CN114159470A - 一种Eze-ECM水凝胶的制备方法 - Google Patents
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Abstract
本发明公开了一种Eze‑ECM水凝胶的制备方法，具体包括如下步骤：A.准备工作；B.猪小肠预处理；C.脱细胞处理；D.凝胶化处理；E.制备Eze‑ECM水凝胶。本发明主要通过提供一种ECM水凝胶的制备方法，使ECM水凝胶能够符合Eze的负载结构，形成具有稳定三维结构的ECM水凝胶，后续能够形成直接注射治疗，相较于如今的脊髓内神经信号网络的重建治疗方案，如口服药和区域注射，本方法的脊髓腔更具有先进性和突破性，可进一步促进损伤区域的恢复。
Description
技术领域
本发明涉及医疗药物制备工艺，特别涉及一种Eze-ECM水凝胶的制备方法。
背景技术
脊髓损伤后药物的使用具有局限性。一方面，口服药物不易透过血-脊髓屏障，且具有存留时间过短、代谢速率过快的缺点，导致其在应用时出现瓶颈；另一方面，传统局部注射药物无法在脊髓损伤指定部位释放。因此，新型载药方式的研究，对于脊髓损伤后的神经再生与功能修复至关重要。
依折麦布(Eze)是一种新型的选择性胆固醇吸收抑制剂，可通过抑制NPC1L1降低胆固醇水平而改善高血脂症，据报道，Eze与辛伐他汀联用能够改善大鼠脊髓损伤后的神经功能，减轻内皮细胞炎症反应，其它相关研究和论文也说明了，Eze能够显著降低脊髓损伤急性期细胞凋亡，促进神经元损伤的修复，改善脊髓损伤后大鼠的运动功能恢复。
在现有技术中，由于ECM具有强大的促进再生的能力，在中枢神经系统的发育、成熟以及损伤修复的过程中起着重要的调控作用，从而在结合Eze和ECM后即可得到具有强修复作用的Eze-ECM水凝胶，然而结合过程中依旧需要具有稳定三维结构的ECM水凝胶并在此基础上负载Eze，否则无法达到最佳的治疗效果，因此制备合格的Eze-ECM水凝胶是如今脊髓损伤后药物的使用趋势。
发明内容
本发明要解决的技术问题是克服现有技术的缺陷，提供一种Eze-ECM水凝胶的制备方法。
为了解决上述技术问题，本发明提供了如下的技术方案：
本发明一种Eze-ECM水凝胶的制备方法，具体包括如下步骤：
A.准备工作，将刮肠机和操作台杀菌消毒，猪小肠放置于恒温箱保存，将0.1％的过氧乙酸溶液，PBS缓冲液，0.1M盐酸-胃蛋白酶液，0.1M氢氧化钠均放置于阴凉处存放；
B.猪小肠预处理，以猪小肠为原料，通过刮肠机刮去猪小肠的浆膜层、肌层和粘膜层，留置小肠粘膜下层及粘膜固有层；
C.脱细胞处理，将步骤B中留置的小肠粘膜下层及粘膜固有层均放置于0.1％的过氧乙酸溶液中浸泡1.5～2.5h，并采用PBS缓冲液清洗沥干水分，随后放置于低温箱中低温冷冻干燥，初步获得ECM粉末；
D.凝胶化处理，将步骤C中ECM粉末溶解于0.1M盐酸-胃蛋白酶液中，在室温状态下搅拌消化46h～50h，采用0.1M氢氧化钠滴定溶液至PH7.2～7.6，之后在37℃恒温箱中放置0.8～1.2h，获得可注射的ECM水凝胶；
E.制备Eze-ECM水凝胶，称取一定量的Eze，将其放入至药品磨粉机中磨粉，称取一定量的Eze加入ECM水凝胶溶液中，低温搅拌至溶解，最后置于4℃下静置24h，获得Eze-ECM水凝胶。
作为本发明的一种优选技术方案，所述步骤C中，放置于0.1％的过氧乙酸溶液中浸泡时间为2h，所述0.1％的过氧乙酸溶液液面没出浸泡物5cm。
作为本发明的一种优选技术方案，所述步骤D中，在室温状态下搅拌消化时间为48h，所述氢氧化钠滴定溶液的PH值为7.4，所述37℃恒温箱中放置时间为1h。
作为本发明的一种优选技术方案，所述步骤E中，Eze-ECM水凝胶的配比调制结果为含10mg/mlEze的10％(w/v)ECM水凝胶。
作为本发明的一种优选技术方案，所述盐酸-胃蛋白酶液和氢氧化钠的单位均为1mg/ml，所述阴凉存放处的温度不得低于20℃。
与现有技术相比，本发明的有益效果如下：
本发明主要通过提供一种ECM水凝胶的制备方法，使ECM水凝胶能够符合Eze的负载结构，形成具有稳定三维结构的ECM水凝胶，后续能够形成直接注射治疗，相较于如今的脊髓内神经信号网络的重建治疗方案，如口服药和区域注射，本方法的脊髓腔更具有先进性和突破性，可进一步促进损伤区域的恢复。
具体实施方式
以下本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例1
本发明提供一种Eze-ECM水凝胶的制备方法，具体包括如下步骤：
A.准备工作，将刮肠机和操作台杀菌消毒，猪小肠放置于恒温箱保存，将0.1％的过氧乙酸溶液，PBS缓冲液，0.1M盐酸-胃蛋白酶液，0.1M氢氧化钠均放置于阴凉处存放；
B.猪小肠预处理，以猪小肠为原料，通过刮肠机刮去猪小肠的浆膜层、肌层和粘膜层，留置小肠粘膜下层及粘膜固有层；
C.脱细胞处理，将步骤B中留置的小肠粘膜下层及粘膜固有层均放置于0.1％的过氧乙酸溶液中浸泡1.5～2.5h，并采用PBS缓冲液清洗沥干水分，随后放置于低温箱中低温冷冻干燥，初步获得ECM粉末；
D.凝胶化处理，将步骤C中ECM粉末溶解于0.1M盐酸-胃蛋白酶液中，在室温状态下搅拌消化46h～50h，采用0.1M氢氧化钠滴定溶液至PH7.2～7.6，之后在37℃恒温箱中放置0.8～1.2h，获得可注射的ECM水凝胶；
E.制备Eze-ECM水凝胶，称取一定量的Eze，将其放入至药品磨粉机中磨粉，称取一定量的Eze加入ECM水凝胶溶液中，低温搅拌至溶解，最后置于4℃下静置24h，获得Eze-ECM水凝胶。
步骤C中，放置于0.1％的过氧乙酸溶液中浸泡时间为2h，0.1％的过氧乙酸溶液液面没出浸泡物5cm。
步骤D中，在室温状态下搅拌消化时间为48h，氢氧化钠滴定溶液的PH值为7.4，37℃恒温箱中放置时间为1h。
步骤E中，Eze-ECM水凝胶的配比调制结果为含10mg/mlEze的10％(w/v)ECM水凝胶。
盐酸-胃蛋白酶液和氢氧化钠的单位均为1mg/ml，阴凉存放处的温度不得低于20℃。
具体的，细胞外基质材料，即ECM材料，其免疫源性低，含有最天然的细胞外微环境，是种子细胞生长的理想材料，ECM含有胶原、糖胺聚糖、蛋白多糖、纤维蛋白等大量活性因子，这些成分相关交错形成紧密的网络结构，不仅支撑着细胞结构，还能调节细胞增殖、迁移和分化，ECM活性因子不仅可以调节脊髓损伤中的炎症反应，而且能参与周围神经生长和星形细胞相关物质的再生，支持并促进轴突生长，因此，ECM具有强大的促进再生的能力，在中枢神经系统的发育、成熟以及损伤修复的过程中起着重要的调控作用。
本方法使用时主要将制备完成的Eze-ECM水凝胶于目标区域的脊髓腔或脊髓附近注射即可，20日一次，具体实验步骤如下：
选取SPF级大鼠，月龄均为3-9个月，性别均为雄性，体重为250-500g，针对同一区域脊髓进行生物建模处理，于CT机中扫描脊髓损伤数据并保存，按照每组10只分为服药组、普通注射组和Eze-ECM水凝胶注射组，并分别在同一环境中喂养观察；
由于脊髓损伤的最佳治疗期为发病的半年内，因此实验周期选为4-6个月，在周期内解剖不同组的小鼠观察神经轴突的恢复面积，且取2到6个月的平均数；并且按照组别区分不同的小鼠可完全恢复运动个数，形成最终的判断依据，完全恢复运动的标准为大鼠与造模前并无异样，可正常自由行走，即为无神经功能缺损，同时结合Berderson神经功能缺损体征评分法判断。
其实验结果表格如下所示：
表1：轴突恢复速率
表2：完全恢复运动数量
由结果可得知，Eze-ECM水凝胶注射组针对于脊髓损伤的治疗较为明显，能够区别于口服药物和普通注射的方式，直接指定脊髓损伤区域注射即可，治疗效果先进，与此同时，本制备方法所制备的工艺简单，流程简便，无需复杂的药品调配，大批量生产时更加方便，性价比更高。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种Eze-ECM水凝胶的制备方法，其特征在于，具体包括如下步骤：
A.准备工作，将刮肠机和操作台杀菌消毒，猪小肠放置于恒温箱保存，将0.1％的过氧乙酸溶液，PBS缓冲液，0.1M盐酸-胃蛋白酶液，0.1M氢氧化钠均放置于阴凉处存放；
B.猪小肠预处理，以猪小肠为原料，通过刮肠机刮去猪小肠的浆膜层、肌层和粘膜层，留置小肠粘膜下层及粘膜固有层；
C.脱细胞处理，将步骤B中留置的小肠粘膜下层及粘膜固有层均放置于0.1％的过氧乙酸溶液中浸泡1.5～2.5h，并采用PBS缓冲液清洗沥干水分，随后放置于低温箱中低温冷冻干燥，初步获得ECM粉末；
D.凝胶化处理，将步骤C中ECM粉末溶解于0.1M盐酸-胃蛋白酶液中，在室温状态下搅拌消化46h～50h，采用0.1M氢氧化钠滴定溶液至PH7.2～7.6，之后在37℃恒温箱中放置0.8～1.2h，获得可注射的ECM水凝胶；
E.制备Eze-ECM水凝胶，称取一定量的Eze，将其放入至药品磨粉机中磨粉，称取一定量的Eze加入ECM水凝胶溶液中，低温搅拌至溶解，最后置于4℃下静置24h，获得Eze-ECM水凝胶。
2.根据权利要求1所述的一种Eze-ECM水凝胶的制备方法，其特征在于，所述步骤C中，放置于0.1％的过氧乙酸溶液中浸泡时间为2h，所述0.1％的过氧乙酸溶液液面没出浸泡物5cm。
3.根据权利要求1所述的一种Eze-ECM水凝胶的制备方法，其特征在于，所述步骤D中，在室温状态下搅拌消化时间为48h，所述氢氧化钠滴定溶液的PH值为7.4，所述37℃恒温箱中放置时间为1h。
4.根据权利要求1所述的一种Eze-ECM水凝胶的制备方法，其特征在于，所述步骤E中，Eze-ECM水凝胶的配比调制结果为含10mg/mlEze的10％(w/v)ECM水凝胶。
5.根据权利要求1所述的一种Eze-ECM水凝胶的制备方法，其特征在于，所述盐酸-胃蛋白酶液和氢氧化钠的单位均为1mg/ml，所述阴凉存放处的温度不得低于20℃。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3165233A1 (en) * | 2015-08-28 | 2017-05-10 | Latvijas Universitate | Biomaterial for treatment of acute and chronic skin wounds |
| US20190015552A1 (en) * | 2016-01-13 | 2019-01-17 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Vascular Extracellular Matrix Hydrogel |
| WO2020197326A1 (ko) * | 2019-03-28 | 2020-10-01 | 연세대학교 산학협력단 | 마이크로채널 네트워크가 형성된 하이드로젤 구조체를 포함하는 인공 조직 모델 |
| CN112089890A (zh) * | 2020-08-28 | 2020-12-18 | 广东乾晖生物科技有限公司 | 脱细胞基质水凝胶及其制备方法和应用 |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3165233A1 (en) * | 2015-08-28 | 2017-05-10 | Latvijas Universitate | Biomaterial for treatment of acute and chronic skin wounds |
| US20190015552A1 (en) * | 2016-01-13 | 2019-01-17 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Vascular Extracellular Matrix Hydrogel |
| WO2020197326A1 (ko) * | 2019-03-28 | 2020-10-01 | 연세대학교 산학협력단 | 마이크로채널 네트워크가 형성된 하이드로젤 구조체를 포함하는 인공 조직 모델 |
| CN112089890A (zh) * | 2020-08-28 | 2020-12-18 | 广东乾晖生物科技有限公司 | 脱细胞基质水凝胶及其制备方法和应用 |
Non-Patent Citations (2)
| Title |
|---|
| XIAOFANG WANG PHD等: "Effects of Combination of Ezetimibe and Rosuvastatin on Coronary ArteryPlaque in Patients with Coronary Heart Disease" * |
| 付小兵主编, 湖北科学技术出版社 * |
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