CN116949169B - Smek1在缺血性脑卒中诊治中的应用 - Google Patents
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Abstract
本发明属于生物医药和分子生物学技术领域，具体涉及SMEK1在缺血性脑卒中诊治中的应用。本发明首次报道了SMEK1在缺血性脑卒中后呈现波动变化，并在相当长的一段时间里呈现下降趋势。小胶质细胞SMEK1的下降会导致其分泌更多的促炎因子，使小胶质细胞向M1型方向极化。SMEK1过表达被证明具有改善小鼠缺血性脑卒中后神经损伤的作用。此外，本发明研究发现SMEK1可通过线粒体代谢重编程来调节小胶质细胞的极化方向，通过促进SMEK1的表达可以减轻缺血性脑卒中后的炎症反应。总之，本发明为缺血性脑卒中发生发展提供了新的机制研究，并为缺血性脑卒中患者提供了有前景的治疗策略，因此具有良好的实际应用之价值。
Description
技术领域
本发明属于生物医药和分子生物学技术领域，具体涉及SMEK1在缺血性脑卒中诊治中的应用。
背景技术
本发明背景技术中公开的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
脑卒中(Stroke)已成为仅次于缺血性心脏病的世界第二大死因，中国第一大死因。其中缺血性脑卒中占80％。缺血性脑卒中(Ischemic stroke)是指由于脑的供血动脉狭窄或闭塞、脑供血不足导致的脑组织坏死的总称，其特点是高发病率、高致残率和高死亡率。
然而，由于急性缺血性脑卒中(Acute ischemic stroke,AIS)后的溶栓治疗时间窗狭窄且存在再灌注损伤等不良反应，目前对于AIS的治疗仍有限。AIS后数分钟至数小时发生的神经炎症和免疫反应与AIS后的脑损伤密切相关，许多关于卒中的临床前和临床研究表明，中枢神经***的免疫调节可能是AIS的一种可行的替代治疗策略。
发明内容
针对上述现有技术，本发明的目的在于提供SMEK1在缺血性脑卒中诊治中的应用。本发明通过研究发现，蛋白磷酸酶4调节亚基3a(SMEK1)在缺血性脑卒中后呈现波动变化，且在相当长的一段时间里呈现下降趋势。小胶质细胞SMEK1的下降会导致其分泌更多的促炎因子，使小胶质细胞向M1型方向极化。SMEK1过表达被证明具有改善缺血性脑卒中后神经损伤的作用。此外，本发明研究发现SMEK1可通过线粒体代谢重编程来调节小胶质细胞的极化方向。通过促进SMEK1的表达可以减轻缺血性脑卒中后的炎症反应。基于上述研究成果，从而完成本发明。
具体的，本发明技术方案如下：
本发明的第一个方面，提供检测SMEK1编码基因和/或其表达产物的试剂在制备用于筛查、(辅助)诊断、检测、监测或预测缺血性脑卒中的进展的产品中的应用。
本发明的第二个方面，提供一种用于筛查、(辅助)诊断、检测、监测或预测缺血性脑卒中的进展的***，所述***包括：
获取模块，其被配置为：获取受试者SMEK1编码基因和/或其表达产物的表达水平；
评估模块，其被配置为：根据获取模块获得的SMEK1编码基因和/或其表达产物的表达水平判断所述受试者的患病情况。
本发明的第三个方面，提供一种计算机可读存储介质，其上存储有程序，该程序被处理器执行时实现如本发明第二方面所述***的功能。
本发明的第四个方面，提供一种电子设备，包括存储器、处理器及存储在存储器上并可在处理器上运行的程序，所述处理器执行所述程序时实现如本发明第二方面所述***的功能。
本发明的第五个方面，提供SMEK1作为靶点在制备和/或筛选缺血性脑卒中药物中的应用。
可以利用候选药物使用前和使用后对SMEK1的影响，从而确定候选药物是否可以用于预防或治疗缺血性脑卒中。
本发明的第六个方面，提供促进SMEK1基因及其表达产物和/或使其活性提高的物质在如下1)-4)至少一种中的应用：
1)制备改善缺血性脑卒中介导的神经损伤的产品；
2)制备减轻缺血性脑卒中介导的炎症反应的产品；
3)抑制缺血性脑卒中后小胶质细胞向促炎方向(M1型)极化或制备抑制缺血性脑卒中后小胶质细胞向促炎方向极化的产品；
4)制备缺血性脑卒中预防和/治疗的产品。
其中，所述缺血性脑卒中可以为急性缺血性脑卒中。
所述产品可以为药物或实验试剂，所述实验试剂可供基础研究使用，诸如构建缺血性脑卒中相关细胞或动物模型，从而对缺血性脑卒中等相关疾病的发生发展机制等进行研究。
本发明的第七个方面，提供一种缺血性脑卒中治疗的方法，所述方法包括：对受试者施用促进SMEK1编码基因及其表达产物表达和/或使其活性提高的物质。
上述一个或多个技术方案的有益技术效果：
上述技术方案首次报道了SMEK1在缺血性脑卒中后呈现波动变化，并在相当长的一段时间里呈现下降趋势。小胶质细胞SMEK1的下降会导致其分泌更多的促炎因子，如IL-1β，iNOS和TNF-α，使小胶质细胞向M1型方向极化。SMEK1过表达被证明具有改善小鼠缺血性脑卒中后神经损伤的作用。此外，本发明研究发现SMEK1可通过线粒体代谢重编程来调节小胶质细胞的极化方向。通过促进SMEK1的表达可以减轻缺血性脑卒中后的炎症反应，起到保护、治疗的作用。
上述技术方案为缺血性脑卒中发生发展提供了新的机制研究，并为缺血性脑卒中患者提供了有前景的治疗策略，因此具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是本发明实施例MCAO/OGD后小鼠脑组织中Smek1的表达随时间变化而波动。其中，A和B示MCAO/OGD的处理时间。C和D示MCAO建模成功的验证。E示后续实验的取材部位。F和G示MCAO后脑组织SMEK1的mRNA表达和蛋白质印迹。H和I示MCAO后脑组织总SMEK1和小胶质细胞内SMEK1的免疫荧光染色。
图2是本发明实施例MCAO/OGD后小鼠源小胶质细胞内Smek1的表达随时间变化而波动。其中，A和B示MCAO后小胶质细胞内SMEK1表达的流式细胞染色结果。C，D，E，F示BV2细胞OGD/R后SMEK1的mRNA表达和蛋白质印迹。G示BV2细胞OGD/R后SMEK1的免疫荧光染色。H和I示BV2细胞敲低/过表达SMEK1的mRNA水平和蛋白质水平的验证。J示敲低SMEK1的BV2细胞OGD后的SMEK1和IL-1β的蛋白质印迹。
图3是本发明实施例MCAO/OGD后小胶质细胞内Smek1减少与炎症相关。其中，A和I示敲低/过表达SMEK1的BV2细胞在未进行OGD/R刺激时IL-1β和IL-10的mRNA表达水平。B，C，D，E，F，G，H分别示敲低SMEK1的BV2细胞在OGD/R刺激后CD86，IL-1β，iNOS，TNF-α，CD206，IL-10和TGF-β的mRNA表达水平。J，K，L，M，N，O，P分别示过表达SMEK1的BV2细胞在OGD/R刺激后CD86，IL-1β，iNOS，TNF-α，CD206，IL-10和TGF-β的mRNA表达水平。
图4是本发明实施例小胶质细胞中的Smek1参与保护缺血导致的神经损伤。其中，A和B示全身过表达SMEK1转基因小鼠的mRNA水平和蛋白质水平的验证。C示过表达转基因小鼠在MCAO前，MCAO第一天，第二天和第三天的mNSS的评分。D示过表达转基因小鼠在MCAO前，MCAO第一天，第二天和第三天的脑皮质血流情况。E，F，G，H，I，J，K示过表达转基因小鼠MCAO后小胶质细胞表达炎症因子的流式细胞染色结果。
图5是本发明实施例敲低SMEK1后BV2细胞的转录组测序结果。其中，A示对样本急性聚类分析，提示shNC组及shSmek1组内生物学重复的样本相关性高。B和C示转录组测序结果中上调基因有594个，下调基因515个。D示GP_BP中富集到多个与细胞因子相关的生物学过程，提示敲低Smek1影响BV2细胞免疫/炎症功能。E示GP_BP中富集到多个与脂质合成相关的生物学过程。G，H，I和F分别示GSEA功能分析提示SMEK1敲低与脂肪酸代谢，胆固醇代谢和丙酮酸盐代谢相关。J和K分别示敲低/过表达SMEK1后BV2细胞表达PDK3的mRNA水平和蛋白质印迹。
图6是本发明实施例小胶质细胞中缺失Smek1后可导致线粒体代谢重编程。其中，A和B示敲低/过表达SMEK1后BV2细胞OGD后表达HIF-1α的mRNA水平和蛋白质印迹。C，D，E示敲低/过表达SMEK1后BV2细胞的FAOBlue脂肪酸氧化检测结果。F和G分别示敲低/过表达SMEK1后BV2细胞胞外和胞内的乳酸水平。H示敲低/过表达SMEK1后BV2细胞的丙酮酸脱氢酶活性。I示敲低/过表达SMEK1后BV2细胞的丙酮酸脱氢酶和磷酸化丙酮酸脱氢酶的蛋白质印迹。
图7是本发明实施例小胶质细胞中缺失Smek1后可导致线粒体代谢重编程。其中，A示敲低/过表达SMEK1后BV2细胞的乳酸脱氢酶活性。B和C示敲低/过表达SMEK1后BV2细胞的OCR水平。D，E，F，G，H分别示敲低SMEK1后BV2细胞的基础呼吸，最大呼吸，质子漏，ATP产生和备用呼吸量的变化。I，J，K分别示过表达SMEK1后BV2细胞的基础呼吸，质子漏和ATP产生的变化。L和M示敲低/过表达SMEK1后BV2细胞的ECAR水平。N，O，P，Q示敲低SMEK1后BV2细胞的基础糖酵解，基础质子流出速率，糖酵解的质粒流出速率和加入2-DG后的酸化的变化。
图8是本发明实施例小胶质细胞中缺失Smek1后可导致线粒体代谢重编程。其中，A和B示过表达SMEK1后BV2细胞的基础糖酵解和加入2-DG后的酸化的变化。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
现结合具体实例对本发明作进一步的说明，以下实例仅是为了解释本发明，并不对其内容进行限定。如果实施例中未注明的实验具体条件，通常按照常规条件，或按照试剂公司所推荐的条件；下述实施例中所用的试剂、耗材等，如无特殊说明，均可从商业途径得到。
如前所述，中枢神经***的免疫调节可能是AIS的一种可行的替代治疗策略。而小胶质细胞是脑内的常驻免疫细胞，在缺血性脑卒中发生后第一时间被激活并迁移到梗死核心区及半暗带区域。小胶质细胞的活化通常被认为有两种途径，一种是通过“经典途径“活化为促炎型小胶质细胞，即M1型；另一种是通过”替代途径“活化为抑炎型小胶质细胞，即M2型。M1型小胶质细胞的表面标志为CD86，并分泌IL-1β、TNF-α、IFN-γ、IL-6、iNOS、MMP-9、MMP-3等促炎因子；M2型小胶质细胞的表面标志为CD206，并分泌IL-10、TGF-β、***、血管内皮生长因子(VEGF)等的抑炎因子。在缺血性脑卒中发生后，脑内微环境的改变使小胶质细胞向两个极端进行活化，M1/M2型小胶质细胞的比例失衡在后续炎症反应中发挥作用。近年来，代谢重编程逐渐受到研究者们的重视。代谢重编程是小胶质细胞免疫反应的关键驱动因素，处于促炎状态的小胶质细胞优先使用糖酵解来产生能量，而处于抗炎状态的细胞主要由氧化磷酸化和脂肪酸氧化供能。因此，我们想能否通过小胶质细胞的代谢重编程来影响其向M2型极化，进而在缺血性脑卒中后的炎症反应中发挥神经保护作用。
蛋白磷酸酶4(PP4)是一种高度保守的丝氨酸/苏氨酸磷酸酶，是一种由催化亚基和调节亚基组成的蛋白质复合物。MEK1抑制剂(SMEK1)是PP4酶的调节亚基，调节PP4催化亚基的活性，通过未知机制导致其靶底物的去磷酸化。PP4参与生物体内的许多细胞过程，并调节多种细胞信号通路，包括核因子κB(NF-κB)、c-Jun N-末端激酶、凋亡信号、胰岛素受体底物蛋白4和雷帕霉素靶点。最近的研究结果表明，PP4是T细胞增殖和免疫反应的重要调节剂，通过去磷酸腺苷活化蛋白激酶(AMPK)。此外，由于调节性T细胞功能受损，CD4-CrePP4fl/fl小鼠会自发发生直肠脱垂和结肠炎(类似于克罗恩病)。作为PP4的一个亚单位，探索SMEK1在缺血性脑卒中后炎症反应中的作用有重要的理论和临床意义。
有鉴于此，本发明的一个典型具体实施方式中，提供检测SMEK1编码基因和/或其表达产物的试剂在制备用于筛查、(辅助)诊断、检测、监测或预测缺血性脑卒中的进展的产品中的应用。
本发明研究发现，SMEK1在缺血性脑卒中后呈现波动变化，并在相当长的一段时间里呈现下降趋势。从而表明SMEK1与缺血性脑卒中的发生发展密切相关。
本发明中，所述SMEK1编码基因的表达产物显然可以是SMEK1蛋白，其为蛋白磷酸酶4(PP4)的调节亚基。
所述缺血性脑卒中的进展包括但不限于缺血性脑卒中患者的肢体瘫痪、感觉缺失、反射消失以及认知功能障碍。
在本发明中，所述缺血性脑卒中具体可以为急性缺血性脑卒中。
所述检测SMEK1编码基因和/或其表达产物的试剂包括但不限于基于测序方法、基于定量PCR方法或基于探针杂交方法检测SMEK1编码基因转录的试剂；或基于免疫检测方法(诸如免疫组织化学、ELISA、胶体金试纸条、蛋白芯片)检测SMEK1蛋白表达情况的试剂。
所述产品可以为试剂盒、检测装置或检测设备等，在此不做具体限定。
本发明的又一具体实施方式中，提供一种用于筛查、(辅助)诊断、检测、监测或预测缺血性脑卒中的进展的***，所述***包括：
获取模块，其被配置为：获取受试者待测样品SMEK1编码基因和/或其表达产物的表达水平；
评估模块，其被配置为：根据获取模块获得的SMEK1编码基因和/或其表达产物的表达水平判断所述受试者的患病情况。
其中，所述缺血性脑卒中为急性缺血性脑卒中。
所述受试者可以是人或非人哺乳动物(如小鼠等)，所述待测样品包括但不限于外周血以及脑相关组织及细胞(如小胶质细胞、神经元、星形胶质细胞及少突胶质细胞等)。
本发明的又一具体实施方式中，提供一种计算机可读存储介质，其上存储有程序，该程序被处理器执行时实现如上述***的功能。
本发明的又一具体实施方式中，提供一种电子设备，包括存储器、处理器及存储在存储器上并可在处理器上运行的程序，所述处理器执行所述程序时实现如上述***的功能。
本发明的又一具体实施方式中，提供SMEK1作为靶点在制备和/或筛选缺血性脑卒中药物中的应用。
可以利用候选药物使用前和使用后对SMEK1的影响，从而确定候选药物是否可以用于预防或治疗缺血性脑卒中。
具体的，筛选缺血性脑卒中药物的方法包括：
1)采用候选物质处理表达和/或含有所述SMEK1的体系；设置不采用候选物质处理的平行对照；
2)完成步骤1)后，检测体系中所述SMEK1的表达水平；与平行对照相比，如果采用候选物质处理的体系中所述SMEK1的表达量显著提高，所述候选物质可作为候选的缺血性脑卒中药物。
本发明的又一具体实施方式中，所述体系可为细胞体系、溶液体系、组织体系、器官体系或动物体系。
本发明的又一具体实施方式中，所述细胞体系中的细胞可以为小胶质细胞、神经元细胞、星形胶质细胞及少突胶质细胞；
本发明的又一具体实施方式中，所述组织体系中的组织可以为大脑皮层、黑质、纹状体、丘脑和脑干被盖部；
本发明的又一具体实施方式中，所述器官体系中的器官可以为大脑；
本发明的又一具体实施方式中，所述动物体系中的动物可以为哺乳动物，如
大鼠、小鼠、豚鼠、兔、猴、人等。
本发明的又一具体实施方式中，提供促进SMEK1基因及其表达产物和/或使其活性提高的物质在如下1)-4)至少一种中的应用：
1)制备改善缺血性脑卒中介导的神经损伤的产品；
2)制备减轻缺血性脑卒中介导的炎症反应的产品；
3)抑制缺血性脑卒中后小胶质细胞向促炎方向(M1型)极化或制备抑制缺血性脑卒中后小胶质细胞向促炎方向极化的产品；
4)制备缺血性脑卒中预防和/治疗的产品。
其中，所述缺血性脑卒中可以为急性缺血性脑卒中。
根据本发明，“预防和/或治疗”的概念表示任一适用于治疗缺血性脑卒中相关疾病的措施，或者对于这种表现的疾病或所表现出来的症状进行预防性治疗，或者避免这种疾病的复发，例如在结束了治疗时间段之后的复发或对已经发作的疾病的症状(如运动功能障碍以及认知功能障碍等)进行治疗，或者预先介入性的防止或抑制或减少该类疾病或症状的发生。
所述促进SMEK1编码基因及其表达产物的物质包括但不限于采用基于基因特异性Mimics技术上调SMEK1表达和/或促进其活性的物质；如上调SMEK1表达的启动子或者慢病毒；同时也包括化合物类促进剂。
所述产品可以为药物或实验试剂，所述实验试剂可供基础研究使用，诸如构建缺血性脑卒中相关细胞或动物模型，从而对缺血性脑卒中等相关疾病的发生发展机制等进行研究。
根据本发明，当所述产品为药物时，所述药物还包括至少一种药物非活性成分。
本发明的又一具体实施方式中，所述药物非活性成分包括药学上可接受的载体、赋形剂和/或稀释剂。例如药学上相容的无机或有机酸或碱、聚合物、共聚物、嵌段共聚物、单糖、多糖、离子和非离子型表面活性剂或脂质、药理上无害的盐例如氯化钠、调味剂、维生素例如维生素A或维生素E、生育酚或维生素原、抗氧化剂，例如抗坏血酸，以及用于延长药物活性成分或配方的使用和保存时间的稳定剂和/或防腐剂，和其它现有技术中公知的常用非药物活性成分或助剂和添加剂，以及它们的混合物。
所述药物的给药剂型可以是固体口服制剂、液体口服制剂或注射剂。
所述药物的给药剂型进一步可以是片剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小水针、注射用冻干粉针、大输液或小输液。
本发明的又一具体实施方式中，所述药物施用对象可以是人和非人哺乳动物，如小鼠、大鼠、豚鼠、兔、狗、猴、猩猩等。
本发明的又一具体实施方式中，提供一种缺血性脑卒中治疗的方法，所述方法包括：对受试者施用促进SMEK1编码基因及其表达产物表达和/或使其活性提高的物质。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中为注明具体条件的试验方法，通常按照常规条件进行。
实施例
实验方法：
1.1动物和短暂性大脑中动脉阻塞手术(tMCAO)
本研究中使用的所有小鼠均为购自维通利华(中国北京)的C57BL/6j雄性小鼠。在特定的无病原体条件下饲养，具有固定的12小时光/暗周期，并可获得充足的食物和水。我们已经尽量减少使用老鼠的数量和它们的痛苦。
8周-10周龄C57BL/6j雄性小鼠(21-25g)，麻醉小鼠后，取仰卧位于动物体式显微镜下，用小动物剃毛器备皮，消毒，之后分离左侧颈总动脉、颈外动脉和颈内动脉。在颈总动脉近心段剪一切口，将线栓(深圳瑞沃德，21-25g规格)沿颈总动脉***颈内动脉，最后送至大脑中动脉开口处并阻断其血流。手术完成后将小鼠放于动物加热毯上，将其体温维持在37.0±0.5℃。阻塞60分钟后，将线栓取出，完成再灌注。对假手术组小鼠进行了相同的手术过程，但是并未***线栓。分别在手术后1天、3天、7天处死小鼠(图1A)。
1.2转基因小鼠的构建
在ROSA26位点***CAG Pr-loxP-Stop-loxP-SMEK1 CDS-HA Tag-WPRE-pA，基于CRISPR/Cas9开发的EGE***制备C57BL/6J模式小鼠，将ROSA26-Smek1f/f小鼠与Ubc-Cre-ERT2小鼠交配交配获得ROSA26-Smek1f/fUbc-Cre-ERT2小鼠。
1.3脑皮质血流和2,3,5-氯化三苯基四氮唑(TTC)染色
使用变焦激光散斑血流成像***(英国Moor仪器公司，MoorFLPI-2)对SHAM组和MCAO组小鼠测量其脑皮质血流。简单来说，就是麻醉小鼠后，沿其头部中线切开以暴露颅骨，然后将其放置在成像仪镜头下，观察其血流情况(图1D)。
通过2,3,5—氯化三苯基四氮唑(TTC)染色(北京索莱宝，G3005)确定梗死体积。将脑组织取出后固定在一个平面上并于-20度冰箱速冻20分钟，然后用刀片将其切成4-6片。加入TTC染料于37°孵箱中放置40分钟，期间进行翻面，染色后观察梗死体积(图1C)。
1.4免疫荧光染色
对脑组织冰冻切片用0.3％ Triton X-100(北京索莱宝，T8200)在室温下通透30分钟，再用10％驴血清(北京索莱宝，SL050)在室温下封闭一小时。用小鼠源IBA-1(武汉赛维尔，GB12105)，兔源SMEK1(美国Sigma，HPA002568)一抗于4°冰箱中孵育过夜。用PBS冲洗后，将冰冻切片在室温下用相应的二抗于黑暗环境中染色2小时。最后用DAPI(上海碧云天，P0131)染细胞核。石蜡切片在染色前需要进行脱蜡和抗原修复过程，其余步骤与冰冻切片相同。
1.5小胶质细胞分离和流式细胞术
对小鼠进行心脏灌注后取脑，将脑组织直接研磨并过滤出悬液，将悬液和100％percoll(北京索莱宝，P8370)配成30％percoll。将含有悬液的30％percoll缓慢加至70％percoll液面上，利用密度梯度离心法(700g，25分钟，降速最低)将细胞分离，吸取中间白膜层于离心管中备用。单细胞悬液里加入Zombie yellow dye-BV605(美国Biolegend，b337269)染料在室温下染色10分钟，再加入CD45-APC-CY7流式抗体(美国Biolegend，103116)、CD11b-BV421(美国Biolegend，101236)染料在4℃染色30分钟。用Foxp3固定液(美国赛默飞，00-5523-00)在室温下固定45分钟，Foxp3洗液(美国赛默飞，00-5523-00)冲洗两遍。加入TNF-α-PE流式抗体(美国Biolegend，504104)、iNOS-PECy7流式抗体(美国eBioscience，25-5920-82)、IL-10-APC流式抗体(美国Biolegend，505010)和兔源SMEK1抗体(美国Sigma，HPA002568)室温下孵育45分钟，用山羊抗兔488-FITC(英国abcam，ab150073)室温下染色45分钟。最后用300ul 0.5％多聚
甲醛(武汉赛维尔，G1101)重悬细胞，上机检测。所有染料的用量均按相应的说明书进行使用。
1.6转录组测序分析
收集敲低SMEK1的BV2小胶质细胞并用PBS洗涤一次，然后在TRIzol中室温裂解5分钟，并在-80℃冷冻。样品送至北京诺禾致源公司进行文库准备和测序。fastq格式的原始数据首先通过内部Perl脚本处理为低质量的读取。通过Hisat2(v2.0.5)定位到参考基因组后，根据每个基因的读取数和长度计算FPKM。此外，使用DESeq2 R软件包(版本1.20.0)对两组进行差异表达分析。为了控制错误发生率，采用Benjamini和Hochberg的方法调整p值，认为padj≤0.05、|log2(fold change)|≥1具有统计学意义。使用clusterProfiler R软件包(3.8.1)对差异表达基因进行基因本体(GO)富集分析。
1.7细胞培养和慢病毒转染
小鼠BV2小胶质细胞(武汉博士德，CX0103)在Dulbecco改良Eagle培养基(DMEM，美国Gibco，C11995500BT)中培养，在该培养基中补充10％胎牛血清(美国Gibco，A3161001C)，1％青霉素-链霉素(美国Gibco，15070063)。细胞在37℃，5％的二氧化碳培养箱中孵育，培养基每2天更换一次。
对于SMEK1在BV2细胞中的敲除或过表达，用以下引物扩增靶基因片段：49830FW-65237:GCGAATTCGAAGTATACCTCGAGGC；49830RW-65238:CGATCGCAGATCCTTGGATCC。然后将该片段克隆到慢病毒载体PGMLV-hU6-MCS-CMV-Puro-WPRE和lenti-CMV-MCS-PGK-Puro(上海，Genomeditech)中。在成功构建后，将含有相应靶片段或阴性对照的慢病毒转染到BV2细胞中。转染48小时后吸弃培养基，再培养48小时，用嘌呤霉素(2.5μg/ml；上海MCE，HY-B1743A/CS-6857)筛选敲除或过表达SMEK1的BV2细胞。使用RT-qPCR和Western Blots进行验证(图2H，I)。
1.8氧糖剥夺(OGD)实验
为了在体外模拟缺血性脑卒中，对细胞进行OGD/R处理。简言之，用无葡萄糖DMEM(美国Gibco，11966025)代替正常培养基，并在37℃的乏氧培养箱(94％氮气和5％二氧化碳)中培养3小时，以模拟OGD损伤。然后将培养基换为含有正常葡萄糖的DMEM培养基并将细胞转移到正常培养箱中分别复氧1h、6h、12h、21h和24h(图1B)。
1.9Seahorse实验
将敲低/过表达SMEK1的BV2细胞以2×105个细胞/ml培养2天，然后在Seahorse 24孔微孔板中以4×104个细胞/孔重新接种。使用Seahorse XF细胞Mito压力测试试剂盒(美国Agilent Technologies，103015-100)、Seahorse XF糖酵解速率测定试剂盒(美国Agilent Technologies，103344-100)和Seahorse XF24细胞外通量分析仪测量细胞的代谢状态。为了测定耗氧量，我们将线粒体复合物抑制剂(oligomycin[1μM]、FCCP[1μM]、鱼藤酮/抗霉素A[0.5μM])依次加入细胞培养微孔板中。为了测量糖酵解速率，我们将鱼藤酮/抗霉素A[0.5μM]和2-脱氧-D-葡萄糖[50mM]依次加入细胞培养微孔板中。使用Seahorse XF24分析仪测量细胞代谢的关键参数。每个样品至少进行三次重复分析。用Wave软件(美国，Agilent Technologies)对结果进行分析。
1.10乳酸水平测定
测定乳酸水平用的试剂盒为L-乳酸(L-LA)含量检测试剂盒(北京索莱宝，BC2235)。对细胞或上清的处理和实验步骤按照试剂盒说明书进行。使用微孔板读取器(美国BioTek，Epoch)在570nm处测量吸光度。
1.11丙酮酸脱氢酶活性和乳酸脱氢酶活性检测
测定丙酮酸脱氢酶活性和乳酸脱氢酶活性用的试剂盒分别为丙酮酸脱氢酶(PDH)活性检测试剂盒(BC0385，索莱宝，北京)和乳酸脱氢酶(LDH)活性检测试剂盒(BC0685，索莱宝，北京)。对细胞的处理和实验步骤按照试剂盒说明书进行。使用微孔板读取器(美国BioTek，Epoch)分别在605nm、450nm处测量吸光度。
1.12FAOBlue脂肪酸氧化检测
我们使用FAOBlue脂肪酸氧化检测试剂(日本Funakoshi，FDV-0033)对BV2细胞进行染色来检测其脂肪酸氧化水平。简单来说，就是将该试剂加入到培养基中并在37℃环境下培养2小时。用共聚焦显微镜(美国Nikon，LU-N4)在405nm波长下观察图像或用PB450通道在流式细胞仪(美国贝克曼，A00-1-1102)上记录平均荧光强度。
1.13实时荧光定量PCR
对于BV2细胞系总RNA的提取，我们使用的是RNA快速提取试剂盒(北京天根，DP451)。对于脑组织总RNA的提取，我们使用的是TRIzol试剂(美国赛默飞，15596026)。提取步骤分别按照相应的说明书进行。然后使用逆转录试剂盒(日本Takara，RR047A)按照说明书将所得的RNA逆转录成cDNA。用FastStart Universal SYBR Green Master Mix(南京诺维赞，Q321-02)在实时PCR检测***(美国Bio-Rad，CFX96 Optics Module)上进行qPCR反应。用于扩增的引物如下：
1.14蛋白印迹
将脑组织和培养的细胞用添加了PMSF(北京索莱宝，P0100)和磷酸酶抑制剂(上海碧云天，P1081)的RIPA裂解液(上海碧云天，P0013B)进行裂解。蛋白质在10％ SDS-PAGE凝胶(上海雅酶，PG112)上电泳分离，然后转移到PVDF膜(德国默克，NO.ISEQ00010)上。用快速封闭液(上海Genefist，GF1815)封闭后，将PVDF膜与一抗一起在4℃环境下孵育过夜。使用的一抗包括SMEK1(1:2000，美国Sigma，HPA002568)，β-actin(1：20000，武汉三鹰，66009-1-Ig)，β-tubulin(1:1000，武汉三鹰，10068-1-AP)，PDH(1:6000，武汉三鹰，18068-1-AP)，P-PDH(1:2000，英国Abcam，ab177461)，HIF-1α(1:1000，英国Abcam，ab179483)，PDK3(1:500，武汉三鹰，12215-1-AP)，IL-1β(1:1000，美国CST，31202)，CD206(1:1000，美国CST，24595)。洗涤后，将PVDF膜与一抗来源种属合适的二抗在室温下孵育2小时。最后，使用超敏ECL化学发光检测试剂盒(武汉三鹰，PK10003)检测免疫印迹。用化学发光全自动成像***(美国Bio-Rad，Chemi Doc)观察蛋白质条带。
1.15小鼠行为学检测
采用改良神经***严重程度评分(mNSS)测试，对小鼠的运动、感觉、反射和平衡功能进行综合测试，以评估整体神经功能缺损。分数从0到18分，其中0分代表正常，18分代表最严重的缺陷。分别在术后1天、2天和3天评估功能缺陷。实验结果：
我们首先检测了MCAO后小鼠脑组织中总mRNA和蛋白质中的SMEK1表达情况。结果显示Smek1在MCAO后第1天达到峰值，在第3-7天表达减少，其中MCAO后第3天的SMEK1表达下降至最少(图1F，G)。免疫荧光染色证明了MCAO后第三天的小鼠脑组织内Smek1表达减少(图1H)。进一步，我们利用免疫荧光染色和流式细胞术证明了MCAO后第3天小胶质细胞内SMEK1的表达减少(图1I，图2A,B)。同样的，体外检测小鼠源BV2小胶质细胞在OGD后mRNA和蛋白质中的SMEK1表达情况。结果显示BV2细胞在OGD后随复氧时间延长出现Smek1表达先升高后降低的趋势，其中OGD3h/R21h的Smek1表达下降至最少(图2C，D，E，F)。免疫荧光染色证明了BV2细胞在OGD3h/R21h的Smek1表达减少(图2G)。我们的研究结果证明了MCAO后小胶质细胞内Smek1的表达随时间变化呈先增多后减少的趋势，并且在相当长的一段时间里呈减少趋势。我们推测小胶质细胞内的Smek1可能参与了缺血性脑卒中后的免疫调控。
为了确定小胶质细胞内Smek1是否参与了缺血性脑卒中后的免疫调控，我们利用慢病毒转染技术构建了敲低/过表达Smek1的BV2细胞稳转细胞系。有趣的是，敲低Smek1组BV2细胞在未进行乏氧刺激的情况下，促炎因子IL-1β的表达便出现了增多(图3A)。过表达Smek1组BV2细胞在未进行乏氧刺激的情况下，抑炎因子IL-10的表达出现增多(图3I)。与阴性对照组(shNC)相比，Smek1敲低组(shSMEK1)BV2细胞在OGD3h/R21h后，CD86，IL-1β，iNOS和TNF-α表达增多(图2J，图3B，C，D，E)，CD206，IL-10和TGF-β表达减少(图3F，G，H)。Smek1过表达组(oe-SMEK1)BV2细胞在OGD3h/R21h后较阴性对照组(oeNC)来说，CD206，IL-10和TGF-β表达增多(图3N，O，P)，CD86，IL-1β，iNOS和TNF-α表达减少(图3J，K，L，M)。以上结果表明小胶质细胞内Smek1参与了缺血性脑卒中后的免疫调控并发挥神经保护作用。
为了研究小胶质细胞SMEK1过表达对缺血性脑卒中后炎症的影响，我们对8只SMEK1过表达小鼠进行腹腔注射他莫昔芬(100mg/kg，美国Sigma，T5648)诱导SMEK1的过表达，对另外8只SMEK1过表达小鼠腹腔注射玉米油作为对照，然后对它们进行MCAO手术。分别在MCAO后1天、2天和3天进行mNSS评分和脑皮质血流成像。在MCAO后第三天处死小鼠并提取梗死区的单个核细胞进行流式染色，同时，留取对侧脑组织提取RNA和蛋白质验证SMEK1的表达情况。我们通过RT-qPCR和Western Blots验证了SMEK1的过表达(图4A，B)。与对照组小鼠相比，SMEK1过表达组小鼠的mNSS评分显著降低(图4C)，且在MCAO第三天时差异最显著，这表明SMEK1对缺血性脑卒中后的神经恢复具有潜在的神经保护作用。变焦激光散斑血流成像***显示，与对照组小鼠相比，SMEK1过表达组小鼠的脑血流恢复情况更好(图4D)。流式细胞染色显示，过表达SMEK1的小胶质细胞在MCAO后表达IL-10增多(图4J，K)，TNF-α和iNOS减少(图4F，G，H，I)。以上结果表明，小胶质细胞中的SMEK1参与保护缺血导致的神经损伤。
为了揭示Smek1的作用机制，我们对敲低Smek1的BV2小胶质细胞进行了转录组测序分析。与阴性对照组相比，敲低Smek1组BV2细胞中共有594个基因上调，515个基因下调(图5B，C)，这表明Smek1的敲低改变了小胶质细胞的基因表达谱。随后，我们对这些差异表达基因进行了基因本体论(GO)分析。结果显示Smek1敲低与脂肪酸代谢、胆固醇代谢和丙酮酸盐代谢密切相关(图5G，H，I)。我们还发现与糖代谢相关的指标丙酮酸脱氢酶激酶3(PDK3)在敲低Smek1后出现了显著增多。
我们通过实验证明了敲低Smek1后PDK3的表达显著增多，过表达Smek1后PDK3表达减少(图5J，K)。为了进一步阐明敲低SMEK1后为何PDK3的表达会增多，我们查阅文献发现缺氧通过上调缺氧诱导因子-1α(HIF-1α)可诱导PDK3的表达，且PDK3的表达与HIF-1α的表达呈正相关。接下来，我们便检测了乏氧后HIF-1α的表达情况。结果显示敲低Smek1后HIF-1α的表达增多。过表达Smek1后HIF-1α的表达减少(图6A，B)。该结果与PDK3的变化一致。
为了证明敲低小胶质细胞内的Smek1后，其线粒体代谢发生了改变，我们分别对细胞的脂肪酸氧化(FAO)水平和细胞内外的乳酸(LA)含量进行了检测。结果显示Smek1敲低后FAO水平出现了下降，细胞内外的乳酸含量都有了明显的增加。过表达Smek1后FAO水平升高，细胞内外的乳酸含量减少(图6C，D，E，F，G)。接下来，我们对参与细胞代谢的受PDK3调节的丙酮酸脱氢酶(PDH)进行了检测。结果显示敲低Smek1后PDH的活性下降，磷酸化丙酮酸脱氢酶(P-PDH)表达增多。过表达Smek1后PDH活性升高，P-PDH表达减少(图6H，I)。同样的，我们还对乳酸脱氢酶(LDH)的活性进行了检测，结果显示敲低Smek1后LDH活性升高，过表达Smek1后LDH活性降低(图7A)。为了进一步了解敲低/过表达小胶质细胞SMEK1后细胞代谢的改变情况，我们使用Seahorse XF24细胞外通量分析仪分别检测了能够反映线粒体氧化磷酸化水平和糖代谢水平的耗氧率(OCR)和细胞外酸化率(ECAR)。结果显示敲低BV2细胞SMEK1后增强了糖酵解水平(图7L)，增强了其基础糖酵解，基础质子流出速率，糖酵解的质粒流出速率和加入2-DG后的酸化(图7N，O，P，Q)，减弱了线粒体氧化磷酸化水平(图7B)，减弱了其基础呼吸，最大呼吸，质子漏，ATP产生和备用呼吸量(图7D，E，F，G，H)。过表达BV2细胞SMEK1后增强了线粒体氧化磷酸化水平(图7C)，增强了其基础呼吸，质子漏和ATP产生(图7I，J，K)，减弱了糖酵解水平(图7M)，减弱了其基础糖酵解和加入2-DG后的酸化(图8A，B)。该结果说明敲低小胶质细胞Smek1后，细胞的代谢状态发生了改变，糖酵解水平升高，而线粒体氧化磷酸化水平下降。相反，过表达Smek1后，小胶质细胞的线粒体氧化磷酸化水平升高，而糖酵解水平下降。这些结果表明小胶质细胞中缺失Smek1后可导致线粒体代谢重编程。
本发明证明了SMEK1缺乏会导致缺血性脑卒中后小胶质细胞向促炎方向极化进而加重炎症。SMEK1通过线粒体代谢重编程调节小胶质细胞向促炎方向极化。我们的结论是，SMEK1对于减轻缺血性脑卒中后的炎症反应至关重要。因
此，SMEK1表达增多可能会减轻缺血性脑卒中的炎症，并有可能成为缺血性脑卒中的一种可行的治疗手段。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
Claims (4)
1.检测SMEK1编码基因和/或其表达产物的试剂在制备用于筛查、诊断、检测、监测或预测缺血性脑卒中的进展的产品中的应用。
2.如权利要求1所述应用，其特征在于，所述缺血性脑卒中的进展包括缺血性脑卒中患者的肢体瘫痪、感觉缺失、反射消失以及认知功能障碍；
所述缺血性脑卒中具体为急性缺血性脑卒中。
3.如权利要求1所述应用，其特征在于，所述检测SMEK1编码基因和/或其表达产物的试剂包括基于测序方法、基于定量PCR方法或基于探针杂交方法检测SMEK1编码基因转录的试剂；或基于免疫检测方法检测SMEK1蛋白表达情况的试剂；
所述产品为试剂盒、检测装置或检测设备。
4.促进SMEK1基因表达和/或使其表达产物活性提高的物质在如下1）-4）至少一种中的应用：
1）制备改善缺血性脑卒中介导的神经损伤的产品；
2）制备减轻缺血性脑卒中介导的炎症反应的产品；
3）制备抑制缺血性脑卒中后小胶质细胞向促炎方向极化的产品；
4）制备缺血性脑卒中预防和/治疗的产品；
所述促进SMEK1基因表达及使其表达产物活性提高的物质包括上调SMEK1表达的启动子或者慢病毒。
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