CN106511367A - C41分子在制备治疗多种不同病原模式分子介导炎症反应药物中的应用 - Google Patents
C41分子在制备治疗多种不同病原模式分子介导炎症反应药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种C41分子在制备治疗多种不同病原模式分子介导炎症反应药物中的应用。所述应用是基于C41分子靶向作用于UNC93B蛋白，对内膜型Toll样受体和NOD样受体介导的炎症反应具有多重免疫抑制效应，从而干预多种不同病原模式分子引导的炎症反应，由此，对过敏性疾病和自身免疫性疾病等具有辅助治疗作用。
Description
技术领域
本发明涉及医药生物领域，特别涉及一种C41分子在制备治疗多种不同病原模式分子介导炎症反应药物中的应用。
背景技术
致病微生物感染机体后，通过特定的模式识别受体(PRRs)与病原相关分子模式(PAMPs)结合，快速激活固有免疫系统。重要的PRRs包括Toll样受体(TLRs)和NOD样受体(NLRs)。
TLRs主要表达于固有免疫细胞，如树突状细胞，巨噬细胞。根据TLRs在细胞的定位分为两大类：一类包括TLR1、2、4、5、6、11，它们主要表达于膜表面，识别如脂质、脂蛋白和蛋白质等微生物的膜组成成分；另一类包括TLR3、7、8、9，它们主要位于细胞内的囊泡，如内质网、核内体等，主要识别病原微生物的核酸。TLR不仅在抵御病原微生物入侵的固有免疫应答中发挥重要作用，而且它连接固有免疫和适应性免疫反应。
NLRP3炎症小体是由NLRs家族成员NLRP3蛋白、接头蛋白ASC及caspase-1形成的一个多蛋白复合物，其活化后能促进IL-1β、IL-18等多种炎性细胞因子成熟与分泌，在炎症发生过程中起关键作用。
C41是申请人前期通过生物信息学筛选出的一种特定序列核酸片段，其序列见CN105541947A。目前，受国家自然科学基金(No.81373133)和重庆市重庆市前沿与应用基础研究计划重点项目(cstc2013jjB0128)的资助，进一步深入研究TLR9阻断剂c41分子的作用机制。
发明内容
本发明的目的是提供一种C41分子在制备治疗多种不同病原模式分子介导炎症反应药物中的应用，所述应用是基于C41分子靶向作用于UNC93B蛋白，对内膜型Toll样受体和NOD样受体介导的炎症反应具有多重免疫抑制效应，从而干预多种不同病原模式分子引导的炎症反应，由此，对过敏性疾病和自身免疫性疾病等具有辅助治疗作用。
本发明的技术方案是：
C41分子在治疗抑制先天免疫系统中内膜型受体介导的过敏性疾病和自身免疫性疾病药物中的应用。
进一步，C41分子靶向作用于UNC93B蛋白，从而对内膜型Toll样受体和NOD样受体介导的炎症反应具有多重免疫抑制效应。
进一步，C41分子在制备缓解内膜型Toll样受体诱导的前炎症因子释放及NOD样受体介导的炎症小体形成的药物中的应用。
进一步，C41分子在制备由多种不同病原模式分子通过内膜型受体诱发炎症性疾病的药物中的应用。
申请人通过免疫荧光技术观察了CpG-c41的内化过程，扑捉到C41与一种关键转运蛋白-UNC93B1，在胞质中发生共定位。UNC93B是一种多跨膜结构域蛋白，通过与识别核酸的TLRs之间物理性相互作用，控制TLRs的转运，从而影响下游信号传递。同时细菌RNA必须依赖于UNC93B活化Caspase-1。申请人的实验表明，当内膜型TLRs和NLRs活化后，UNC93B蛋白参与内化转运，但是，在此过程中，C41通过靶向UNC93B蛋白，阻断多种胞内受体介导的信号传递，从而达到多重免疫抑制效应。
本发明不仅揭示了C41在先天免疫过程中的多重免疫抑制效应，也提示了靶向UNC93B，能够干预多种不同病原模式分子介导的炎症反应。这对于未来治疗内膜型TLRs及NLRs过度活化相关的过敏性疾病及自身免疫性疾病，提供了新的治疗靶点和策略。
下面的实施例可详细地说明本发明，但不以此限制本发明。
附图说明
图1为所述C41特异性抑制骨髓巨噬细胞TLR3、7/8，9活化；
图2为所述核酸片段抑制经RAW264.7细胞NLR介导的炎症小体的形成和活化；
图3为所述核酸片段与UNC93B蛋白共定位。
具体实施方式
本发明所述C41序列和制备方法见申请人申请号为201610015280.2的专利申请。
实施例1 C41特异性抑制骨髓巨噬细胞TLR3、7/8，9活化
1.1主要材料、试剂与设备：
1)实验动物：Balb/c小鼠(华阜康生物科技股份有限公司，中国)。
2)M-CSF from mouse：Sigma
3)细胞培养基：DMEM(Gibco，Inc.，USA)。
4)Elisa试剂盒：抗鼠TNF-a、IL-6(eBioscience，Inc.，USA)。
5)细胞培养箱：FORMA 3111.
6)酶标仪：全波长多功能酶标仪(Thermo Scientific，USA).
1.2实验方法：取自Balb/c小鼠股骨骨髓细胞，加入96孔板(200μL/孔)，M-CSF终浓度50ng/ml，置37℃，5％CO2培养箱培养2天后使用PBS洗掉未贴壁细胞，再加入含50ng/mlM-CSF的DMEM培养基培养至第4天，更换不含M-CSF的DMEM培养基后分为以下组：DMEM组，TLR3激动剂Poly(I：C)(5μl，终浓度100μg/ml)组，TLR4激动剂LPS(5μl，终浓度100ng/ml)组，TLR7/8激动剂R848(5μl，终浓度0.2μg/ml)组，TLR9激动剂CpG-ODN1826(5μl，终浓度1μM)组，以上各组均设8复孔。然后，各组分出4复孔，加培养液5μl，作为安慰剂；各组剩余的4复孔，加入本发明所述c41分子5μl(终浓度2μM)作为治疗组。继续培养24h后，收集上清，进行Elisa检测，以450nm处测定的各孔吸光度(OD450)值，获取TNF-a、IL-6浓度。
1.3实验结果：安慰剂组，TLR3、4、7、9正常活化，刺激骨髓巨噬细胞细胞释放大量TNF-a和IL-6，；治疗组，在本发明所述药物c41分子作用下，TLR3、7、9的TNF-a、IL-6水平受到显著抑制(P＜0.01)，但是TLR4未见有抑制效应。所以，该药物能特异性有效抑制内模型TLRs活化。如附图1所示。
实施例2 C41抑制RAW264.7细胞经NLR介导的炎症小体的形成和活化
2.1主要材料、设备：
1)细胞株：RAW264.7(ATCC，USA)。
2)细胞培养基：DMEM(Gibco，Inc.，USA)。
3)细胞培养箱：FORMA 3111.
4)化学发光成像系统：ChemioDoc XRS+(USA)
2.2实验方法：
2.2.1主要试剂与作用浓度
试剂
| 试剂 | 终浓度 | 剂量/孔 | 来源 |
| C41 | 10μM | 20μl | 生工 |
| Poly(I：C) | 100μg/ml | 20μl | Ivivogen |
| LPS | 100ng/ml | 20μl | Sigma |
| R848 | 1μg/ml | 20μl | Ivivogen |
| CpG-1826 | 3μM | 20μl | 生工 |
| ATP | 5mM | 20μl | Sigma |
抗体
2.2.2分组与刺激
1)RAW264.7细胞的分离与培养
2)细胞铺板，6孔板，4X106/ml，2ml/每孔，并按下表所分组加工作液，12h后加c41和各TLR激动剂，4h后加ATP，0.5h后取样。
| 安慰剂组 | DMEM | DMEM+Poly(I：C) | DMEM+LPS | DMEM+R848 | DMEM+1826 |
| 治疗组 | C41 | C41+Poly(I：C) | C41+LPS | C41+R848 | C41+1826 |
2.2.3检测：
1)取样：将培养基吸除，加入PBS洗2次，最后加1ml PBS放于冰上20min，细胞大部分脱落。使用细胞刮，将细胞刮取下来，1000转/min 5min，吸除PBS加入120μl细胞裂解液，冰浴30min。12000转/min 4℃15min，留上清。
2)检测蛋白浓度：使用BCA法检测蛋白浓度，确定上样量(上样总蛋白为25μg)；
3)煮蛋白：105℃，5min；
4)配胶：配13.5％的分离胶；
5)上样：按照确定的上样量加入到上层胶孔道，并且加Marker。
上层胶70V 30min，下层胶120V 90min；
6)转膜：250mA 90min；
7)封闭：5％BSA封闭1h；
8)孵一抗：4℃摇床过夜。
9)洗膜：5min，3次
10)孵二抗：1.5h
11)洗膜：15min，4次；
12)曝光：
2.3实验结果：
相对于安慰剂组，LPS，R848，CpG-1826能够有效上调NLRP3的表达，并且能够活化炎症小体产生Caspase10，并导致IL-1beta分泌；治疗组，在本发明所述药物c41分子作用下，R848，CpG-1826作用组的NLRP3、Caspase10(图2A)以及IL-1beta(图2B)水平受到显著抑制(P＜0.01).由于不同于R848，CpG-1826只作用于内膜受体，而LPS还可作用于膜上受体，所以c41对LPS作用组未见有抑制效应。所以，c41药物能特异性抑制病原模式分子经内膜型NLR介导的炎症小体活化。
实施例3 C41与UNC93B共定位。
3.1主要材料、试剂与设备：
1)细胞株：RAW264.7(ATCC，USA)。
2)细胞培养基：DMEM(Gibco，Inc.，USA)。。
3)细胞培养箱：FORMA 3111.
4)激光共聚焦培养皿：20mm(NEST，USA)
5)激光共聚焦显微镜：SLM 780(德国)
6)C41-cy3：生工生物
7)兔抗小鼠UNC93B抗体：Abcam
3.2实验方法：
1)RAW264.7细胞的分离与培养；
2)细胞铺板，激光共聚焦培养皿，1×105/ml，0.5ml/每孔，过夜后加CpG-1826，终浓度1μM；同时加c41-FAM，终浓度2μM。
1.5h吸除培养基，PBS洗两遍；
3)多聚甲醛固定10min，PBS洗2遍；
4)0.1％Triton穿孔5min，PBS洗3次，5min/次；
5)室温5％BSA封闭1h；
6)加浓度为20μg/ml UNC93B和Tubulin一抗50μl，37℃2h，PBS洗3次，5min/次；
7)加山羊抗兔二抗，37℃，50min，PBS洗3次，15min/次；
8)激光共聚焦下拍照。
3.3实验结果：
由于c41能够抑制多种PRRs介导的活化，而这些不同的PRRs下游的信号通路又不同，提示c41抑炎环节与下游信号通路无关，且应当处于内化转运的过程中。于是，我们通过激光共聚焦显微镜，观察了与内化转运相关蛋白，包括网格蛋白、肌动蛋白、UNC93B1蛋白等，以探明c41的抑制功能是否与这些蛋白相关。
该结果显示，无刺激性的c41单独入胞后，我们并未观察到任何共定位(图3A)，有意思的是，在使用内膜型TLRs激动剂刺激之后，UNC93b在胞浆中的分布有所扩散，并与C41发生显著的共定位(图3B)，如图3所示。相比而言，c41与对照组tubulin蛋白的无共定位(图3C)。UNC93B1是近年发现的新蛋白，对其功能的认识尚不全面，以往报道证实UNC93B1是一种伴侣蛋白能够调节所有胞内TLRs运输至溶酶体中，然后依赖于MyD88/TRIF的途径传递信号。由此说明，c41单独入胞后，不会激发UNC93b的运动，但是，在内膜型PRRs受刺激后，UNC93B发生动态调用，才发生与c41的特异性结合，从而干扰刺激信号向下游传递。
Claims (4)
1.C41分子在治疗抑制先天免疫系统中内膜型受体介导的过敏性疾病和自身免疫性疾病药物中的应用。
2.根据权利要求1所述的应用，其特征在于：C41分子靶向作用于UNC93B蛋白，从而对内膜型Toll样受体和NOD样受体介导的炎症反应具有多重免疫抑制效应。
3.根据权利要求1所述的应用，其特征在于：C41分子在制备缓解内膜型Toll样受体诱导的前炎症因子释放及NOD样受体介导的炎症小体形成的药物中的应用。
4.根据权利要求1所述的应用，其特征在于：C41分子在制备由多种不同病原模式分子通过内膜型受体诱发炎症性疾病的药物中的应用。
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| CN109646678A (zh) * | 2017-10-12 | 2019-04-19 | 中国科学院上海生命科学研究院 | Sun2蛋白、其制药用途及药物 |
| CN110251657A (zh) * | 2019-06-14 | 2019-09-20 | 中山大学 | Ebv brlf1及其功能性小肽在抑制炎症小体活性中的应用 |
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