CN115197913A - 一种原代角膜内皮细胞培养液及其应用 - Google Patents
一种原代角膜内皮细胞培养液及其应用 Download PDFInfo
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Abstract
本发明公开了一种原代角膜内皮细胞培养液及其应用。该原代角膜内皮细胞培养液为含碱性成纤维细胞生长因子、透明质酸、半胱氨酸蛋白酶9（Caspase‑9）抑制剂、还原型谷胱甘肽、受体作用蛋白激酶3（RIP3）抑制剂、胎牛血清、青链霉素双抗的高糖DMEM培养基。所得原代角膜内皮细胞培养液用于原代细胞的培养，经原代角膜内皮细胞培养液培养的原代角膜内皮细胞生存时间显著延长。
Description
技术领域
本发明涉及生物学试剂技术领域，具体涉及一种原代角膜内皮细胞培养液及其应用。
背景技术
研究显示，细胞株由于长期的传代，已经丢失了组织细胞的一些特性，包括遗传信息和表型信息等，用于体外药物毒性测试有一定局限性。
新药进行非临床安全性评价前使用原代肝细胞进行候选化合物肝毒性评价可对化合物进一步筛选，可能降低非临床安全性评价和临床研究中失败的风险。
原代细胞由于传代间隔时间限制，只能用于短期的体外药物毒性测试。使用原代细胞制作的3D细胞生存周期较长，因此有的研究者开发3D细胞并用于体外药物长毒测试。然而，3D细胞在形成过程中原代细胞通过多代增殖而成，代数越多，其遗传和表型信息越有可能发生变化。
三代以内且生存时间长的原代细胞可能成为体外药物长毒性测试的优选。
发明内容
本发明的目的在于提供一种原代角膜内皮细胞培养液，其能够减缓原代角膜内皮细胞的衰老与凋亡，延长三代内原代角膜内皮细胞的生存时间。
为实现上述目的，本发明提供了一种原代角膜内皮细胞培养液，为包括碱性成纤维细胞生长因子、透明质酸、半胱氨酸蛋白酶9（Caspase-9）抑制剂、还原型谷胱甘肽、受体作用蛋白激酶3（RIP3）抑制剂、胎牛血清、青链霉素双抗的高糖DMEM培养基。
所述碱性成纤维细胞生长因子浓度为50-200ug/L。
所述透明质酸钠为0.5-2g/L。
所述Caspase-9抑制剂为Z-LEHD-FMK或Z-LEHD-FMKTFA，Z-LEHD-FMK或Z-LEHD-FMKTFA的浓度为0.5uM-2.5uM。
所述还原型谷胱甘肽的浓度为60-120mg/L。
RIP3抑制剂为GSK840、GSK843、GSK2593074A、GSK872和HS1371中的任意一种，GSK840浓度为0.25-0.5uM, GSK843浓度为0.5-1.5uM, GSK2593074A浓度为0.5-1.5nM,GSK872浓度为0.5-1.5uM,HS1371浓度为1-5uM。
所述胎牛血清浓度为10-20%。
所述青链霉素双抗浓度为1-2%。
本发明所述原代角膜内皮细胞培养液用于原代内皮细胞的培养。
与现有技术相比，本发明具有如下有益效果：
本发明征对原代角膜内皮细胞的特点，在DMEM中添加了利于角膜内皮细胞存活的碱性成纤维细胞生长因子和透明质酸钠，添加了凋亡和坏死相关因子抑制剂以及抗氧化剂还原型谷胱甘肽，从而促进了原代角膜内皮细胞的生长与存活，同时抑制了原代角膜内皮细胞的凋亡与坏死。
使用本发明所述的原代角膜内皮细胞培养液培养对使用CN106497863B专利方法获得的原代角膜内皮细胞进行培养，三代内每代细胞生存时间达到2-3周。
具体实施方式
下面对本发明的具体实施方式进行详细描述，但应当理解本发明的保护范围并不受具体实施方式的限制。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可以从商业途径得到。
实施例1：原代角膜内皮细胞培养液的配制
Z-LEHD-FMK溶于DMSO配制成终浓度为0.5mM的溶液，还原型谷胱甘肽溶于去离子水配制成10mg/ml的溶液，GSK843溶于DMSO配制成0.5mM的溶液。
准备100ml容量瓶，加入60ml的DMEM, 然后加入胎牛血清10ml，混匀；接着加入青链霉素双抗溶液（100×）1ml,混匀。加入0.5mg/ml的碱性成纤维细胞生长因子溶液1ml。加入50mg/ml的透明质酸钠1ml,混匀。加入10mg/ml的还原型谷胱甘肽1ml，混匀。加入0.5mM的Z-LEHD-FMK溶液100ul，混匀。最后加入0.5mM的GSK843溶液100ul,混匀。
用DMEM培养基定容至100ml,颠倒混匀，即得原代角膜内皮细胞培养液。
表1.原代角膜内皮细胞培养液的配方
| 试剂 | 用量 | 最终浓度 |
| GSK843（0.5mM） | 100ul | 0.5uM |
| Z-LEHD-FMK(0.5mmM) | 100ul | 0.5uM |
| 还原型谷胱甘肽(10mg/ml) | 1ml | 100mg/L |
| 透明质酸钠（50mg/ml) | 1ml | 0.5g/L |
| 碱性成纤维细胞生长因子（0.5mg/ml） | 1ml | 50ug/L |
| 青链霉素双抗（100×） | 1ml | 1% |
| FBS | 10ml | 10% |
| DMEM | 85.8 | 85.8% |
实施例2：原代角膜内皮细胞培养液的配制
Z-LEHD-FMKTFA溶于DMSO配制成终浓度为2.5mM的溶液，还原型谷胱甘肽溶于去离子水配制成10mg/ml的溶液，HS1371溶于DMSO配制成5mM的溶液。
准备100ml容量瓶，加入50ml的DMEM, 然后加入胎牛血清5ml，混匀；接着加入青链霉素双抗1ml(100×),混匀。加入0.5mg/ml的碱性成纤维细胞生长因子溶液2ml。加入50mg/ml的透明质酸钠2ml,混匀。加入10mg/ml的还原型谷胱甘肽1.2ml，混匀。加入2.5mM的Z-LEHD-FMKTFA溶液100ul，混匀。最后加入5mM的HS1371溶液100ul,混匀。
用DMEM培养基定容至100ml,颠倒混匀，即得原代角膜内皮细胞培养液。
表2.原代角膜内皮细胞培养液的配方
| 试剂 | 用量 | 最终浓度 |
| HS1371（5mM） | 100ul | 5uM |
| Z-LEHD-FMKTFA(2.5mM) | 100ul | 2.5uM |
| 还原型谷胱甘肽(10mg/ml) | 1.2ml | 120mg/L |
| 透明质酸钠（50mg/ml） | 2ml | 1g/L |
| 碱性成纤维细胞生长因子（0.5mg/ml） | 2ml | 100ug/L |
| 青链霉素双抗（100×） | 2ml | 2% |
| FBS | 15ml | 15% |
| DMEM | 77.6ml | 77.6% |
实施例3：原代角膜内皮细胞培养液培养大鼠原代角膜内皮细胞
分别使用表1和表2制备的原代角膜内皮细胞培养液培养CN106497863B专利方法获得的大鼠原代角膜内皮细胞，同时用含10%FBS和1%青链霉素双抗的DMEM作为对照。
三种培养液培养的大鼠原代角膜内皮细胞生存时间及细胞状态见表3。
表3. [0026]配方原代角膜内皮细胞培养液、[0031]配方原代角膜内皮细胞培养液、对照培养液培养原代角膜内皮细胞的生存时间和细胞活率
以上所述，仅为本发明较佳的实施方式，但本发明的保护范围并不局限于此实施实例。任何在本发明的精神和原则之内做出任何修改，等同替换和改进等，均应包含在本发明的保护范围内。
Claims (8)
1.一种原代角膜内皮细胞培养液，其特征在于，包括碱性成纤维细胞生长因子、透明质酸钠、半胱氨酸蛋白酶9（Caspase-9）抑制剂、还原型谷胱甘肽、受体作用蛋白激酶3（RIP3）抑制剂、胎牛血清、青链霉素双抗的高糖DMEM培养基。
2.根据权利要求1所述原代角膜内皮细胞培养液，其特征在于所述碱性成纤维细胞生长因子浓度为50-200ug/L。
3.根据权利要求1所述原代角膜内皮细胞培养液，其特征在于所述透明质酸钠为0.5-2g/L。
4.根据权利要求1所述原代角膜内皮细胞培养液，其特征在于所述Caspase-9抑制剂为Z-LEHD-FMK或Z-LEHD-FMKTFA，Z-LEHD-FMK或Z-LEHD-FMKTFA的浓度为0.5uM-2.5uM。
5.根据权利要求1所述原代角膜内皮细胞培养液，其特征在于所述还原型谷胱甘肽的浓度为60-120mg/L。
6.根据权利要求1所述原代角膜内皮细胞培养液，其特征在于RIP3抑制剂为GSK840、GSK843、GSK2593074A、GSK872和HS1371中的任意一种，GSK840浓度为0.25-0.5uM, GSK843浓度为0.5-1.5uM, GSK2593074A浓度为0.5-1.5nM, GSK872浓度为0.5-1.5uM,HS1371浓度为1-5uM。
7.根据权利要求1所述原代角膜内皮细胞培养液,其特征在于所述胎牛血清浓度为10-20%。
8.根据权利要求1所述原代角膜内皮细胞培养液,其特征在于所述青链霉素双抗浓度为1-2%。
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