CN113694057B - 硫化氢缓释有机供体adt-oh在制备神经系统疾病治疗药物中的应用 - Google Patents
硫化氢缓释有机供体adt-oh在制备神经系统疾病治疗药物中的应用 Download PDFInfo
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Abstract
本发明涉及硫化氢缓释有机供体ADT‑OH药物在神经前体细胞分化中的应用，该物质能够诱导神经前体细胞更多的定向分化为神经元和少突胶质细胞，而更少的分化为星型胶质细胞，且促进神经元的轴突生长，同时可使神经前体细胞的死亡数减少，为移植神经前体细胞修复受损神经提供了方向，ADT‑OH将有很大可能成为临床上治疗神经系统疾病的新药物靶点。
Description
技术领域
本发明涉及生物细胞学和再生医学领域，尤其涉及一种硫化氢缓释有机 供体ADT-OH在制备神经系统疾病治疗药物中的应用。
背景技术
神经干细胞又称神经前体细胞(NPCs)，是一种具有多向分化潜能的细 胞。在中枢神经系统(CNS)的发育过程中，脑室管膜下区是NPCs主要分布 的区域。NPCs可以通过对称分裂来维持自身细胞数量，也可通过不对称分 裂来产生神经元、星形胶质细胞和少突胶质细胞。在哺乳动物神经系统中， 神经元是终末分化的细胞，本身不能重新分裂并产生新的细胞，所以神经元 死亡后基本无法得以补充。成年哺乳动物神经系统中神经干细胞数量少，一 般不足以补偿神经损伤或疾病引起的神经元死亡。正是因为神经元的这一缺 陷，神经系统损伤或疾病会导致不可逆的感觉、运动或认知功能丧失。因此 如何产生新的神经元并使其重新形成功能性回路是当前神经科学研究以及 未来神经损伤修复与再生临床治疗中的一个重要目标。
神经损伤后，一些神经胶质细胞，如星型胶质细胞，在损伤后能重新被 激活并快速分裂，从数量上得以补充。但是这些新生成的星型胶质细胞往往 是疤痕组织的主要成分，对神经损伤后的轴突再生起抑制作用。少突胶质细 胞是轴突髓鞘细胞，其在损伤后一般由前体细胞分裂分化得以补充。但是损 伤后的神经组织对新髓鞘的形成有一定的抑制作用，所以内源少突胶质细胞 不能完全满足新髓鞘的形成。因此，修复损伤的神经系统并恢复功能需要： 1)补充新的神经元，2)补充新的少突胶质细胞，和3)减少形成疤痕组织 的星型胶质细胞。
神经前体细胞移植修复受损神经系统是神经修复领域的一个重要研究 方向。但是当前本领域的研究表明，移植到受损部位的神经前体细胞会大量 分化为星型胶质细胞从而形成胶质瘢痕，达不到修复受损神经的效果。因此， 如何把移植的外源干细胞诱导成为神经元或少突胶质细胞是目前神经科学 领域的一个重要研究方向。
硫化氢(H2S)是继一氧化氮和一氧化碳之后已知的第三种内源性气体信 号分子，表达于哺乳动物多个器官，尤其是大脑。H2S对肝脏及其他器官的 缺血再灌注损伤具有保护作用。此外，它还参与不同的生理和病理过程，调 节中枢小胶质细胞从m1型激活状态极化到m2激活状态，从而减少中枢神 经系统的炎症反应，加强损伤脑的再生和修复。由于H2S具有保护细胞的特 性，可以通过人工调节内源性H2S或生物合成H2S供体体外给药来恢复细胞或器官系统的生理功能。因此，人们的兴趣越来越集中于确定新的H2S 供体。由于H2S的浓度在短时间内激增有致死性，因此需要缓慢释放率的供 体。5-(对羟基苯基)-1,2-二硫酮-3-硫酮(ADT-OH)是一种能在体内缓慢释放 H2S的有机供体。与NaHS等无机H2S供体相比，ADT-OH能以可控的速率 缓慢、永久释放H2S，且效果优于普通无机H2S供体。ADT-OH在体内释放 H2S分子，对谷氨酸诱导的氧化应激具有神经保护作用。然而，ADT-OH对 神经前体细胞分化的潜在影响尚不清楚。
发明内容
为解决上述技术问题，本发明提供了一种硫化氢缓释有机供体ADT-OH 的新用途，该物质可使神经前体细胞的死亡数减少，同时能够诱导神经前体 细胞更多的定向分化为神经元和少突胶质细胞，而更少的分化为星型胶质细 胞，为移植神经前体细胞修复受损神经提供了方向，ADT-OH将有很大可能 成为临床上治疗神经系统疾病的新药物靶点。
本发明要求保护硫化氢缓释有机供体ADT-OH在制备神经系统疾病治 疗药物中的应用。
进一步地，硫化氢缓释有机供体ADT-OH用于神经系统的修复，其中， ADT-OH诱导神经前体细胞分化为神经元和少突胶质细胞，抑制神经前体细 胞分化为星型胶质细胞。
本发明还要求保护硫化氢缓释有机供体ADT-OH在神经前体细胞体外 分化中的应用。
进一步地，硫化氢缓释有机供体ADT-OH用于诱导神经前体细胞分化 为神经元。
进一步地，硫化氢缓释有机供体ADT-OH用于促进神经元轴突生长。
进一步地，硫化氢缓释有机供体ADT-OH用于诱导神经前体细胞分化 为少突胶质细胞。
进一步地，硫化氢缓释有机供体ADT-OH用于抑制神经前体细胞分化 为星型胶质细胞。
进一步地，神经前体细胞为胚胎期大脑皮质神经前体细胞，具体为脑室 管膜下区神经前体细胞。本发明采用胚胎期的脑室管膜下区神经前体细胞， 因为成年哺乳动物神经系统中神经干细胞数量很少，一般不足以补偿神经损 伤或疾病引起的神经元死亡。成年哺乳动物脑室管膜下区的少量神经前体细 胞也可分化为神经元和胶质细胞，但是由于其数量的不足难以达到实现修复 神经损伤的效果。
所述的脑室管膜下区神经前体细胞又称神经干细胞，是一种能够自我更 新并具有多向分化潜能的细胞，既可增殖亦可分化为神经元和胶质细胞，在 胚胎发育第12天之前，神经前体细胞进行大量增殖形成一个细胞池，为后 期大脑皮质的发育提供条件。为排除伦理问题，当应用于人体时，本发明使 用的是经伦理审查批准获得的人神经前体细胞，包括商业化的人神经前体细 胞(如NHNP–Human Neural Progenitor Cells,HopCellTM等)。特别指出， 本发明所使用的神经前体细胞均不能发育成完整个体。
一种诱导神经前体细胞分化的方法，包括以下步骤：
将神经前体细胞在含有ADT-OH的神经前体细胞培养基中培养1-3d， 随后在含有ADT-OH的分化培养基中诱导培养5-8d；
含有ADT-OH的神经前体细胞培养基中ADT-OH的浓度为70-90μmol/L， 含有ADT-OH的分化培养基中ADT-OH的浓度为70-90μmol/L，神经前体细 胞培养基为DMEM-F12培养基中含有B27、bEGF和FGF，分化培养基为 DMEM-F12培养基中含有FBS和B27。
进一步地，取胚胎期脑室管膜下区脑组织，用胰酶消化后用神经前体细 胞培养基终止消化，得到游离神经前体细胞后，取沉淀部分，加入神经前体 细胞培养基后接种于细胞培养板中继续培养3-4d，得到上述神经前体细胞；
优选地，ADT-OH的浓度为80μmol/L。
借由上述方案，本发明至少具有以下优点：
为了解决目前存在的如何诱导神经前体细胞定向分化及存活这一难题， 本发明创新性地通过ADT-OH药物对体外培养的神经前体细胞进行处理， 提供了一种诱导神经前体细胞定向分化和保护细胞存活的药物和方法，达到 了神经前体细胞更多的分化为神经损伤修复所需要的神经元和少突胶质细 胞，更少的分化为星型胶质细胞，并且神经前体细胞的死亡数减少，为采用 神经前体细胞移植修复受损神经奠定了基础。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技 术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配 合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施 例并结合附图，对本发明作进一步详细的说明。
图1为ADT-OH对神经元分化的影响结果，其中，A为Tuj1细胞染色 图，B为神经元分化比率统计图，C为神经元轴突长度定量结果；
图2为ADT-OH对少突胶质细胞分化的影响结果，其中，A为MBP细 胞染色图，B为少突胶质细胞分化比率统计图；
图3为ADT-OH对星型胶质细胞分化的影响结果，其中，左图为GFAP 细胞染色图，右图为星型胶质细胞分化比率统计图；
图4为ADT-OH对星型胶质细胞分化的影响结果，其中，A为caspase3 细胞染色图，B为神经前体细胞凋亡统计图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术 人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的 限定。
实施例1
(1)成年ICR小鼠雌雄按2:1的比例随机交配。每天晚上8点，雄性和 雌性小鼠进行合笼。第2天，如果雌性小鼠在早上8点有阴栓，则说明交配 成功，将这一天记录为小鼠怀孕的第一天。当小鼠怀孕14.5天(E14.5)时，可 用于神经前体细胞提取。
将怀孕14.5天的C57BL/6J小鼠用4％的水合氯醛进行腹腔注射麻醉， 用无菌的手术剪和手术镊子打开孕鼠腹腔，刨开孕鼠子宫取出胎鼠大脑，在 体式显微镜下分离脑室管膜下区的胎鼠脑组织，将组织用0.05％的胰酶在 37℃水浴锅中消化10min后用神经前体细胞培养基(DMEM-F12培养基 39.2ml，1X的B27 800ul,0.2mg/ml的bEGF 4ul,0.2mg/ml的FGF8ul)终止 消化，接着将组织轻轻吹散成单个细胞，将细胞于700rcf离心5分钟后弃掉 上清液，加入神经前体细胞培养基后接种于24孔细胞培养板中进行培养， 待细胞生长3-4天增殖到足够数目时进行下一步检测。
培养的原代神经前体细胞待细胞增殖长到一定大小的神经球时，将神经 球吹散成单个细胞后接种于铺有黏附圆玻片的24孔细胞培养板中，细胞分 别在加有ADT-OH(80μmol/L)和对照溶质DMSO的神经前体细胞培养基 中生长24小时后，将神经前体细胞培养基分别换为加有ADT-OH和DMSO 的分化培养基(DMEM/F12 39ml,0.5％的FBS,1X的B27 800ul)诱导神经 前体细胞分化。之后进行下一步检测。
培养的细胞用4％的PFA固定15分钟后，用0.01M的PBS洗3遍后用 Tuj1,GFAP,MBP,caspase3抗体进行免疫荧光染色。用含有DAPI的封片剂 (Vector Labs,H-1400)封片。在蔡司荧光显微镜下观察并拍照。使用ImageJ 和adobe photoshop分别计数Tuj1阳性神经元、GFAP阳性星形胶质细胞、 MBP阳性少突胶质细胞和DAPI阳性细胞。计算阳性细胞占所有DAPI阳性 细胞的比例。
(2)细胞在分化培养基中分化5天后用4％的PFA固定细胞，用神经 元的特异性抗体Tuj1进行细胞免疫荧光染色，染色结束后在荧光显微镜下 进行图片采集，统计每组神经元占所有DAPI阳性细胞的比率。
结果如图1所示。统计结果显示，和DMSO对照组相比较，ADT-OH 组有更多的细胞分化成了神经元(图1A和B)，并且神经元的轴突更长(图 1A和C)。以上数据显示，ADT-OH可促进神经前体细胞向神经元的分化 和轴突的生长。
(3)细胞在神经前体细胞的分化培养基中分化5天后用4％的多聚甲醛 固定细胞，用少突胶质细胞的特异性抗体MBP进行免疫荧光染色，统计 MBP阳性细胞占所有DAPI阳性细胞的比率。
结果如图2所示。统计结果显示，和DMSO对照组相比较，ADT-OH 组有更多的细胞分化成了少突胶质细胞(图2A和B)，以上数据显示， ADT-OH可促进神经前体细胞向少突胶质细胞分化。
(4)细胞在神经前体细胞的分化培养基中分化8天后用4％的多聚甲醛 固定细胞，用星型胶质细胞的特异性抗体GFAP进行免疫荧光染色，统计 GFAP阳性细胞占DAPI阳性所有细胞的比率。
结果如图3所示。统计结果显示，和DMSO对照组相比较，ADT-OH 组有较少的细胞分化成了星型胶质细胞，以上数据显示，ADT-OH可抑制神 经前体细胞向星型胶质细胞的分化。
实施例2
原代分离的神经前体细胞培养3-4天后，将神经球吹散成单个细胞，接 种于铺有黏附玻片的24孔细胞培养板中，细胞分别在加有ADT-OH (80μmol/L)和对照溶质DMSO的神经前体细胞培养基中生长3天后进行 细胞凋亡特异性抗体caspase3染色，荧光显微镜下采集图片后统计caspase3 阳性细胞占所有DAPI阳性细胞的比率，统计结果显示，和DMSO对照组 相比较，ADT-OH组有较少的细胞在凋亡(图4A和B)，以上数据显示， ADT-OH可保护神经前体细胞去凋亡。
综上，和DMSO对照组相比，ADT-OH组神经元分化比率增加了57.69％， 少突胶质细胞分化比率增加了87.5％,而星型胶质细胞分化比率降低了 69.2％。此外，ADT-OH也可有效阻止神经前体细胞凋亡，和DMSO对照组 相比，ADT-OH组细胞凋亡减少了44.8％。
实施例3
采用重物坠落撞击小鼠脊髓背侧，建立小鼠脊髓损伤模型，具体操作如 下：麻醉小鼠，将其背部正中切开，切除椎板，显露脊髓硬膜；之后将重物 从某一高度自由坠落，打击于脊髓上，致脊髓损伤。之后在体式显微镜下将 经ADT-OH药物处理的神经前体细胞移植到脊髓损伤部位，一个月后检测 损伤部位神经前体细胞的分化、细胞存活及神经轴突生长情况来评估在脊髓 修复上的效果。
结果显示，植入经ADT-OH药物处理的神经前体细胞一个月后，小鼠 脊髓损伤部位的神经元和少突胶质细胞的数量大大增加，且星型胶质细胞的 数量明显低于神经元或少突胶质细胞，轴突也有一定伸长，脊髓功能损伤得 到显著改善。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的 限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出 其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而 由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (7)
1.一种诱导神经前体细胞分化的方法，其特征在于，包括以下步骤：
将神经前体细胞在含有ADT-OH的神经前体细胞培养基中培养1-3d，随后在含有ADT-OH的分化培养基中诱导培养5-8d；
所述含有ADT-OH的神经前体细胞培养基中ADT-OH的浓度为70-90μmol/L，所述含有ADT-OH的分化培养基中ADT-OH的浓度为70-90μmol/L，所述神经前体细胞培养基为DMEM-F12培养基中含有B27、bEGF和FGF，所述分化培养基为DMEM-F12培养基中含有FBS和B27。
2.根据权利要求1所述的方法，其特征在于：该方法诱导神经前体细胞分化为神经元。
3.根据权利要求1所述的方法，其特征在于：该方法促进神经元轴突生长。
4.根据权利要求1所述的方法，其特征在于：该方法诱导神经前体细胞分化为少突胶质细胞。
5.根据权利要求1所述的方法，其特征在于：该方法抑制神经前体细胞分化为星型胶质细胞。
6.根据权利要求1所述的方法，其特征在于：所述神经前体细胞为胚胎期脑管膜下区神经前体细胞。
7.根据权利要求1所述的方法，其特征在于：取胚胎期脑室管膜下区脑组织，用胰酶消化后用神经前体细胞培养基终止消化，得到游离神经前体细胞后，取沉淀部分，加入神经前体细胞培养基后接种于细胞培养板中继续培养3-4d，得到所述神经前体细胞。
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