CN114763529A - 罗沙司他在增强bmsc在缺血环境中的生存能力的应用 - Google Patents
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Abstract
针对骨髓间充质干细胞在移植进入缺血脑组织后的存活率以及增殖能力低的问题，本发明提供了罗沙司他在改善骨髓间充质干细胞移植后存活率以及增殖能力的新用途。
Description
技术领域
本发明属于药物新用途技术领域，具体涉及罗沙司他在早增强BMSC在缺血环境中的生存能力的应用。
背景技术
缺血性脑卒中是国内严重的社会健康问题，年龄标化的死亡率为56.5/100000人。当脑内微血管发生阻塞时，脑组织因缺血而梗死伴有或不伴有功能损伤，这被称为缺血性脑卒中。脑内血供的缺乏致使炎症，细胞毒性，氧化应激等病理反应的发生，最终导致严重的继发性颅脑损伤。当前，缺血性脑卒中的治疗主要为：t-PA溶栓以及机械取栓。但这两种方法都不能挽救已经梗死的脑组织。骨髓间充质干细胞移植已被明确证实能够用于治疗脑卒中，改善改善脑缺血后神经功能预后，但存在移植后存活率低的问题。因此，急需研发提高骨髓间充质干细胞移植后存活率低的调节剂。
罗沙司他（Roxadustat，FG-4592）是一种可口服的强效可逆性低氧诱导因子脯氨酰羟化酶抑制剂，于2018年在中国上市用于慢性肾脏病透析患者的贫血治疗。罗沙司他能够短暂稳定并激活 HIF 转录因子，启动适应性转录反应，从而在含氧量正常的条件下，模拟了在人体低氧环境下才发生的促红细胞的生成反应，包括产生内源性促红细胞生成素以及通过减少铁调素进行铁动员，最终诱导红细胞生成，改善贫血。结构式如下：
发明内容
本发明罗沙司他预处理后，骨髓间充质干细胞在厌氧条件下的细胞活力以及增殖能力显著增加。在体内实验中，构建了大鼠脑缺血模型，随后将预处理后的骨髓间充质干细胞移植进入脑内，发现干细胞的存活率及增殖能力显著增强。
为实现上述目的，罗沙司他在早增强BMSC在缺血环境中的生存能力的应用。
进一步地，在移植前对干细胞进行罗沙司他预处理24小时。
进一步地，罗沙司他浓度为10μmol/L。
本发明还提供了罗沙司他在提高骨髓间充质干细胞在缺血环境的存活率的应用。
本发明还提供了罗沙司他在提高骨髓间充质干细胞在缺血环境的增值能力的应用。
本发明的有益之处在于：
实验结果证实FG-4592（10umol/L）处理24小时糖氧剥夺处理后的BMSCs的细胞活力提高1.3倍，增殖能力提高2.3倍。FG-4592处理后的BMSCs脑内移植进入脑缺血大鼠后的存活率在移植后第7天提高1.5倍，增殖能力提高1.4倍。
附图说明
图1为罗沙司他提高糖氧剥夺处理后的BMSCs细胞活力的示意图。
图2为罗沙司他提高糖氧剥夺处理后的BMSCs增殖能力的示意图。
图3为罗沙司他提高体内移植后的BMSCs的存活率及增殖能力的示意图。
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清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例1罗沙司他提高糖氧剥夺处理后的BMSCs的细胞活力
1实验材料
本发明使用的罗沙司他购自MCE公司，无糖培养基购自Gibco公司,CCK-8试剂盒购自MCE公司
2.实验方法
首先将细胞接种到96孔板中，每孔1*105个细胞，培养24小时。分别用完全培养基稀释FG-4592成不同浓度（0.1；5；10；50uM）并加入96孔板中，孵育24小时。PBS清洗细胞三次，更换无糖培养基，并放入厌氧培养箱培养24小时。10ul CCK-8溶液添加到每个孔中，在培养箱中下孵育2小时。通过spectramax 190在450nm处检测吸光度。
3.实验结果
FG-4592在10 μmol/L的浓度时明显提高糖氧剥夺处理后的BMSCs的细胞活力，约1.3倍
实施例2罗沙司他提高糖氧剥夺处理后的BMSCs的增殖能力。
实验材料
Ki-67抗体购自abcam公司
实验方法
FG-4592（10 μmol/L）处理BMSCs24小时，PBS清洗细胞三次，更换无糖培养基，并放入厌氧培养箱培养24小时。将细胞进行固定，破膜，封闭，孵育ki-67抗体（增值指标）以及相应荧光二抗，并进行拍摄。
实验结果
FG-4592（10 μmol/L）明显提高糖氧剥夺处理后的BMSCs的增殖能力，约2.3倍。
实施例三罗沙司他提高体内移植后的BMSCs的存活率及增殖能力
实验材料
SD大鼠购自浙江维通利华公司，栓线购自北京西浓公司
实验方法
线栓法阻塞大脑中动脉构建稳定的脑缺血模型，转染绿色荧光蛋白慢病毒构建稳定表达绿色荧光蛋白的BMSCs。借助立体定位技术移植FG-4592预处理的BMSCs进入梗死周围区域。将移植后第7天的大鼠脑子取出，并制备冰冻切片。对GFP，ki-67免疫荧光染色验证BMSCs移植后的存活率及增殖能力。
实验结果
罗沙司他处理后的BMSCs脑内移植后的存活率在移植后第7天提高1.5倍，增殖能力提高1.4倍。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明，因此无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
Claims (5)
1.罗沙司他在增强BMSC在缺血环境中的生存能力的应用。
2.根据权利要求1所述的罗沙司他在增强BMSC在缺血环境中的生存能力的应用其特征在于，在移植前对干细胞进行罗沙司他预处理24小时。
3.根据权利要求2所述的罗沙司他在增强BMSC在缺血环境中的生存能力的应用，其特征在于，罗沙司他浓度为10μmol/L。
4.罗沙司他在提高骨髓间充质干细胞在缺血环境的存活率的应用。
5.罗沙司他在提高骨髓间充质干细胞在缺血环境的增值能力的应用。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550409A (zh) * | 2009-04-29 | 2009-10-07 | 浙江大学 | 一种增强骨髓间充质干细胞移植后存活率的处理方法 |
| CN105802907A (zh) * | 2016-04-20 | 2016-07-27 | 浙江大学 | 提高老年人骨髓间充质干细胞移植后生存能力的方法 |
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