CN110511240B - 一种髓细胞触发受体2的内源性配体及其应用 - Google Patents
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Abstract
本发明公开了一种髓细胞触发受体2的内源性配体及其应用，属于药物研发技术领域，所述髓细胞触发受体2的内源性配体为鞘氨醇‑1‑磷酸，本发明揭示了S1P在不依赖于S1PRs受体的前提下，通过激活TREM2增强小胶质细胞的吞噬功能，首次揭露了S1P是TREM2的内源性配体，初步提示了S1P调节小胶质细胞吞噬功能的新机制。
Description
技术领域
本发明涉及药物研发技术领域，特别是涉及一种髓细胞触发受体2的内源性配体及其应用。
背景技术
小胶质细胞是中枢神经系统中固有的免疫效应细胞。在神经系统的正常发育中，小胶质细胞发挥对凋亡神经元的迅速清除，对神经元突触的修剪，分泌细胞因子等调节神经元的存活等功能；在病理情况下，小胶质细胞是最早发生反应的免疫细胞之一。如缺血性脑卒中等脑损伤时，伴随着大量神经元死亡，死亡的神经元释放诸多危险分子如核酸，蛋白质，脂质，进而导致炎症反应，这些具有神经毒性的细胞碎片若得不到有效的清除，会减弱损伤后神经元的可塑性，引发二次炎症、加剧损伤。因此，调节小胶质细胞的吞噬功能，是减轻神经损伤的重要途径之一。
髓细胞触发受体2(Triggering receptor expressed on myeliod cells，TREM2)是I型单跨膜蛋白，是免疫球蛋白受体家族的一员。TREM2高表达于巨噬细胞，树突细胞，破骨细胞以及小胶质细胞。在中枢神经系统内，TREM2特异地表达于小胶质细胞。由于缺乏胞内段，TREM2须通过其共受体12kDa的DNAX活化蛋白(DAP12)实现信号的传导，最终发挥调节吞噬、促进细胞生长、抑制凋亡、调节炎症等功能。迄今，虽然已发现诸多物质能与TREM2结合引发下游信号，如磷脂类，核酸，蛋白聚糖，热休克蛋白60和载脂蛋白等，但其内源性的配体仍未确定。
鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)是一种具有广泛生物学效应的鞘脂类物质，可调节细胞分化、存活、凋亡、增殖和血管生成等过程。S1P具有增强巨噬细胞的吞噬功能，但是否通过作用于小胶质细胞的TREM2、促进吞噬未见报道。
发明内容
本发明的目的是提供一种髓细胞触发受体2的内源性配体，明确鞘氨醇-1-磷酸(S1P)与髓细胞触发受体2(TREM2)的关系，确定S1P是TREM2的激活剂。
为实现上述目的，本发明提供了如下方案：
本发明提供了一种髓细胞触发受体2的内源性配体，所述内源性配体为鞘氨醇-1-磷酸。
进一步的，所述鞘氨醇-1-磷酸的分子式是C18H38NO5P，结构式为
本发明还提供所述的内源性配体在调节细胞吞噬、调节炎症、促进细胞生长、抑制细胞凋亡中的应用。
进一步的，所述内源性配体在增强小胶质细胞吞噬功能中的应用。
进一步的，所述内源性配体通过激活髓细胞触发受体2增强小胶质细胞的吞噬功能。
本发明用S1P处理转染了TREM2-DAP12嵌合蛋白的CHO稳转株，发现S1P刺激后能增强原本不具有吞噬功能的CHO细胞对吞噬燃料的吞噬，表明S1P通过TREM2引起小胶质细胞的吞噬功能增强。
本发明通过液质联用(LC-MS/MS)和微量热涌动(MST)的方法进一步证明，S1P直接与TREM2结合，表明S1P是TREM2的一个内源性配体。神经元与小胶质细胞共培养时，氧糖剥夺-再灌注损伤后，S1P处理能增强小胶质细胞对神经元碎片的吞噬。小胶质细胞上仅表达S1PR2和S1PR4，鉴于S1PR4与S1P的亲和力很低，因此需排除S1P对S1PR2的作用。结果发现，敲低S1PR2不影响小胶质细胞的吞噬增强，进一步从功能角度说明S1P通过TREM2受体发挥促吞噬功能。
本发明的有益效果：
本发明揭示了S1P是TREM2的一种内源性配体，S1P通过作用于TREM2增强小胶质细胞的吞噬功能，促进对病理损伤情况下细胞碎片的清除。本发明揭示了S1P调控小胶质细胞功能的新作用和新机制。
附图说明
图1为稳定转染TREM2-DAP12嵌合蛋白的CHO细胞内吞吞噬染料情况，其中图1a是稳转株的验证；图1b是无刺激或S1P，LPS处理2小时和4小时，普通CHO细胞和稳转株的吞噬情况，其中LPS是作为阳性参照；图1c是图1b的统计结果；
图2为液质联用结果，显示BV2细胞来源的TREM2能够与S1P发生结合；
图3为微量热涌动检测结果，其中hTREM2，rTREM2，mTREM2分别代表人源性，大鼠源性和小鼠源性的TREM2重组蛋白，结果显示各源性的TREM2均能与S1P发生结合，进一步表明TREM2与S1P的结合关系；
图4为免疫荧光染色结果，其中，图4a：WB验证小胶质细胞上进行TREM2RNA干扰(RNA interference，RNAi)表达的效率；图4b：进行Iba1与NeuN免疫荧光双染色，反映小胶质细胞的吞噬情况；图4c：统计图4b中出现吞噬的小胶质细胞的数量百分比，结果显示S1P促进氧糖剥夺-复灌损伤后小胶质细胞对神经元碎片的吞噬，且TREM2敲低后S1P的促吞噬作用显著降低；
图5为Iba1与CD68免疫荧光双染色结果。图5a：免疫荧光染色原图；图5b：基于荧光染色、统计CD68表达量。结果显示S1P增强OGD处理后小胶质细胞的吞噬功能；
图6为应用RNAi降低S1PR2表达后、评价S1P通过何种途径增强小胶质细胞吞噬功能。图6a：WB验证S1PR2表达的干扰效率，选取S1PR2-430进行后续RNAi实验；图6b：正常和OGD处理后的小胶质细胞，给予S1P后进行Iba1与CD68免疫荧光染色；图6c：对图6b中小胶质细胞CD68表达量进行统计、比较RNA干扰S1PR2表达后对吞噬功能的影响，结果显示S1PR2RNAi对S1P的促吞噬作用无显著影响，排除S1PR2的作用。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：稳转株的构建
构建CD8 leader sequence-TREM2-DAP12嵌合蛋白质粒，将CD8前导段序列，TREM2的胞外及跨膜区域和DAP12的胞内区域的嵌合mRNA序列，装载进pGEFP-N1载体中，得到质粒，并扩增，质粒序列如表1所示。用Lipofectamine 2000转染入CHO细胞，转染完成后用含G418的培养基筛选培养2天，消化并梯度稀释，种入96孔板，保证绝大多数孔内只有单个细胞。待细胞长成团，消化后种入24孔板，待24孔板中成团，再消化种入6孔板，如此重复扩增，整个过程都用含G418的培养基进行选择性筛选。扩增后进行免疫荧光实验，如图1a，验证稳转株构建是否成功。
表1 CD8 leader sequence-mTREM2-DAP12嵌合蛋白质粒序列
(注：“Atg”为CD8前导段序列，“ctc”为TREM2胞外及跨膜区序列，“cag”为DAP12胞内区域序列)
实施例2：稳转株吞噬实验
将稳转细胞和普通CHO细胞按10万/孔的密度种入24孔板，细胞贴壁后使用5mM活细胞胞浆染料Cell tracker进行染色，随即给予3μl/100μl红色荧光标记的酵母偶联的pHrodo吞噬染料和20μM S1P或10μg/ml LPS(阳参)处理，或S1P处理的同时加入2μM cytoD以阻断吞噬(阴参)，在2h或4h时进行拍摄。如图1所示，未转染的细胞基本没有出现吞噬情况，符合CHO细胞不具有吞噬功能的特性；转染后的细胞在给与S1P或LPS后，与未给药的转染细胞相比，2h时出现一定的吞噬增强，差别具有统计学意义，到4h时吞噬增强更明显；对LPS刺激的转染细胞给予吞噬抑制剂cytoD后，细胞吞噬显著减少并降低至未给药刺激组以下的水平，一方面说明稳转株转染后具有吞噬功能，另一方面也提示S1P能通过刺激TREM2引起促吞噬作用。
实施例3：液质联用
稳转株铺2块大皿，长满后分别给予20μM S1P或正常培养基培养，作用2小时后用PBS洗3遍，弃净后每皿加入400μl匀浆缓冲液进行裂解，匀浆缓冲液配方见表2。刮刀刮取细胞后收集于1.5ml EP管，放入-80℃超低温冰箱进行冰冻，冰冻完全后取出融化，如此反复冻融5次促进细胞裂解。冻融完成后进行玻珠研磨，研磨40下后冰上静置5min，如此重复4次。于4℃，12000rpm离心15min，取上清，加入免疫沉淀用TREM2抗体3μl孵育过夜，继而加入100μl proteinA+G beads/ml细胞裂解液上清，4℃旋转过夜以捕获免疫沉淀复合物。经4℃，8000rpm离心2min收集beads并弃上清，用1ml裂解液洗涤beads3次，10min/次。将beads重悬于50mM NH4HCO3中，加入2倍beads体积的NH4HCO3溶液，混匀后沸水煮10min，离心收集溶液。向溶液中加入200μl色谱纯级冰甲醇，混匀并于4℃，12000rpm离心30min，取上清以去沉淀，再于10kD Millipore超滤离心管，7500g离心15min以去除部分杂蛋白干扰，所得样品委托南京医科大学分析测试中心进行检测。
表2 匀浆缓冲液配方
如图2所示，未给与S1P处理的BV2细胞，由于本身S1P的存在，TREM2上有一定的S1P结合，而给与S1P处理后，TREM2上结合的S1P更多，因此可以定性得出结论，TREM2能够与S1P结合。
实施例4：微量热涌动
以pET-24a(+)为载体，构建C端His-tag标记的各种源性的TREM2蛋白表达质粒，小鼠源性TREM2所用序列参照表1，人源性和大鼠源性TREM2所用序列分别参照表3和表4。将重组质粒转化入BL(DE3)大肠杆菌：取新鲜制备的感受态细胞100μl，加入10μl重组质粒，轻弹混匀后冰上放置30min；42℃热激80s，快速转移至冰浴中，冷却5min；加入400μl 37℃的LB培养基，转移至37℃孵箱温育1h复苏细胞；取适量转化产物涂布于Kanamycin+的LB平板上，37℃孵箱倒置培养16h，挑取单菌落扩增培养至400ml培养液的OD600达到0.8左右，加入终浓度1mmol/L的IPTG继续培养8h，诱导目的蛋白的表达。将培养液转移至离心管，4℃下5000rpm离心5min收集菌体，用预冷的tris buffer重悬菌体，5000rpm再次离心5min收集菌体，所用试剂配方如表5和表6所示，用His标签蛋白纯化试剂盒纯化收集目的蛋白，并使用Monolith His-Tag Labeling Kit对带有His标签的目的蛋白进行荧光标记，按说明书检测TREM2与S1P的结合情况。如图3所示，S1P与人，大鼠，小鼠源性的TREM2具有结合能力，KD值分别为83.6μM，47.7μM，72.8μM，证明S1P是TREM2的配体。
表3 hTREM2质粒序列
表4 rTREM2质粒序列
表5 LB培养基配方
表6 tris buffer配方
实施例5：实验分组及给药
分别提取原代神经元和小胶质细胞，原代神经元用Neurobasal+B27+1％双抗培养于24孔板(20万个/孔)，隔天半换液，培养一周至细胞成熟；原代小胶质细胞用10％GibcoFBS+DMEM+1％双抗培养，每三天换一次液，培养约一周至细胞成熟。细胞成熟后将小胶质细胞加入神经元中共培养(2万个/孔)，换用Neurobasal:10％Gibco FBS+DMEM+1％双抗＝3:1的培养基培养。分为正常组，正常给药组，正常敲低组，敲低给药组，以及氧糖剥夺-复灌(OGD/R)损伤后各时间点(3h，5h，7h，9h，12h)的这四组。敲低转染后48h进行OGD处理3h，复灌时换正常培养基或10nM的S1P处理，于各时间点0.01MPBS洗涤后用多聚甲醛固定1h。
实施例6：免疫荧光染色
细胞固定后用0.01M PBS洗3次，弃净后加入封闭液(含5％山羊血清和0.1％的Triton X-100)室温封闭1h。滴加一抗(抗体滴度见表7)，4℃孵育过夜。次日取出后用0.01MPBS洗净一抗，洗3次，每次5min，滴加相应的荧光二抗(抗体滴度见表8)，室温避光孵育1h，0.01M PBS洗3次，每次5min。滴加5ug/ml的Hoechst溶液避光反应20min，0.01M PBS洗3次，每次5min，摄片。
表7 免疫荧光染色一抗
表8 免疫荧光染色二抗
如图4所示，未进行OGD损伤时小胶质细胞极少有吞噬现象，Con组造模后至3h小胶质细胞对神经元碎片的清除都较少，5h略有增加，在7h可以看到明显的清除作用，随后清除作用逐时减弱；给与S1P处理后3h起就有明显的清除，一直到12h都有明显的清除作用；TREM2敲低后各组小胶质细胞的清除作用明显减弱。
如图5所示，CD68作为巨噬细胞特异性溶酶体标记物，可以反映小胶质细胞的激活水平以及吞噬能力。未OGD损伤时，小胶质细胞CD68表达很少，造模后Con组在5h出现明显表达增加，持续至7h，到9h时明显减少，给与S1P处理后3h已出现明显的CD68表达增加，一直持续至12h仍有高水平CD68表达；TREM2敲低后CD68的表达明显减少。这些结果均表明S1P增强了小胶质细胞对神经元碎片的吞噬清除，且TREM2具有重要作用。
敲低S1PR2后给予S1P处理，对共培养的小胶质细胞的吞噬功能没有明显影响，排除了S1PR的作用，结果见图6，TREM2和S1PR2的敲低序列见表9。
表9 siRNA序列
本发明通过液质联用(LC-MS/MS)和微量热涌动(MST)的方法进一步证明，S1P直接与TREM2结合，且亲和力较强，表明S1P是TREM2的一个内源性配体。神经元与小胶质细胞共培养时，氧糖剥夺-再灌注损伤后，S1P处理能增强小胶质细胞对神经元碎片的吞噬。小胶质细胞上仅表达S1PR2和S1PR4，鉴于S1PR4与S1P的亲和力很低，因此需排除S1P对S1PR2的作用。结果发现，敲低S1PR2不影响小胶质细胞的吞噬增强，进一步从功能角度说明S1P通过TREM2受体发挥促吞噬功能。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (3)
1.一种髓细胞触发受体2的内源性配体在髓细胞触发受体2激活剂中的应用，其特征在于，所述内源性配体为鞘氨醇-1-磷酸，鞘氨醇-1-磷酸直接与髓细胞触发受体2结合，结合后触发髓细胞触发受体2介导的促细胞吞噬作用；
所述鞘氨醇-1-磷酸的结构式为
2.根据权利要求1所述应用，其特征在于，所述的内源性配体在制备调节细胞吞噬、调节炎症、促进细胞生长、抑制细胞凋亡药物中的应用。
3.根据权利要求2所述应用，其特征在于，所述内源性配体在制备增强小胶质细胞吞噬功能药物中的应用。
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