CN116672462A - 多肽在制备中枢神经系统缺氧性疾病防治药物中的应用 - Google Patents
多肽在制备中枢神经系统缺氧性疾病防治药物中的应用 Download PDFInfo
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Abstract
本发明公开了TAT‑MK‑ELE多肽在制备中枢神经系统缺氧性疾病防治药物中的应用。为了模拟MK‑4的作用，本发明设计并合成了TAT‑MK‑ELE多肽，同时研究证明了TAT‑MK‑ELE多肽对缺氧少突胶质细胞具有保护作用，一方面可显著提高缺氧少突胶质细胞的活性，同时另一方面可显著抑制缺氧少突胶质细胞内促炎因子的表达，为临床上以少突胶质细胞为靶点防治缺氧导致的中枢神经系统疾病提供参考。
Description
技术领域
本发明属于生物医药技术领域，具体涉及TAT-MK-ELE多肽在制备中枢神经系统缺氧性疾病防治药物中的应用。
背景技术
少突胶质细胞是中枢神经系统有髓神经纤维髓鞘的形成细胞和脑白质的重要组成细胞。少突胶质细胞除了以上功能外，还具有营养和保护轴突，为中枢神经系统提供神经营养因子和生长因子、产生轴突生长抑制因子等作用。临床上少突胶质细胞与中枢神经系统疾病如多发性硬化症、脊髓损伤、脑白质损伤等有着重要联系，同时少突胶质细胞和神经元一样对缺氧极为敏感，缺氧造成的中枢神经系统细胞损伤和死亡是各种脑疾病的发生和发展的病理基础，如缺氧是导致新生儿脑白质损伤的最主要原因。
但目前临床上针对治疗少突胶质细胞缺氧性损伤疾病的药物疗效并不显著。因此，研究少突胶质细胞的缺氧损伤机制和寻找缺氧少突胶质细胞的保护剂将会对脑白质损伤、脱髓鞘脑病等中枢神经系统缺氧性损伤具有重要意义。
发明内容
本发明的目的是提供TAT-MK-ELE多肽在制备中枢神经系统缺氧性疾病防治药物中的应用。
所述TAT-MK-ELE多肽的的氨基酸序列为：
GRKKRRQRRRPQGGSGHSGELETAFADAWRTPTTTVIEM(seq_1)。
其中GRKKRRQRRRPQ为促进多肽进入细胞内的TAT序列，GGSGHSG为连接肽，ELETAFADAWRTPTTTVIEM为目标序列。
具体氨基酸人工序列：Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro GlnGly Gly Ser Gly His Ser Gly Glu Leu Glu Thr Ala Phe Ala Asp Ala Trp Arg ThrPro Thr Thr Thr Val Ile Glu Met。
经过实验证明，TAT-MK-ELE多肽可显著提高缺氧少突胶质细胞的活性，同时该多肽可显著抑制缺氧少突胶质细胞内促炎因子的表达。
本发明提供的TAT-MK-ELE多肽能够通过改善缺氧少突胶质细胞中促炎因子的表达，进而起到了保护缺氧少突胶质细胞的作用，这对于在设计并制备以少突胶质细胞为靶点的防治中枢神经系统缺氧性疾病药物中良好的应用前景。
附图说明
图1为TAT-MK-ELE多肽对缺氧少突胶质细胞活力影响的结果。其中，#p<0.05vsDMSO处理正常氧含量组；*p<0.05vs DMSO处理缺氧组。
图2为TAT-MK-ELE多肽对缺氧少突胶质细胞促炎因子IL-1βmRNA表达影响的结果。其中，N+D:DMSO处理正常氧含量组；N+M:MK-4处理正常氧含量组；N+T:TAT-MK-ELE处理正常氧含量组；H+D:DMSO处理缺氧组；H+M:MK-4处理缺氧组；H+T:TAT-MK-ELE处理缺氧组。#p<0.05vs N+D组；*p<0.05vs H+D组。
图3为TAT-MK-ELE多肽对缺氧少突胶质细胞促炎因子IL-6mRNA表达影响的结果。其中，N+D:DMSO处理正常氧含量组；N+M:MK-4处理正常氧含量组；N+T:TAT-MK-ELE处理正常氧含量组；H+D:DMSO处理缺氧组；H+M:MK-4处理缺氧组；H+T:TAT-MK-ELE处理缺氧组。#p<0.05vs N+D组；*p<0.05vs H+D组。
图4为TAT-MK-ELE多肽对缺氧少突胶质细胞促炎因子Tnf-αmRNA表达影响的结果。其中，N+D:DMSO处理正常氧含量组；N+M:MK-4处理正常氧含量组；N+T:TAT-MK-ELE处理正常氧含量组；H+D:DMSO处理缺氧组；H+M:MK-4处理缺氧组；H+T:TAT-MK-ELE处理缺氧组。#p<0.05vs N+D组；*p<0.05vs H+D组。
具体实施方式
甲基萘醌-4(menaquinone-4,MK-4)又称为维生素K2(vitamin K2,VK2)，在除肝脏以为器官中，VK2均将转换成MK-4。同时，MK-4是脑中维生素K主要活性形式。研究表明：MK-4可以促进胚胎后期皮层、海马和纹状体的神经元细胞存活，MK-4能够抑制脂多糖诱导的小胶质细胞炎症反应，MK-4可降低发育中胚胎皮层神经元和少突胶质前体细胞氧化应激损伤。这些研究表明了MK-4在抗氧化应激和抗炎中起到一定作用。已经有研究证实，MK-4对少突胶质细胞的缺氧性损伤具有保护作用。为了模拟MK-4的作用，寻找到一种可以大量合成并临床上操作简便的药物，发明人设计并合成了TAT-MK-ELE多肽，同时研究证明了TAT-MK-ELE多肽对缺氧少突胶质细胞具有保护作用，为临床上以少突胶质细胞为靶点防治缺氧导致的中枢神经系统疾病提供参考。
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
一、实验材料
TAT-MK-ELE(强耀生物)、大鼠少突胶质细胞Oln-93(北京中科质检生物技术有限公司)、DMEM-F12(Thermo)、胎牛血清(Gibco)、Cell Counting Kit-8(CCK-8,DojindoLaboratories)、TRI Reagnet(Sigma)、SYBR Green PCR Kit(Thermo)。
二、实验方法
1、TAT-MK-ELE多肽溶液配制
多肽冻干粉溶解于无菌水中，储存浓度为1mg/mL，100μL分装，至于-80℃保存。
2、细胞培养和缺氧模型的建立
Oln-93细胞培养在含有10％的胎牛血清的DMEM-F12培养基中，待细胞长满进行传代。将Oln-93细胞接种于培养板，分为正常氧含量组和缺氧组。正常氧含量组用含10％FBS的DMEM-F12培养基，置于5％ CO2/95％空气、37℃的培养箱中。缺氧组采用DWS H35低氧工作站(5％ CO2/95％ N2，37℃)加无糖无血清的培养条件建立缺氧少突胶质细胞模型，缺氧12h。
3、CCK-8细胞活性检测
Oln-93细胞以每孔104细胞接种于96孔板，每孔100μL细胞悬液，置于培养箱。次日，细胞分为正常氧含量组和缺氧组，并换成相应的培养基；在培养基中加入0.01％ DMSO或者8μmol/L MK-4或者20μg/mL TAT-MK-ELE多肽，预处理细胞20min；接着将细胞置于正常氧含量培养箱或低氧工作站，培养12h。终止孵育，每孔加入10μL CCK-8溶液，培养箱内孵育2h。用Tecan M200酶标仪测定在450nm处各孔的吸光度。每种浓度设置3个复孔，实验重复3次。
4、炎症因子mRNA的表达变化的检测
0.01％ DMSO或者8μmol/L MK-4或者30μg/mL TAT-MK-ELE多肽预处理细胞20min后，分别正常氧或缺氧孵育12h。用TRI Reagnet提取各实验组细胞的总RNA，用逆转录试剂盒合成cDNA。利用Primer 3software version 1.0设计白介素-1beta(interleukin-1beta，IL-1β)，白介素-6(interleukin-6，IL-6)、肿瘤坏死因子-alpha(tumor necrosisfactor alpha，TNF-α)，和β-actin的引物(表1)。
表1.实时定量RT-PCR引物序列
三、实验结果
1、TAT-MK-ELE多肽提高缺氧少突胶质细胞的活性
MK-4可以保护少突胶质细胞，对缺氧少突胶质细胞的活性有显著提高的作用。在分别用0.01％ DMSO或者8μmol/L MK-4或者20μg/mL TAT-MK-ELE多肽，预处理Oln-93细胞20min，然后将细胞缺氧处理12h。结果显示缺氧12h后，Oln-93细胞活性显著下降，但是MK-4、TAT-MK-ELE多肽可以显著提高缺氧少突胶质细胞的活性(图1)；更重要地是，TAT-MK-ELE多肽提高缺氧少突胶质细胞活性的效果要优于MK-4(图1)。
2、TAT-MK-ELE多肽抑制缺氧少突胶质细胞内促炎因子的表达
为了进一步理解小分子多肽TAT-MK-ELE对缺氧少突胶质细胞保护作用的机制，用实时定量RT-PCR检测了TAT-MK-ELE多肽对缺氧少突胶质细胞内促炎因子的影响。结果显示，当Oln-93细胞缺氧12h后，胞内促炎细胞因子IL-1β、IL-6、Tnf-α的mRNA水平明显升高；但当用8μmol/L MK-4或者30ug/ml TAT-MK-ELE多肽处理缺氧细胞，其胞内IL-1β,IL-6,Tnf-α的mRNA水平相较于DMSO处理缺氧组则明显降低(图2-图4)；更重要地是，TAT-MK-ELE多肽在抑制缺氧细胞促炎因子表达的效果要优于MK-4(图2-图4)。此结果说明，缺氧可以导致少突胶质细胞内促炎因子的表达增加，引起细胞的炎症反应，但TAT-MK-ELE多肽可以有效降低缺氧引起的少突胶质细胞炎症反应，进而达到保护细胞的作用。
Claims (5)
1. TAT-MK-ELE多肽在制备中枢神经系统缺氧性疾病防治药物中的应用，其特征在于，
所述TAT-MK-ELE多肽的的氨基酸序列为：
GRKKRRQRRRPQGGSGHSGELETAFADAWRTPTTTVIEM。
2.根据权利要求1所述的应用，其特征在于，所述TAT-MK-ELE多肽提高缺氧少突胶质细胞的活性。
3.根据权利要求1所述的应用，其特征在于，所述TAT-MK-ELE多肽抑制缺氧少突胶质细胞内促炎因子的表达。
4.一种中枢神经系统缺氧性疾病防治药物，其特征在于，以TAT-MK-ELE多肽为有效活性成分。
5.根据权利要求4所述的中枢神经系统缺氧性疾病防治药物，其特征在于，还包括药学上可接受的载体。
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