CN114452376A - 胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用 - Google Patents
胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用。IGFBP2干预可以显著的提高髓鞘化水平，并且改善应激脱髓鞘引起的精神症状。
Description
技术领域
本发明涉及医药技术领域，尤其涉及一种胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用。
背景技术
髓鞘是包裹在神经细胞轴突外面的一层膜，即髓鞘由施万细胞和髓鞘细胞膜组成。其作用是绝缘，防止神经电冲动从神经元轴突传递至另一神经元轴突。脱髓鞘是指髓鞘形成后发生的髓鞘损坏。脱髓鞘疾病是一类病因不相同、临床表现各异、但有类同特征的获得性疾患，其特征的病理变化是神经纤维的髓鞘脱失而神经细胞相对保持完整。由于神经冲动的正常传导依赖于轴突周围髓鞘的绝缘特性，髓鞘功能障碍或髓鞘的改变会损害学习和适应行为。研究数据表明，在整个生命周期中，即使髓鞘的微小变化也会对参与认知和行为的回路和系统层面上的机制产生影响。髓鞘的功能可能比以前假设的更广泛：功能失调或适应不良的髓鞘形成可能会导致神经退行性疾病和神经精神疾病，这些疾病迄今被认为具有神经元基础。
许多人类和动物研究报告表明，中枢神经系统特定脑区的白质脱髓鞘可能是精神障碍疾病的重要病理生理特征。白质作为重要的神经基质连接分布式大脑系统。神经影像学研究揭示了暴露于压力的人类白质的异常微观结构。据报道，白质脱髓鞘的改变与压力适应不良和恢复能力有关。同时，来自焦虑症患者的证据强烈表明前额叶皮层相关通路的白质脱髓鞘与特质焦虑评分呈负相关。同时，在具有焦虑样及抑郁样行为的各种动物应激模型中也观察到了mPFC中髓鞘的丢失。我们推测，当髓鞘损伤得到及时有效的修复时，发生焦虑症、抑郁症及认知损伤等精神障碍的风险会降低。
胰岛素生长因子结合蛋白2(IGFBP2)作为IGF结合蛋白中的六大成员之一，是一种具有自分泌和旁分泌的生长因子。研究表明，IGFBP2不仅参与大脑的生长发育，还参与调节神经元的可塑性、调节空间学习记忆和信息处理等高级认知功能。此外，IGFBP2在中枢系统中广泛存在，并且在发育中的脑中高度表达，在初代星形胶质细胞和神经元中分泌。其在外周中的浓度较低，IGFBP2的分子量为36kD，可以穿过血脑屏障(blood-brain barrier，BBB)。但是目前并没有相关研究证明IGFBP2可以治疗脱髓鞘后的精神障碍。
发明内容
本发明的目的是克服了上述现有技术的缺点，提供一种胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用。
本发明提供的技术方案具体为，胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用。
较佳地，胰岛素样生长因子结合蛋白2在制备提高髓鞘化水平的药物中的应用。其中，提高髓鞘化水平包括在髓鞘化状态和髓鞘化数量方面。
或者，胰岛素样生长因子结合蛋白2在制备治疗脱髓鞘疾病的药物中的应用。
较佳地，所述的神经脱髓鞘性疾病(脱髓鞘性疾病)包括焦虑抑郁症状和认知损伤症状。
本发明还提供了所述的胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用的评价方法，所述的方法包括以下步骤：
步骤1：建立脱髓鞘模型；
步骤2：选择评价标准；
步骤3：评价IGFBP2干预效果。
附图说明
图1a为药物脱髓鞘造模及IGFBP2干预流程图。
图1b为应激脱髓鞘造模及IGFBP2干预流程图。
图2a、2b为通过药物脱髓鞘验证IGFBP2干预对动物髓鞘化水平的影响。
图3a～3e为通过应激脱髓鞘验证IGFBP2干预对动物髓鞘化水平的影响。
图4a～4f为通过药物脱髓鞘验证IGFBP2干预对动物应激脱髓鞘后行为学的影响。
图5a～5h为通过应激脱髓鞘验证IGFBP2干预对动物应激脱髓鞘后行为学的影响。
具体实施方式
为了能够更清楚地描述本发明的技术内容，验证IGFBP2在治疗神经脱髓鞘性疾病中的作用，下面结合具体实施例来进行进一步的描述。
建立脱髓鞘大鼠模型
SD大鼠饲养在12小时光照/黑暗节律环境中，可以自由获取水和食物。所有的行为学实验均在动物的光照节律中进行。
药物脱髓鞘：
首先，利用SD大鼠造模，异氟醚(R510-22，RWD China，4-5％诱导，1-2％维持)用于诱导麻醉。深度麻醉后，使用33号注射器针头(Hamilton)在以下立体坐标下，通过手术将1μL 1％溶血卵磷脂(LPC,Sigma-Aldrich)溶解在盐水中，对照组使用同体积的盐水，将液体注射到mPFC的双侧：+2.7毫米；前囟的中间侧，0.4毫米；和背腹-硬脑膜表面以下，3.3毫米。溶血卵磷脂体积为1μL，单侧注射时间为15min。注射后，针头静置5分钟，使药液完全吸收。慢慢调节Z轴旋钮向上拔针。缝合皮肤以完成脑定位注射。
应激脱髓鞘：
应激方式为：长期不可预期慢性应激(CUMS)，即采用不同的应激方式，进行为期5周的慢性应激，每天一次。具体的应激方式见表1。
表1.CUMS应激方式
| 应激类型 | 描述 | 持续时长 |
| 笼子倾斜 | 笼子倾斜45° | 12小时 |
| 陌生鼠 | 将一只陌生动物放在笼子中 | 12小时 |
| 食物或水剥夺 | 将食物或水拿走 | 12小时 |
| 湿笼 | 100克垫料加200毫升水 | 12小时 |
| 持续照明 | 在夜晚进行照明 | 12小时 |
| 束缚 | 放在管子中束缚 | 2小时 |
| 夹尾巴 | 用夹子在尾巴尖端1厘米处 | 1分钟 |
| 冰水游泳 | 放在6℃的冰水中 | 5分钟 |
IGFBP2的干预
对于药物脱髓鞘的IGFBP2的干预：LPC注射手术4天后脱髓鞘完成，进行IGFBP2的干预。手术8天后进行行为学检测。实验流程如图1a所示。
对于应激脱髓鞘的IGFBP2的干预：应激结束后1天，进行IGFBP2的干预。第2天进行行为学检测。实验流程如图1b所示。
评价IGFBP2干预效果
首先通过免疫组化和电镜实验，观察髓鞘的形态，判断IGCBP2是否逆转药物或者应激引发的脱髓鞘现象。
免疫组化实验：将大鼠固定在单独的解剖板上，首先用1xPBS进行灌注，然后用4％多聚甲醛(PFA)灌注，并在4℃下后固定到4％PFA中过夜。然后用30％蔗糖脱水至脑组织完全沉入50mL管底。OCT包埋后，将大脑保存在-80℃冰箱中。冰冻切片机切30μm冠状切片。mPFC区域切片用PBS洗涤3次，每次5分钟，通过在含有4％山羊血清和0.1％Triton X-100的PBS中室温孵育封闭1小时，随后孵育一抗MBP抗体(1:500)，4℃过夜。PBS和0.1％Triton X-100(PBST)中在室温下洗涤5次，持续5分钟，室温孵育荧光二抗1:1000稀释的Alexa Fluor488山羊抗兔，随后在PBST中在暗室中洗涤5次，每次5分钟。切片用DAPI复染，然后密封切片。所有图像均使用共聚焦显微镜(OLYMPUS)获得，并使用Fuji(NIMH,Bethesda MD,USA)进行分析。
电镜实验：用于测量髓鞘厚度以及mPFC区域中突触后致密体(PSD)的厚度和长度。修剪mPFC大脑区域以包含1mm3区域。在超薄切片机(Leica EM UC7)上以60nm切割切片，然后用醋酸双氧铀和柠檬酸铅染色。使用透射电子显微镜(Tecnai G2 SpiritBiotwin)收集每只大鼠的图像。通过将轴突的直径除以整个有髓纤维的直径来确定G比。Fuji(NIMH,Bethesda MD,USA)用于测量轴突口径、髓鞘纤维直径和PSD的厚度和长度。
如图2a和2b所示，脱髓鞘后，通过免疫组化观察IGFBP2的干预效果。在图2a和2b显示mPFC脑区髓鞘化水平，药物脱髓鞘后，MBP的表达量显著下降，髓鞘化水平显著下降，IGFBP2干预后，髓鞘化水平显著升高。双因素方差分析，***代表P<0.001，**代表P<0.001，*代表P<0.05。
如图3a～3e所示，应激脱髓鞘后，电镜实验结果显示，应激导致脱髓鞘，IGFBP2干预后髓鞘化得到改善；免疫荧光实验结果显示，应激导致MBP的荧光强度显著下降，而IGFBP2的干预使得荧光强度得到恢复。其中，***代表P<0.001，**代表P<0.001，*代表P<0.05，*代表对照组vs应激组；#代表对照组vsIGFBP2干预组。
综上实验数据，支持了IGFBP2干预可以显著的提高髓鞘化，改善脱髓鞘疾病的髓鞘化水平。
进一步通过行为学实验观察IGFBP2干预对脱髓鞘疾病的精神症状的改善。
通过行为学检测验证IGFBP2对脱髓鞘后的精神症状的干预效果。所有的行为学实验均在大鼠光照时间进行。在行为学实验前，将大鼠放入行为学房间适应2小时。各实验过程中，每完成一只老鼠实验，用70％酒精擦拭，避免气味对下只老鼠的影响。行为学前每天Handle大鼠几分钟，至少适应4天。行为学实验采用了评价抑郁样行为的糖水偏好实验、悬尾实验、强迫游泳实验，评价焦虑样行为的旷场实验和高架十字迷宫实验，以及评价认知行为的新物体识别实验和Barnes迷宫实验。
糖水偏好实验：提前两天进行双瓶适应，一瓶饮用水，一瓶1％的蔗糖。第三天进行检测，检测前动物断水6小时，糖水偏好(％)＝(糖水消耗/(糖水+普通水消耗))*100。
旷场实验：将实验动物放入50cm X 50cm的箱子中，摄像时间10分钟，计算其在中心区的时间。
高架十字迷宫实验：将大鼠从中央格面向开放臂方向放入十字迷宫，分别记录进入开放臂的次数、停留的时间、进入闭合臂的次数、停留的时间，记录5分钟。
水迷宫实验：大鼠进行四天的训练，记录大鼠找到平台的时间，并且记录动物最后一天在目标象限的时间。
对于药物脱髓鞘动物，图4a～4d为旷场实验结果，图4e和4f为高架十字迷宫实验结果，其中动物在开放臂的时间比例：开放臂时间/(开放臂时间+闭合臂时间)。采用双因素方差分析，***代表P<0.001，**代表P<0.001，*代表P<0.05。
旷场实验结果显示，脱髓鞘组动物(LPC组)在中心区的时间显著低于对照组，而IGFBP2干预后，脱髓鞘组的动物(IGFBP2干预组)在中心区的时间显著增加。
高架十字迷宫实验结果显示，脱髓鞘组动物(LPC组)在开放臂的时间要显著低于对照组。IGFBP2的干预显著提高了脱髓鞘组动物(IGFBP2干预组)在开放臂的时间。
对于应激脱髓鞘动物，应激后导致mPFC脑区脱髓鞘，如图5a～5c显示旷场实验结果，图5d和5e显示高架十字迷宫实验结果，动物在开放臂的时间比例：开放臂时间/(开放臂时间+闭合臂时间)，图5f显示糖水偏好实验结果，动物的糖水消耗比例：糖水消耗量/(糖水消耗量+水的消耗量)，图5g和5h显示水迷宫实验结果，动物在训练过程种找到平台的时间。采用双因素方差分析，***代表P<0.001，**代表P<0.001，*代表P<0.05。
旷场实验结果显示，在对照动物中，IGFBP2的干预并不会影响其在中心区时间。应激组动物在中心区的时间显著低于对照组，而IGFBP2干预后，应激组的动物在中心区的时间显著增加(P<0.0001)。
高架十字迷宫实验结果显示，应激组动物在开放臂的时间要显著低于对照组。IGFBP2的干预并不会影响对照组动物在开放臂的时间。但是，IGFBP2的干预显著提高了应激组动物在开放臂的时间。
糖水偏好实验结果显示，应激组动物的糖水偏好值显著低于对照组动物，表现出明显的快感缺失行为。IGFBP2的干预并没有影响对照组动物的糖水偏好值，但是显著提高了应激组动物的糖水偏好值。
水迷宫实验结果显示，应激组的动物在训练过程中要花费更多的时间去找到平台，但是在IGFBP2干预后，应激组动物找到平台的时间显著降低。
旷场实验和高架十字迷宫实验结果显示IGFBP2可以有效的改善脱髓鞘引起的焦虑样行为。糖水偏好实验结果显示IGFBP2干预可以有效地改善脱髓鞘引起的抑郁样行为。水迷宫实验结果表明IGFBP2干预可以有效的改善脱髓鞘引起的认知功能障碍。
综上所述，支持了IGFBP2干预可以显著的提高髓鞘化水平，并且改善应激脱髓鞘引起的精神症状。
在此说明书中，本发明已参照其特定的实施例作了描述。但是，很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。因此，说明书被认为是说明性的而非限制性的。
Claims (2)
1.胰岛素样生长因子结合蛋白2在制备治疗神经脱髓鞘性疾病的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，胰岛素样生长因子结合蛋白2在制备提高髓鞘化水平的药物中的应用。
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