CN114736283B - 一种多肽及其在制备预防和/或治疗帕金森药物中的应用 - Google Patents
一种多肽及其在制备预防和/或治疗帕金森药物中的应用 Download PDFInfo
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Abstract
本发明提供了一种多肽及其在制备预防和/或治疗帕金森药物中的应用，属于生物医药技术领域，本发明提供的多肽由封端基团和短肽连接得到，所述短肽的序列为LCPPPSPAVAK，解决了现有技术中缺少利用C9orf72相关的多肽用于制备预防和治疗帕金森的药物的问题。本发明提供的多肽MYR‑C9orff72可通过抑制CDK5磷酸化C9orf72蛋白的S9位点，逆转PD小鼠模型黑质C9orf72蛋白的降解，改善自噬，加速对自噬底物的清除，从而发挥神经保护作用。为PD的药物治疗提供了新的思路和方向，为寻找新的PD药物靶点及设计新的PD临床药物提供了依据。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种多肽及其在制备预防和/或治疗帕金森药物中的应用。
背景技术
帕金森病(PD，Parkinson's disease)是一种常见的进行性发展、隐匿起病的神经退行性疾病。多见于老年人，平均发病年龄为60岁。PD又称为震颤麻痹，临床上主要表现为静止性震颤、运动迟缓、肌强直和姿势步态障碍。PD最重要的病理改变是中脑黑质多巴胺能神经元的退化和死亡，导致纹状体多巴胺含量显著减少，以及神经元中路易小体的形成。
9号染色体开放阅读框72(C9orf72)基因位于9号染色体(9p21)的短臂上，编码11个外显子。C9orf72基因编码的蛋白质存在于各种组织中。在神经系统中，C9orf72蛋白在大脑的各个区域都有表达。这种蛋白质在大脑皮层的神经元以及大脑和脊髓中控制运动的特殊神经元(运动神经元)中含量丰富。近年来的研究发现C9orf72与神经退行性的疾病的发病密切相关，有研究表明，C9orf72可以在阿尔茨海默病(AD)细胞模型中高表达，并且抑制C9orf72表达可提高AD细胞的存活率并抑制其凋亡；ALS和FTD病人的C9orf72蛋白表达量降低，敲减C9orf72会导致内吞作用和自噬功能障碍。C9 FTD/ALS患者的神经元基础自噬受损，对自噬抑制的敏感性增加，表明C9orf72水平的降低会导致神经元细胞受损。由于ALS、FTD和帕金森的临床和病理特征存在重叠，所以我们推测C9orf72蛋白功能缺失引起的自噬障碍也可能与PD有关。
目前尚未见到相关研究利用C9orf72相关的多肽用于制备预防和治疗帕金森的药物。
发明内容
本发明的目的在于提供一种多肽及其在制备预防和/或治疗帕金森药物中的应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种多肽，由封端基团和短肽连接得到，所述短肽的序列如SEQ IDNO.1所示。
优选的，所述封端基团为肉豆蔻酸。
本发明还提供了所述多肽在制备预防和/或治疗帕金森药物中的应用。
本发明还提供了所述多肽在制备保护神经的药物中的应用。
本发明的技术效果和优点：
本发明提供了一种多肽，由封端基团和短肽连接得到，所述短肽的序列如SEQ IDNO.1所示，所述封端基团为肉豆蔻酸。本申请的多肽可以挽救MPP+所致的神经元细胞死亡，可以有效减轻MPTP所致急性PD小鼠的运动行为学障碍，显著改善了MPTP所致的小鼠黑质多巴胺能神经元丢失以及挽救MPTP对小鼠造成的自噬底物堆积和自噬功能障碍。为PD的药物治疗提供了新的思路和方向，为寻找新的PD药物靶点及设计新的PD临床药物提供了依据。
附图说明
图1为Myr-C9orf72肽对MPP+处理的原代神经元细胞活力的影响结果；
图2为Myr-C9orf72对MPTP所致小鼠运动行为学功能障碍的影响结果；
图3为TH酶免疫荧光染色结果；
图4为TH+神经元计数及统计分析结果；
图5为C9orf72、p62、α-synuclein、LC3的Westernbottting结果；
图6为C9orf72、p62、α-synuclein、LC3的相对含量的统计分析结果。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1设计多肽
MYR-C9orff72肽：包含C9orf72蛋白Ser9位点(C9orf72蛋白4-14位氨基酸)，序列为LCPPPSPAVAK的十一个氨基酸的多肽，并在短肽的N-末端共价结合肉豆蔻酸(Myristicacid，MYR)，用以提高其穿透细胞膜的能力。
MYR-Scramble：MYR-C9orff72肽的乱序肽(Scramble)，序列为PASAPVPLPCK，在短肽的N-末端同样结合肉豆蔻酸(Myristic acid，MYR)。
委托GLBiochem(shanghai)Ltd公司合成。
实施例2实验分组
空白组、200μM MPP+组、0.1μM MYR-Scramble肽+MPP+组、1μMMYR-Scramble肽+MPP+组、10μM MYR-Scramble肽+MPP+组、0.1μMMYR-C9orf72肽+MPP+组、1μM MYR-C9orf72肽+MPP+组、10μMMYR-C9orf72肽+MPP+组、0.1μM MYR-Scramble肽组、1μM MYR-Scramble肽组、10μMMYR-Scramble肽组、0.1μM MYR-C9orf72肽组、1μMMYR-C9orf72肽组、10μM MYR-C9orff72肽组。
MPP+处理：MPP+组加入200μM MPP+，处理完后放入培养箱继续培养24h；
MYR-Scramble肽+MPP+组先加入对应量的MYR-Scramble肽预处理24h，再加入MPP+，处理完后放入培养箱继续培养24h；
MYR-C9orf72肽+MPP+组先加入对应量的MYR-C9orf72肽预处理24h，再加入MPP+，处理完后放入培养箱继续培养24h；
MYR-Scramble肽组分别加入对应量的MYR-Scramble肽，处理完后放入培养箱继续培养24h；
MYR-C9orff72肽组分别加入对应量的MYR-C9orf72肽，处理完后放入培养箱继续培养24h。
实施例3对MPP+处理的原代神经元细胞活性的影响
用实施例2中的实验组对原代神经元细胞(原代神经元细胞采用常规方法分离自SD大鼠，SD大鼠购自桂林医学院动物实验中心)进行处理，实验组分别处理完成后，弃掉培养液，取新鲜完全培养液与CCK-8按100μL：10μL混匀，每孔加入110μL混合液，继续培养4h。用酶标仪在450nm波长下测定每孔的OD值。
细胞存活率(％)＝(实验组A﹣空白组A)/(对照组A﹣空白组A)×100％
统计结果，如图1所示(数据以均数±SEM形式表示，n＝3。与空白组相比，***P＜0.001，**P＜0.01。)。
由图1可知，不同浓度的Myr-C9orf72肽处理均可挽救MPP+所致的神经元死亡，且与空白组和对照组相比具有统计学意义(P＜0.001)，说明Myr-C9orf72肽对MPP+所致的神经元细胞死亡有挽救作用。
实施例4对MPTP所致的小鼠的运动行为学障碍的影响
涉及的实验动物：健康C57BL/6J小鼠，雄性，8周龄，体重25±3g，购自湖南斯莱克景达实验动物有限公司，动物使用许可证号：SCXK桂2019-0004，实验动物生产许可证号：SCXK(湘)2020-0005。每笼6只饲养于温度23±2℃，相对湿度60±10％的动物饲养室，饲养条件为室温(23±1℃)，人工光照/昼夜循环(12h-12h)，及时添食、换水，换垫料。于给药前适应1周。所有动物实验均经桂林医学院动物伦理委员会审查和批准。
将8周龄C57雄性小鼠分4组，每组8只，分别为对照组(Control)、MPTP组、MPTP+Myr-C9orf72肽模型组(C1)、MPTP+MYR-Scramble肽模型组(C2)。
对照组注射0.9％生理盐水，MPTP组25mg/kg腹腔注射MPTP，注射5天；
模型组：每天上午按25mg/kg腹腔注射MPTP；下午MPTP+Myr-C9orf72肽模型组和MPTP+MYR-Scramble肽模型组分别腹腔注射治疗多肽和乱序多肽2mg/kg，持续5天；第6-10天MPTP+Myr-C9orf72肽模型组和MPTP+乱序多肽模型组分别腹腔注射Myr-C9orf72肽和Myr-Scramble肽2mg/kg。
运动行为学检测方法：
转棒实验：正式实验开始前三天将待测小鼠放入实验室适应三天，每天半小时至一小时。其中每隔一定时间去抓小鼠尾巴使其适应。实验开始前半小时将所有小鼠放入实验室适应环境。设置转棒转动模式“匀加速运动”最小速度2r/min，最大速度20r/min。正式实验开始前将小鼠放在转轮上适应1-2分钟，若其掉下时时间不足30秒，再放上，至其学会为止，共学习三次。正式实验开始时，先用酒精喷洒转轮，再搽干，之后将小鼠放上，背对实验人员，开始实验5min。若小鼠滞留时间不足30秒，记为30秒，每只小鼠测3次，取最大值。
旷场实验：旷场四壁贴上不同形状及颜色纸板标记；正式实验开始前三天将待测小鼠放入实验室适应三天，每天半小时至一小时；实验开始前半小时将所有小鼠放入实验室适应环境；打开“动物行为学”系统，点击“实验”的下拉菜单中的“新建”选择实验类型“自发活动”动物“小鼠”并命名选中新建的实验；点击“添加动物”可以用耳标号命名并设置不同组；选中新建的实验点击“系统设置”，选择视频存储路径，右键下拉菜单点击“添加实验箱”并命名活动区域；向旷场四壁及底部喷洒75％酒精，再擦干。将小鼠轻柔抓取，小心放在旷场中央。同时点击“录像”先让其适应3分钟，再录像10分钟。查看“实验结果”并“导出”实验结果。记录导出的各指标，用来分析小鼠运动能力。
转棒试验中掉落滚轴的时间及旷场实验中运动距离结果如图2所示(n＝8/组。**P＜0.01，*P＜0.05。数据以平均值±SEM表示)。
由图2可知，与空白组相比，MPTP处理的模型组小鼠在转棒试验中掉落滚轴的时间及旷场实验中运动距离均明显缩短，且有显著差异(P＜0.01)；然而，Myr-C9orf72肽+MPTP模型组与MPTP模型组比较，掉落滚轴的时间及运动距离均明显增高，有统计学差异(P＜0.05)，说明Myr-C9orf72肽可以有效减轻MPTP所致急性PD小鼠的运动行为学障碍。
实施例5对MPTP所致的小鼠中脑黑质多巴胺能神经元丢失的影响实验分组情况同实施例4。
通过免疫荧光TH染色及计数评价小鼠中脑DA能神经元的损伤，TH酶免疫荧光染色如图3所示，TH+神经元计数及统计分析结果如图4所示(n＝3/组。***P＜0.001，*P＜0.05。数据以平均值±SEM表示)。
由图3和图4可知，与空白组相比，MPTP模型组小鼠中脑黑质TH阳性细胞数表达明显减少(P＜0.001)；Myr-C9orf72肽+MPTP模型组小鼠中脑黑质TH阳性细胞数表达增高(P＜0.05)，说明Myr-C9orf72肽给药显著改善了MPTP所致的小鼠黑质多巴胺能神经元丢失。
实施例6对MPTP对小鼠中脑黑质区C9orf72、p62、α-synucelin和LC3蛋白表达的影响
采用Westernblotting检测方法，具体过程如下：
1)配置SDS-PAGE凝胶：选取配套的内外玻板，用洗涤剂清洗干净，然后冲洗干净；再用双蒸水冲洗，玻板上端朝下，至于37℃温箱中烤干；分别在不同的管内配置实验所需的分离胶和浓缩胶；取出烤干的玻板，用玻板夹夹好，置于制胶架上固定好；向分离胶中加入APS和TEMED，充分混匀，倒入玻板中，再加入异丙醇；30min后分离胶凝固后，倒掉异丙醇，双蒸水清洗充分，用滤纸吸干水分残留，向浓缩胶中加入APS和TEMED，充分混匀，倒入玻板中，再插入梳子。
(2)电泳：配置1×running buffer，700ml；将制好的胶用电泳夹夹好，倒入1×running buffer；上样，在第一个样品孔的左侧，加5μl的protein ladder，多余的孔用1×SDS-PAGE loading buffer补齐；放入电泳槽，80V恒压跑胶；当蛋白进入分离胶，110V恒压跑胶。
(3)转膜：配制1×trans buffer 500ml，4℃预冷；裁剪好与胶相应大小的PVDF膜，用甲醇激活2-3min，之后转移至1×trans buffer中；将SDS-PAGE胶切下放入1×transbuffer中；从正极到负极的顺序依次为：海绵，滤纸，PVDF膜，SDS-PAGE胶，滤纸，海绵。将转膜夹放入转膜槽中，倒入全部的1×trans buffer；整个转膜槽至于冰水混合物中，200mA恒流转膜2h。
(4)封闭：将膜取出至于，5％牛奶(1×TBST为溶剂)封闭1h。
(5)孵育一抗：将PVDF膜至于乘有一抗稀释液的50mL离心管，蛋白面朝上，4℃孵育过夜。
(6)孵育二抗：将PVDF膜至于1×TBST中洗10min，3次；向20ml的1×TBST中加入1μl的HRP标记的Goat Anti-Rabbit IgG或Goat Anti-Mouse IgG；将PVDF膜至于1×TBST中洗10min，3次。
(7)ECL显色、曝光：将保鲜膜平铺在曝光夹上；配置ECL发光液；将PVDF膜至于保鲜膜上，倾斜弃去PVDF膜上的1×TBST；加上ECL发光液，均匀铺满PVDF膜，计时3-4min；倾斜并用滤纸吸去ECL发光液，盖上保鲜膜，擦拭使其平整；取出胶片，至于曝光夹内一定时间(根据条带的强度决定)，至于显影液中显影，然后再将胶片至于定影液中定影；水冲洗胶片，晾干；用扫描仪扫描胶片后，用Image J分析软件分析条带灰度值。
其中，蛋白样本上样量为10μL；各一抗的稀释比例分别为：小鼠抗β-actin(1:5000)，兔抗C9orf72(1:800)，兔抗α-synuclein(1:800)，兔抗p62(1:1000)，兔抗LC3(1:1000)。
C9orf72、p62、α-synuclein、LC3的Westernbottting如图5所示，C9orf72、p62、α-synuclein、LC3的相对含量的统计分析如图6所示(n＝3/组，**P＜0.01，*P＜0.05。数据以平均值±SEM表示)。
由图5和图6可知，与对照组相比，MPTP模型组小鼠C9orf72明显降低，p62、α-synuclein表达均显著升高，LC3Ⅱ/LC3Ⅰ降低。与MPTP模型组相比，Myr-C9orf72肽模型组C9orf72升高，p62、α-synuclein表达降低，LC3Ⅱ/LC3Ⅰ升高，证明Myr-C9orf72肽可以挽救MPTP对小鼠造成的自噬底物堆积和自噬功能障碍。
由以上实施例可知，本发明提供的多肽MYR-C9orff72可通过抑制CDK5磷酸化C9orf72蛋白的S9位点，逆转PD小鼠模型黑质C9orf72蛋白的降解，改善自噬，加速对自噬底物的清除，从而发挥神经保护作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 桂林医学院
<120> 一种多肽及其在制备预防和/或治疗帕金森药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Leu Cys Pro Pro Pro Ser Pro Ala Val Ala Lys
1 5 10
Claims (2)
1.一种多肽，其特征在于，由封端基团和短肽连接得到，所述短肽的氨基酸序列如SEQID NO.1所示；所述封端基团为肉豆蔻酸。
2.权利要求1所述的多肽在制备治疗帕金森病药物中的应用。
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| CN107802823A (zh) * | 2017-10-27 | 2018-03-16 | 胡军 | Sh2b衔接蛋白1在治疗帕金森病中的功能应用 |
| CN110041408A (zh) * | 2018-01-16 | 2019-07-23 | 华中科技大学 | 一种小分子多肽及其在制备防治帕金森综合症药物中的应用 |
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| CN107802823A (zh) * | 2017-10-27 | 2018-03-16 | 胡军 | Sh2b衔接蛋白1在治疗帕金森病中的功能应用 |
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