CN111909888A - 用于构建屏障加强体外重组表皮模型的ta培养液 - Google Patents
用于构建屏障加强体外重组表皮模型的ta培养液 Download PDFInfo
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- CN111909888A CN111909888A CN202010689504.4A CN202010689504A CN111909888A CN 111909888 A CN111909888 A CN 111909888A CN 202010689504 A CN202010689504 A CN 202010689504A CN 111909888 A CN111909888 A CN 111909888A
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Abstract
本发明提供一种用于构建屏障加强体外重组表皮模型的TA培养液，涉及组织工程技术领域，能够从化学组成及物理结构两方面加强组织工程重建表皮模型的屏障功能，满足皮肤吸收和渗透试验要求，扩大了产品的应用范围。
Description
技术领域
本发明涉及组织工程技术领域，尤其涉及一种用于构建屏障加强体外重组表皮模型的TA培养液。
背景技术
皮肤是人体与外界环境接触的屏障，保护体内各种组织和器官免受物理性、机械性、化学性和病原微生物的侵袭。人体皮肤主要由三部分构成，即表皮层、真皮层和皮下组织。表皮层位于皮肤的最外层，由基底层、棘细胞层、颗粒细胞层和角质层构成。角质层是表皮细胞分化的最后产物，由角质形成细胞及其细胞间脂质基质组成，厚度大约为10μm，是皮肤屏障功能的主要结构，因此角质层的屏障功能一直是组织工程皮肤的主要研究课题，很多研究者在如何提高组织工程皮肤模型角质层屏障方面付诸了大量努力。
近年来，有越来越多的证据表明细胞紧密连接对维持机体表皮正常结构及功能具有重要作用，承担着部分皮肤屏障功能。紧密连接是一种细胞与细胞之间的连接结构，它的主要功能是：将表皮细胞紧密连接，使细胞不易受到破坏；控制分子的细胞旁通路，对分子大小、离子类型、细胞渗透性有选择性，具有细胞间透过屏障功能；维持细胞的极性，分离细胞膜基底部分和胞膜顶部的脂质；紧密连接蛋白还参与信号转导通路；此外，紧密连接可能还参与表皮钙离子梯度(Ca2+)形成和板层小体的极性分泌过程，是皮肤屏障功能的重要组成部分和关键调节因子。
现有的组织工程皮肤模型产品虽然在一般的结构、组成和生物化学方面与人类皮肤具有高度相似性，可用于皮肤毒性测试，但其渗透性相比于人类皮肤仍然要高得多。造成模型屏障功能缺陷的因素除了脂质组成和板层结构上的异常外，可能还有细胞间连接结构的不完整，角质层正常脱落过程受损和非角化的微观病灶的存在，这些方面的缺陷导致现有的重组表皮模型屏障功能不完善，限制了这些皮肤模型在皮肤吸收和渗透实验等其他方面的应用。
具有成纤维细胞真皮层和表皮层的全层皮肤模型虽然在组织结构上更类似于人体天然皮肤，并且能在一定程度上降低渗透性，但真皮层的加入又会引入其他影响，如在模型制备过程中，下层成纤维细胞的来源，数量以及状态的差异性对于表皮细胞的增殖及分化有较大影响，造成模型稳定性差，影响模型对化学物质评判的准确性。另外由于成纤维细胞在体外分化的过程中，会自身分泌的胶原，对外界的胶原支架或者其他生物材料支架进行塑形，易导致真皮部分乃至模型收缩，引起测试物从模型边缘的渗漏，干扰测试结果的评判；最后，成纤维细胞的存在也要求构建体系的培养液必须含有血清，引进了不可预估的安全风险。并且，模型构建工艺繁琐，制备周期长，结构复杂。
发明内容
本发明提供了一种屏障加强的体外重组表皮模型的构建方法，能够从两方面加强组织工程重建表皮模型的屏障功能，满足皮肤吸收和渗透试验要求，扩大了产品的应用范围。
为达到上述目的，本发明采用如下技术方案：
本发明提供过一种屏障加强的体外重组表皮模型的构建方法，该方法包括以下步骤：
1、表皮模型构建培养基配制：
(1)表皮细胞培养液配制：以KC-growth培养液为基础液，每500ml中添加L-谷氨酰胺1.0～1.5mM和牛垂体提取物12～25mg。
(2)表皮细胞TU培养液：在(1)的基础上添加表皮生长因子0.5～5μg、氢化可的松2～50μg、胰岛素1～10mg、腺嘌呤5～25mg、转铁蛋白2.5～6μg、氯化钙0.03～0.2mM。
(3)表皮细胞TA培养液：在(2)基础上，每500ml中添加脂质混合物(L-丝氨酸0.25～5mM、棕榈酸0.4～5μM、亚油酸0.01～0.05mM、精氨琥珀酸0.025～0.05mM、牛血清白蛋白0.01～0.05mM)、巨噬细胞活化肽2(Macrophage-activating Lipopeptide MALP2)2.5～5.0mg、维生素C15～25μg，调整氯化钙浓度为1.0～1.5mM。
2、表皮干细胞分离和培养：
表皮干细胞分离：将人体皮肤组织置于培养皿中，加入75％酒精10mL，迅速进行冲洗，转入PBS溶液中，用剪刀剥离去除皮下疏松结缔组织，用PBS浸洗，将组织块切成0.3cm×0.5cm的大小，加入15ml质量比为1％的Dispase消化液，4℃消化过夜，分离真皮、表皮，收集表皮皮片，剪为碎块，再加入10ml质量比为0.025％的EDTA-胰酶消化液，37℃消化10分钟，获得分离成单个细胞的表皮细胞溶液，用200目不锈钢筛网过滤表皮碎片，1000rpm离心7分钟，去掉上层清液，用PBS洗涤下层细胞，然后1000rpm离心7分钟，倒掉上清液，加入表皮细胞培养液，按2～4×106/瓶接种到IV型胶原包被的培养瓶中，37℃，5％CO2培养箱中孵育10-15min，吸出培养基，更换新鲜表皮细胞培养液继续培养，隔48h换一次液。
3、组织工程表皮构建：
将步骤2中获得的表皮干细胞用胰酶消化液消化，用表皮细胞TU培养液制成密度为1×106～6×106个/ml的表皮干细胞分散液，取200μl接种于含有聚碳酸酯滤膜的细胞培养小室中，震荡均匀，在37℃，5％CO2培养箱中孵育20～30min。将接种后的细胞培养小室转入表皮模型培养模具中，小室外的培养皿中加入新鲜表皮细胞TU培养液，37℃，5％CO2培养箱继续培养24～48h后，吸去细胞小室中的剩余培养基，将细胞培养小室升至气液面，更换表皮细胞TA培养液，37℃5％CO2培养箱继续培养12～14天后可得具有形态分化完全的基底层、棘层、颗粒层、角质层的组织工程表皮模型。
本发明采用表皮干细胞作为种子细胞，聚碳酸脂膜作为支架构建用于渗透试验的屏障加强的重组表皮模型。表皮干细胞增殖和分化能力强，在无真皮层支持的条件下形成的表皮组织形态和结构完整、功能和活性体外维持时间较长；聚碳酸脂膜具有无毒性、生物相容性好的特点，以此为支架材料标准化程度高、批间差异小、生产周期短。本发明中还在气液面培养阶段中添加了脂质混合物和巨噬细胞活化肽2，脂质混合物的添加通过调节表皮细胞脂质合成，改善了角质层的脂质组成和屏障功能，同时巨噬细胞活化肽2通过调节紧密连接蛋白的表达，增强紧密连接渗透屏障，从两方面加强组织工程重建表皮模型的屏障功能，能够满足皮肤吸收和渗透试验要求，扩大了产品的应用范围。
附图说明
图1为本发明实施例提供的皮肤刺激性体外重组表皮模型和屏障加强的重组表皮模型组织学染色结果对比图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行详细地描述。
实施例1
本发明实施例提供一种屏障功能加强的体外重组表皮模型构建方法，具体包括以下步骤：
1、表皮模型构建培养基配制：
表皮细胞培养液配制：以KC-growth培养液为基础液，每500ml中添加L-谷氨酰胺1.0～1.5mM和牛垂体提取物12～25mg。
表皮细胞TU培养液：在(1)的基础上添加表皮生长因子0.5～5μg、氢化可的松2～50μg、胰岛素1～10mg、腺嘌呤5～25mg、转铁蛋白2.5～6μg、氯化钙0.03～0.2mM。
表皮细胞TA培养液：在(2)基础上，每500ml中添加脂质混合物(L-丝氨酸0.25～5mM、棕榈酸0.4～5μM、亚油酸0.01～0.05mM、精氨琥珀酸0.025～0.05mM、牛血清白蛋白0.01～0.05mM)、巨噬细胞活化肽2(Macrophage-activating Lipopeptide MALP-2)2.5～5.0mg、维生素C15～25μg，调整氯化钙浓度为1.0～1.5mM。
2、表皮干细胞分离和培养：
表皮干细胞分离：将人体皮肤组织置于培养皿中，加入75％酒精10mL，迅速进行冲洗，转入PBS溶液中，用剪刀剥离去除皮下疏松结缔组织，用PBS浸洗，将组织块切成0.3cm×0.5cm的大小，加入15ml质量比为1％的Dispase消化液，4℃消化过夜，分离真皮、表皮，收集表皮皮片，剪为碎块，再加入10ml质量比为0.025％的EDTA-胰酶消化液，37℃消化10分钟，获得分离成单个细胞的表皮细胞溶液，用200目不锈钢筛网过滤表皮碎片，1000rpm离心7分钟，去掉上层清液，用PBS洗涤下层细胞，然后1000rpm离心7分钟，倒掉上清液，加入表皮细胞培养液，按2～4×106/瓶接种到IV型胶原包被的培养瓶中，37℃，5％CO2培养箱中孵育10～15min，吸出培养基，更换新鲜表皮细胞培养液继续培养，隔48h换一次液。
3、组织工程表皮构建：
将步骤2中获得的表皮干细胞用胰酶消化液消化，用表皮细胞TU培养液制成密度为1.25×106个/ml的表皮干细胞分散液，取200μl接种于含有聚碳酸酯滤膜的细胞培养小室中，震荡均匀，在37℃，5％CO2培养箱中孵育20～30min。将接种后的细胞培养小室转入表皮模型培养模具中，小室外的培养皿中加入新鲜表皮细胞TU培养液，37℃，5％CO2培养箱继续培养48h后，吸去细胞小室中的剩余培养基，将细胞培养小室升至气液面，更换表皮细胞TA培养液，37℃，5％CO2培养箱继续培养14天后可得具有形态分化完全的基底层、棘层、颗粒层、角质层的组织工程表皮模型。
实施例2
本发明实施例提供一种屏障功能加强的体外重组表皮模型构建方法，具体包括以下步骤：
1、表皮模型构建专用培养基配制：
表皮细胞培养液配制：以KC-growth培养液为基础液，每500ml中添加L-谷氨酰胺1.0～1.5mM和牛垂体提取物12～25mg。
表皮细胞TU培养液：在(1)的基础上添加表皮生长因子0.5～5μg、氢化可的松2～50μg、胰岛素1～10mg、腺嘌呤5～25mg、转铁蛋白2.5～6μg、氯化钙0.03～0.2mM。
表皮细胞TA培养液：在(2)基础上，每500ml中添加脂质混合物(L-丝氨酸0.25～5mM、棕榈酸0.4～5μM、亚油酸0.01～0.05mM、精氨琥珀酸0.025～0.05mM、牛血清白蛋白0.01～0.05mM)、巨噬细胞活化肽2(Macrophage-activating Lipopeptide MALP-2)2.5～5.0mg、维生素C15～25μg，调整氯化钙浓度为1.0～1.5mM。
2、表皮干细胞分离和培养：
表皮干细胞分离：将人体皮肤组织置于培养皿中，加入75％酒精10mL，迅速进行冲洗，转入PBS溶液中，用剪刀剥离去除皮下疏松结缔组织，用PBS浸洗，将组织块切成0.3cm×0.5cm的大小，加入15ml质量比为1％的Dispase消化液，4℃消化过夜，分离真皮、表皮，收集表皮皮片，剪为碎块，再加入10ml质量比为0.025％的EDTA-胰酶消化液，37℃消化10分钟，获得分离成单个细胞的表皮细胞溶液，用200目不锈钢筛网过滤表皮碎片，1000rpm离心7分钟，去掉上层清液，用PBS洗涤下层细胞，然后1000rpm离心7分钟，倒掉上清液，加入表皮细胞培养液，按2～4×106/瓶接种到IV型胶原包被的培养瓶中，37℃，5％CO2培养箱中孵育10～15min，吸出培养基，更换新鲜表皮细胞培养液继续培养，隔48h换一次液。
3、组织工程表皮构建：
将步骤2中获得的表皮干细胞用胰酶消化液消化，用表皮细胞TU培养液制成密度为2.5×106个/ml的表皮干细胞分散液，取200μl接种于含有聚碳酸酯滤膜的细胞培养小室中，震荡均匀，在37℃，5％CO2培养箱中孵育20～30min。将接种后的细胞培养小室转入表皮模型培养模具中，小室外的培养皿中加入新鲜表皮细胞TU培养液，37℃，5％CO2培养箱继续培养48h后，吸去细胞小室中的剩余培养基，将细胞培养小室升至气液面，更换表皮细胞TA培养液，37℃，5％CO2培养箱继续培养12天后可得具有形态分化完全的基底层、棘层、颗粒层、角质层的组织工程表皮模型。
实施例3
体外重组表皮模型在化学品皮肤吸收渗透实验中的用途，其使用方法如下：
1、皮肤预处理
选用健康的人体腹部皮肤，仔细剔除皮下黏膜和脂肪组织，用生理盐水冲洗干净，滤纸吸干皮肤表面生理盐水，解剖镜下选取适当大小未损伤的皮肤备用。体外重组表皮模型(刺激模型)，屏障加强表皮模型(渗透模型)分别在气液面培养10或14天，用来做渗透试验。实验前检查人体皮服和表皮模型的完整性，确保屏障没有任何损伤。
2、经皮渗透实验
(1)采用Franz扩散池进行透皮试验(有效渗透面S为0.196cm2，接收池容积为4.1ml)，将人体皮肤或表皮模型固定于供给室和接收池之间，皮肤角质层面向供给室，真皮层一侧朝向接受池；接收液为PBS液、pH＝7.4，接受液液面要与皮肤内层接触；保持32±1℃恒温水浴，并确保水浴夹层无气泡，开启电磁搅拌器以600rpm/min的速度搅拌。
(2)将样品加至供给室中(一般固体样品添加量为1～5mg/cm2，液体最多添加量为10μL/cm2)于1h、2h、4h、8h、16h、24h取出接收液0.3ml(每次取样后均补加等量的接受液)，将取得的样品进行离心，取上清液，再用0.45μm滤膜过滤。
(3)将过滤液用HPLC测定样品含量。液相色谱条件见下表1。
3、样品对照品标准曲线的制定
精密称量一定质量的对照品置于棕色容量瓶中，加稀释液溶解并稀释至刻度，摇匀，制成对照品储备液，低温避光保存备用。测试时，按一定浓度梯度倍比稀释，色谱条件下进样分析，记录标准品色谱峰面积。以色谱峰面积Y对标准品浓度C(μg·mL-1)进行线性回归。同时实验过程中要排除皮肤溶出物/空白基质在样品保留时间时没有吸收峰，排除样品测定的干扰。
4、计算累积透皮量Q，公式如下：
Q＝[Cn×V+∑Ci×V0]/S(i＝1…n-1)
Q：累积渗透量；S：有效扩散面积；V：接收室中接收液体积；V0：每次取样的体积；Ci：第1次至上次取样时接收液中药物浓度；Cn：该次取样时接收液中药物浓度。此外透皮速率和迟滞时间根据累积渗透量对时间的斜率计算得到，其中将单位面积药物累积透过量(μg/cm2)对时间作图，直线部分的斜率即为稳态通透速率(J,μg/cm2/h)，直线与x轴的交点为时滞(T,h)；药物的渗透系数(Kp,cm/h)则是稳态通透速率(J)与药物在供给液中溶解度(Cs，mg/ml)的比值，渗透系数根据Fick’s第一扩散定律(J＝Kp Cs)计算得到。两种样品在人体皮肤、体外重组表皮模型和屏障加强的体外重组表皮模型上的渗透测试结果见表2，从表中可以看出通过本发明构建的表皮模型，具有更强的屏障功能，对两种渗透标准品(咖啡因和氢化可的松)的渗透性虽然仍高于人体皮肤，但相比于优化前的表皮模型，渗透性显著降低。
表2样品在三种皮肤替代物上的24h体外渗实验结果
参见图1，可以看出，体外重组表皮模型和屏障加强的重组表皮模型组织学染色结果对比图，从HE染色图中可以看出采用本发明构建的屏障加强的重组表皮模型具有更厚的角质层，屏障功能更强。
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限于上述的具体实施方案，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下，在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本发明保护之列。
Claims (2)
1.用于构建屏障加强体外重组表皮模型的TA培养液，其特征在于，所述培养液由以下组分构成：每500mlKC-growth培养液中，添加L-谷氨酰胺1.0~1.5mM、牛垂体提取物12~25mg、表皮生长因子0.5～5μg、氢化可的松2～50μg、胰岛素1～10mg、腺嘌呤 5～25mg、转铁蛋白2 .5～6μg、脂质混合物、巨噬细胞活化肽-2 2.5～5.0mg、维生素C 15～25μg、浓度调整为1.0～1.5mM的氯化钙。
2.根据权利要求1所述的用于构建屏障加强体外重组表皮模型的TA培养液，其特征在于，所述脂质混合物包括L-丝氨酸0 .25～5mM、棕榈酸0 .4～5μM、亚油酸 0 .01～0.05mM、精氨琥珀酸0 .025～0 .05 mM、牛血清白蛋白0.01～0 .05mM。
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