CN114366738A - 雷帕霉素在促进神经干细胞扩增中的用途 - Google Patents
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Abstract
本发明公开了雷帕霉素在促进神经干细胞扩增中的用途。本发明克服了目前雷帕霉素仅作为免疫抑制剂使用的应用局限性，雷帕霉素能够显著增强神经干细胞扩增能力，可用于治疗阿尔茨海默症、帕金森氏病、亨廷顿病、缺血性脑卒中、脑和脊髓损伤等病症。
Description
技术领域
本发明属于生物医药技术领域，具体涉及雷帕霉素在促进神经干细胞扩增中的用途。
背景技术
神经干细胞数量减少是神经退行性疾病或神经损伤难以自我修复的主要原因。成体中神经干细胞数量有限，并且难以从静息状态被激活扩增进而行使功能。神经干细胞体外扩增后的移植治疗为这些患者提供了一种切实可行的候选方案，另外在体内如果能够有效促进神经干细胞增殖对于治疗该类疾病将带来光明前景。自噬是一种保守的分解代谢机制，可降解多余或受损的蛋白质或细胞器，为细胞生长快速提供所需的氨基酸、核苷酸、脂肪酸和能量。自噬对于干细胞增殖和分化等过程是必需的，已经发现自噬在各种干细胞中被高度激活，例如诱导多能干细胞(iPS)、造血干细胞(HSCs)和神经干细胞(NSCs)等，而且自噬在神经发生中也起着至关重要的作用。到目前为止，通过调控自噬来干预神经干细胞增殖的应用产品还很少，因此亟需一种能通过诱导自噬发生的药物来促进神经干细胞的体内外扩增。
雷帕霉素(rapamycin,Rapa)，又名Sirolimus，分子式为C51H79NO13，分子量914.17，化学名为(3S，6R，7E，9R，10R，12R，14S，15E，17E，19E，21S，23S，26R，27R，34aS)-9，10，12，13，14，21，22，23，24，25，26，27，32，33，34，34a-十六氢-9，27-二羟基-3-[(1R)-2-[(1S，3R，4R)-4-羟基-3-甲氧环己基]-1-甲基乙基]-10，21-二甲氧-6，8，12，14，20，26-六甲基-23，27-环氧-3H-吡啶并[2，1-c][1，4]氧杂氮杂三十一环烯-1，5，11，28，29(4H，6H，31H)-戊酮，CAS号53123-88-9，其结构式为如下所示：
雷帕霉素由美国家用化学品公司开发上市，它是一种新型大环内酯类免疫抑制剂，美国食品与药品管理局(FDA)批准雷帕霉素作为口服免疫抑制剂而使用，在临床上可用于器官移植的抗排斥反应和自身免疫性疾病的治疗。但是，到目前为止，雷帕霉素是否能够起到扩增神经干细胞的作用尚未见报道。
雷帕霉素是一种有效且特异性的mTOR抑制剂，通过与免疫亲和素FKBP12形成雷帕霉素-FKBP12复合物并结合于mTOR分子的FRB结构域，变构抑制mTORC1激酶活性。mTORC1在参与控制细胞生长和增殖的信号通路中起中心作用，能影响蛋白质合成、核糖体生物发生和代谢等过程。近年来，研究者们的兴趣集中在它作为抗癌药物的潜力上。然而，雷帕霉素及其类似物(雷帕洛尔)的表现并不显著，这表明靶向mTOR的治疗潜力仍有待开发。雷帕霉素还是一种自噬(autophagy)激活剂，能通过促进自噬为细胞生长提供更快的氨基酸、核苷酸、脂肪酸和能量供应。尽管mTORC1对细胞生长和增殖的调节很重要，但据我们所知，雷帕霉素靶向mTORC1以开启自噬信号进而调控神经干细胞增殖的作用还没有被探索。
发明专利CN105331583A公开了雷帕霉素用于人诱导多能干细胞(iPS)向神经元诱导分化的方法，该专利说明书阐述了雷帕霉素在加速神经元分化进程中的作用。虽然雷帕霉素在上述诱导过程中会经历神经干细胞阶段，但该专利说明书仅仅证明了雷帕霉素可加速其向神经元分化进程，并未涉及到对神经干细胞增殖的作用。而干细胞(包括神经干细胞)的增殖与分化是两个完全不同的发育阶段，受不同的调控机制所调节。该发明专利所阐述的重点是雷帕霉素促进iPS向神经元分化，而发明人发现的是雷帕霉素促进NSCs自身的增殖。二者所针对的细胞不同，所达到的效果不同，所应用的目的也不相同。此外，发明人发现的雷帕霉素可直接应用于体内(因为人体脑内有成体神经干细胞)，而上述专利无法将iPS直接用于体内来诱导。
发明内容
本发明的目的在于提供雷帕霉素在促进神经干细胞扩增中的用途。
雷帕霉素在促进神经干细胞扩增中的用途。
雷帕霉素促进神经干细胞的增殖，同时抑制mTORC1活性并促进自噬的发生。
雷帕霉素作为活性成分在制备促进神经干细胞扩增的药物中的用途。
雷帕霉素促进神经干细胞扩增，进而治疗神经退行性疾病或神经损伤，所述神经退行性疾病为指阿尔茨海默症、帕金森氏病、缺血性脑卒中、亨廷顿病；所述神经损伤为脑损伤、脊髓损伤。
一种在体外促进神经干细胞增殖的药用组合物，包括雷帕霉素或其药学上可接受的盐。
一种在体内促进神经干细胞增殖的药用组合物，包括雷帕霉素或其药学上可接受的盐。
优选的，还包括一种或多种药学上可接受的辅料或载体。
所述辅料或载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂。
所述药用组合物可制成的剂型选自片剂、胶囊剂、泡腾片、颗粒剂、散剂、分散片、口服液、丸剂或注射剂。
在体外分离的神经干细胞培养基中添加50nM的雷帕霉素，作用48小时后检测神经干细胞增殖、mTORC1活性与自噬情况。结果发现，施用雷帕霉素后能够明显增加神经干细胞中PCNA(proliferating cell nuclear antigen)表达(PCNA表达量反映细胞的增殖活性)，同时还可以检测到mTORC1信号通路被抑制，而自噬活性被激活；通过自噬抑制剂Baf A1抑制自噬，则神经干细胞自噬活性及增殖活性均受到抑制。因此，采用雷帕霉素作为药效成分或活性成分仅需给予较少剂量且持续较短时间即可通过mTORC1-自噬通路实现促进神经干细胞扩增的作用。
本发明的有益效果：本发明克服了目前雷帕霉素仅作为免疫抑制剂使用的应用局限性，雷帕霉素能够显著增强神经干细胞扩增能力，可用于治疗阿尔茨海默症、帕金森氏病、亨廷顿病、缺血性脑卒中、脑和脊髓损伤等病症。在体外用雷帕霉素处理神经干细胞48小时能显著促进神经干细胞的增殖，同时抑制mTORC1活性并促进自噬的发生。其中，雷帕霉素诱导的自噬是促进神经干细胞增殖的作用途径。
附图说明
图1是采用Western blot方法检测体外培养的神经干细胞经雷帕霉素处理后对其增殖、mTORC1信号通路活性及自噬活性的影响。
图2是采用EdU细胞增殖试剂盒检测雷帕霉素对神经干细胞增殖的作用。
图3是采用免疫荧光方法检测雷帕霉素对神经干细胞增殖的影响。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
下述实施例中所用的雷帕霉素购自美国Selleck公司，产品目录号：S1039，批次：14，为白色粉末。
实施例1雷帕霉素对神经干细胞体外扩增的作用
1.雷帕霉素药品
(1)雷帕霉素药品的处理：雷帕霉素购自Selleck公司，CAS号：53123-88-9，分子量为914.18。取50mg雷帕霉素粉末，加入0.5469ml二甲基亚砜(DMSO)，震荡使其充分溶解，混匀，此储液终浓度为10mM。分装后保存于-20℃。
(2)神经干细胞的处理：实验采用分离培养的大鼠胚胎期神经干细胞。取第三代神经干细胞，细胞密度为1×106/ml，接种24h后将配制的雷帕霉素添加到培养液中，使终浓度为50nM，轻轻混匀，置于37℃，5％CO2细胞培养箱中继续培养48h。
(3)对照组：对照组为阴性对照，阴性对照组神经干细胞不给予雷帕霉素药物处理，而是给予相同浓度的DMSO。
2.神经干细胞的培养
取孕13.5-14.5天SD大鼠，用2％戊巴比妥钠麻醉，完全麻醉后置于75％酒精中消毒，用无菌外科剪刀依次剪开孕鼠皮肤、皮下组织、肌肉，打开盆腔，暴露子宫，打开子宫后小心取出胎鼠，置100mm培养皿中洗涤残留血液，在解剖显微镜下剥掉脑组织表面的蜕膜组织及血管，分离胎鼠前脑和中脑，置于35mm培养皿中，待所有胎鼠分离完毕，加入1ml0.25％胰蛋白酶消化分离出来的脑组织，终止消化后用40μm滤网过筛，收集滤液于15ml离心管中，1200rpm离心5min。然后弃掉上清，用神经干细胞完全培养液(含2％B27，1％N2，20μg/ml bFGF，20μg/ml EGF，1％Glutamine，1％青霉素-链霉素)重悬细胞沉淀，用细胞计数器计数细胞密度，按1×106/ml密度接种于25cm2细胞培养瓶中，置37℃培养箱内培养。
3.Western Blot实验
实验中，按照常规方法收集并裂解神经干细胞，提取蛋白后进行蛋白免疫印迹分析，检测增殖细胞核抗原PCNA(proliferating cell nuclear antigen)、P70核糖体蛋白S6激酶(ribosomal protein S6 kinase,S6K)及其磷酸化形式pT389-S6K、UNC-51样激酶1(Unc-51like autophagy activating kinase 1,ULK1)及其磷酸化形式pS757-ULK1和自噬标记分子LC3(microtubule-associated protein1 light chain3)的表达变化。
如图1所示，当神经干细胞经过雷帕霉素处理后采用Western Blot进行检测。结果显示：与阴性对照组相比，雷帕霉素明显抑制了mTORC1活性，增加了PCNA的表达与自噬活性(LC3II表达)。
4.EdU增殖实验
根据常规实验方案，在NSCs培养68h后，将神经球暴露于EdU 4h，收取神经球并甩片至载玻片上，使用Cell-Light 5-ethynyl-2-deoxyuridine(EdU)Apollo488试剂盒测量细胞增殖，检测EdU+细胞。
5.Immunofluorescence实验
将神经球甩片至载玻片上，用4％多聚甲醛固定15分钟，在0.3％Triton X-100中孵育过夜。次日，用2％牛血清白蛋白封闭1h，然后在4℃下与Anti-Ki67一抗孵育过夜。第三天，在室温下与二抗孵育1h，然后与Hoechst33342在室温下孵育5min。通过倒置共聚焦显微镜检测Ki67+细胞，评估细胞增殖情况。
为了进一步证明雷帕霉素可以促进神经干细胞增殖，发明人还用EdU实验和免疫荧光实验技术分别检测了代表细胞增殖活性的EdU+细胞和Ki67+细胞比例。结果显示，施加雷帕霉素后EdU+和Ki67+细胞百分比都得到了显著增加，表明雷帕霉素处理后神经干细胞的增殖能力得到了增强(图2，3)。以上结果说明，雷帕霉素具有明显地促进神经干细胞增殖的作用。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (8)
1.雷帕霉素在促进神经干细胞扩增中的用途。
2.根据权利要求1所述的用途，其特征在于，雷帕霉素促进神经干细胞的增殖，同时抑制mTORC1活性并促进自噬的发生。
3.雷帕霉素作为活性成分在制备促进神经干细胞扩增的药物中的用途。
4.一种在体外促进神经干细胞增殖的药用组合物，其特征在于，包括雷帕霉素或其药学上可接受的盐。
5.一种在体内促进神经干细胞增殖的药用组合物，其特征在于，包括雷帕霉素或其药学上可接受的盐。
6.根据权利要求4或5所述的药用组合物，其特征在于，还包括一种或多种药学上可接受的辅料或载体。
7.根据权利要求6所述的药用组合物，其特征在于，所述辅料或载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂。
8.根据权利要求6所述的药用组合物，其特征在于，所述药用组合物可制成的剂型选自片剂、胶囊剂、泡腾片、颗粒剂、散剂、分散片、口服液、丸剂或注射剂。
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