCT-4的表达，具体步骤如下：
取旋转培养系统中培养了4天的微型活性真皮替代物，4％多聚甲醛室温固定30分钟；PBS洗涤3次，每次5分钟，0.1％Triton X-100孵育5分钟并使用1％BSA溶液封闭1小时；以1:100的稀释比加入Alexa Fluor488荧光Anti-Oct4抗体(Abcam公司)于4℃孵育过夜，而后加入DAPI避光孵育5分钟，PBS洗涤3次后置于荧光倒置显微镜下观察并拍照。
1.3 ELISA检测生长因子、炎症因子及趋化因子的表达
取旋转培养系统中培养了4天的微型活性真皮替代物及活性微粒羊膜，DMEM培养基反复清洗后，以无血清培养基继续培养24小时，收集上清后离心去除细胞碎片，并使用0.22μm滤膜过滤后-80℃保存备用。检测时按照ELISA试剂盒操作流程，简单来说：以包被抗体包被孔底，加入待检样本后加入酶标抗体，而后加入底物液显色，加入终止液后立即将96孔板置于酶标仪，读数450mm波长下的OD值，重复3个复孔。以标准品的OD值绘制标准曲线，并应用标准曲线计算所测因子浓度。
2.统计分析
统计分析采用软件SPSS 20.0，计数资料的多组数据之间的比较采用ANOVA分析，两组数据之间的比较采用t检验，P<0.05认为差异有统计学意义。
3.实验结果
图1为本发明的实施例中活性微粒羊膜结合旋转细胞培养系统扩增成纤维细胞的状态图，图1A为培养1天的相差显微镜下照片，图1B为培养1天的荧光显微镜下照片，图1C为培养4天的相差显微镜下照片，图1D为培养4天的荧光显微镜下照片。
如图1所示，旋转培养系统中加入绿色荧光标记的成纤维细胞与活性微粒羊膜，培养1天后相差显微镜下观察(图1A)与荧光显微镜下观察(图1B)，可见成纤维细胞大量粘附于活性微粒羊膜表面；培养4天时可见成纤维细胞在活性微粒羊膜上密布(图1C和图1D)。
图2为本发明的实施例中成纤维细胞在活性微粒羊膜结合旋转体系中培养与普通培养皿中培养的相对增殖活性比较图。
如图2所示，与传统二维培养板相比，旋转体系中成纤维细胞增殖活性明显增高。第7天时培养板中细胞生长逐渐进入平台期，而旋转体系中细胞仍大量增殖。
图3为本发明的实施例中微型活性真皮替代物中羊膜上皮细胞干性标志物OCT-4的表达图。
如图3所示，含成纤维细胞和活性微粒羊膜的微型活性真皮替代物中羊膜上皮细胞仍维持正常干性，培养4天后免疫荧光可见OCT-4高表达。
图4为本发明的实施例中微型活性真皮替代物中生长因子、炎症因子及趋化因子的表达。
如图4所示，微型活性真皮替代物中高表达bFGF、IL-6、IL-8、Laminin、TGF-β1、MCP-1、MIP-1β、IGFBP-2等生长因子、炎症因子及趋化因子；相比于活性微粒羊膜(LMAM)，微型活性真皮替代物(HFB-LMAM)中旁分泌的bFGF、IL-6、IL-8、Laminin、TGF-β1等因子明显增多，而MCP-1、MIP-1β、IGFBP-2等因子的表达在两组之间无明显差异。
上述实施方式为本发明的优选案例，并不用来限制本发明的保护范围。
例如，本实施例中，成纤维细胞来自于儿童的包皮，在实际使用时，成纤维细胞还可以来自于健康人的皮肤。
Claims (8)
1.一种微型活性真皮替代物，其特征在于：以活性微粒羊膜为微载体构建而成。
2.根据权利要求1所述的微型活性真皮替代物，其特征在于：
其中，所述活性微粒羊膜由新鲜羊膜经微粒皮切割机制备成300μm-600μm大小的活性羊膜微粒，该活性羊膜微粒包含完整的羊膜上皮细胞层及羊膜基质层。
3.根据权利要求1所述的微型活性真皮替代物，其特征在于：
其中，所述微型活性真皮替代物包括所述活性微粒羊膜以及成纤维细胞。
4.根据权利要求3所述的微型活性真皮替代物，其特征在于：
其中，所述成纤维细胞来自于包皮或皮肤。
5.一种微型活性真皮替代物的构建方法，其特征在于，包括以下步骤：
制备活性微粒羊膜；
成纤维细胞的分离培养；
旋转培养系统中同时加入培养的所述成纤维细胞以及所述活性微粒羊膜，旋转培养得到微型活性真皮替代物。
6.根据权利要求5所述的微型活性真皮替代物的构建方法，其特征在于：
其中，所述旋转培养系统中同时加入的所述成纤维细胞以及所述活性微粒羊膜的体积质量比为20ml：600mg，
所述成纤维细胞的浓度为1×105个/ml，所述活性微粒羊膜的质量为湿重。
7.根据权利要求5所述的微型活性真皮替代物的构建方法，其特征在于：
其中，所述旋转培养的旋转速度为20rpm。
8.根据权利要求5所述的微型活性真皮替代物的构建方法，其特征在于，还包括：
向旋转培养得到所述微型活性真皮替代物中加入新的活性羊膜微粒，成纤维细胞向新的所述活性羊膜微粒的表面迁移，从而实现成纤维细胞的自动扩增及成纤维细胞-活性微粒羊膜复合皮的一步法同步构建。
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