CN116808216A - Alpk1基因作为脑缺血导致的中枢神经系统疾病的防治靶点的应用 - Google Patents
Alpk1基因作为脑缺血导致的中枢神经系统疾病的防治靶点的应用 Download PDFInfo
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Abstract
本发明提供α‑激酶1基因(ALPK1)作为脑缺血导致的中枢神经系统疾病的防治靶点的应用，即将α‑激酶1作为脑缺血导致的中枢神经系统疾病(如缺血性脑卒中、血管性痴呆)的研究靶点。本发明发现α‑激酶1基因的表达量与缺血性脑损伤导致的中枢神经系统疾病相关，从而为相关的研究和药物筛选提供新的分子靶点和分子机制。
Description
本发明是分案申请，母案的申请号为“202210799044X”，发明名称为“ALPK1基因作为中枢神经系统疾病预防或治疗靶点的应用”，申请日为2022年07月06日。
技术领域
本发明属于医用动物模型构建技术领域，具体涉及α-激酶1(alpha-kinase 1，ALPK1)基因作为脑缺血导致的中枢神经系统疾病的防治靶点的应用。
背景技术
现代生活方式和医学技术使人均寿命得以延长，报告显示，2050年左右，世界六十岁以上老年人数将翻两倍。随着人类寿命的延长，很多老年性疾病发病率也逐渐上升，其中阿尔茨海默症(Alzheimer′s Disease,AD)这一年龄依赖性神经退行性疾病的患者数量逐年增加，是当今社会促进人类健康生活目标的强大阻力，很多老年人饱受其苦。因此，探索衰老进程中发生认知障碍的机制并寻找干预治疗策略，从而减缓甚至预防增龄性过程中认知障碍的发生，是一个亟待突破的课题。
AD发生的最大危险因素是衰老，衰老发生的过程中，个体逐渐呈现一系列机能老化的现象，其中就包括大脑的老化，表现为学习能力不佳、记忆障碍等，且这种神经性退行是不可逆的过程。在衰老过程中，神经元不断丢失，给AD患者日常生活带来极大困扰。研究表明AD呈现年龄的高依赖性，由德国科学家Alois Alzheime在1906年首次提出。随着对AD研究的不断深入，其特征性病理变化也逐渐被探索发现，在其发病过程中，老年斑随着β-淀粉样蛋白聚集而生成，神经纤维在Tau蛋白过度磷酸化中不断缠结，进一步导致神经元损伤丢失等。
认知障碍的发生与年龄呈高度正相关，全世界痴呆患者中60～70％均为老年人。研究发现，老年AD患者肠道微生物群中细菌水平较低，同样的结论在Quigley EMM等的研究中被同样发现，肠道微生物群结构及多样性与年龄相关，不同年龄个体中有较大差异。许多啮齿类动物研究表明，肠道菌群改变可能与个体行为变化有关。
Jeffery IB等在一项对老年群体肠道菌群的研究中发现，衰老的过程中伴随着肠道菌群结构的改变。研究发现肠道菌群中常见的革兰氏阴性菌外膜主要成分脂多糖(Lipopolysaccharides，LPS)可诱导认知功能障碍的发生。Jaeger等研究发现给小鼠腹腔注射LPS后，其更易透过血脑屏障向脑内迁移，而更少的往肠道等转移。并且在研究中，还发现了脑内有Aβ斑块沉积。已有报道将LPS注射到小鼠第四脑室后，其大脑发现了AD特征性病理产物，通过神经行为学实验发现其认知能力下降。综上可发现AD的发生发展与肠道微生物的生长代谢密不可分，肠道微生物的失调而导致AD特征性产物的生成。而且伴随着胃肠道屏障完整性的降低，细菌及其代谢物迁移入血等，最终导致微生物-肠-脑轴(Microbiota-gut-brain，MGB)全面失衡。
对衰老机制研究和抗衰老药物筛选中第一步就是建立最近似人类衰老状态的衰老动物模型，只有这样其模型所表现出的研究结果才更能提供参考意义。在很多研究中，使用自然生长且无外部其他刺激因素饲养到18～24月龄的自然衰老小鼠模型，此时小鼠年纪大致对应56～70岁人类，此模型在构建过程中并不复杂，但其却与衰老人群特点最为相近，如出现神经细胞丢失变性、行为迟缓、认知障碍及感觉降低甚至丧失等。但该模型构建时间长，饲养成本高且死亡率高，因而急需构建一种周期相对短，死亡率低且成功率高的与衰老致认知障碍相关的动物模型。
脑卒中是世界范围内致残和致死的主要疾病之一。根据病因的不同，脑卒中可分为出血性脑卒中和缺血性脑卒中，缺血性脑卒中的发病率高于出血性脑卒中，占卒中的80％以上。缺血性脑卒中发生后，伴随着细胞凋亡、氧化应激、兴奋性毒性和神经炎症等病理生理过程。这些病理生理过程严重的损害了胶质细胞、神经元和内皮细胞，它们相互影响、相互关联，形成恶性循环，损害大脑的神经功能。国内外缺血性脑卒中平均发病年龄60～70岁左右，是老年人群的常见疾病。
发明内容
本发明的目的是提供α-激酶1(alpha-kinase 1，ALPK1)基因作为脑缺血导致的中枢神经系统疾病的防治靶点的应用，即将α-激酶1作为脑缺血导致的中枢神经系统疾病的研究靶点。
本发明首先提供α-激酶1(alpha-kinase 1，ALPK1)基因的一种用途，即作为中枢神经系统疾病预防或治疗靶点的应用，所述的中枢神经系统疾病为脑缺血导致的相关疾病。
本发明再一个方面还提供ALPK1基因的抑制剂或拮抗剂在制备用于预防或治疗脑缺血导致的中枢神经系统疾病的制品中的应用。
本发明还提供一种筛选用于预防或治疗脑缺血导致的中枢神经系统疾病的药物的方法，所述的方法是使用构建的动物模型进行筛选。
所述的动物模型，是通过上调ALPK1基因表达量构建的动物模型。
本发明发现α-激酶1(alpha-kinase 1，ALPK1)基因的表达量与脑缺血导致的中枢神经系统疾病相关，从而为相关的研究和药物筛选提供新的分子靶点、分子机制和动物模型。
附图说明
图1：ALPK1敲减对脑缺血小鼠神经功能的影响。(A)小鼠神经行为学评分。(B)小鼠海马CA1区NeuN免疫荧光染色代表图(×400)及阳性表达定量分析。实验数据表示为mean±SD。采用双因素方差分析进行统计，与对应的Sham组进行比较，**P<0.01，*P<0.05；与sgNC/2VO组比较，##P<0.01。
图2：小鼠脑缺血后ALPK1蛋白及mRNA水平变化情况图，其中左侧的图为小鼠脑缺血后ALPK1蛋白变化代表图，右侧两幅图分别为小鼠脑缺血后ALPK1的mRNA及蛋白水平变化统计图。每组n＝6，实验数据表示为mean±SD。与Sham组比较，**P<0.01。
图3：ALPK1敲除对脑缺血小鼠神经行为功能及脑水肿的影响，左侧图为各组小鼠改良神经行为功能缺损评分，右侧图为各组小鼠脑水肿统计图。每组n＝6，实验数据表示为mean±SD。与WT/Sham组比较，**P<0.01；与WT/tMCAO组比较,##P<0.01。
具体实施方式
本发明提供了ALPK1基因的一个新的用途，确定其表达水平与脑缺血导致的缺血性脑损伤相关联，可作为脑缺血导致的中枢神经系统疾病防治的新靶点。
所述的小鼠ALPK1基因，其一种具体的NCBI编号为NC_000069.7，但还可以选择其它ALPK1的同源基因进行操作。
下面结合具体实施例和附图对本发明进行详细的描述。
实施例1：敲减ALPK1对短暂全脑缺血小鼠(2VO)的神经保护作用
采用Crispr/Cas9基因技术敲除小鼠ALPK1慢病毒购自于上海吉凯基因医学科技股份有限公司。慢病毒的滴度分为sgNC(4×108TU/ml)，sg-ALPK1-2(6×108TU/ml)。
实验采用SPF级2月龄雄性C57BL/6小鼠36只，购自成都达硕实验动物有限公司。小鼠饲养于SPF级屏障系统，环境温度控制在24℃～26℃，相对湿度控制在50％～70％，12h交替昼夜照明，自由饮水摄食。
1)慢病毒注射
小鼠腹腔注射戊巴比妥钠溶液进行麻醉，麻醉后将小鼠固定在脑立体定位仪上，剪开头部皮肤组织，暴露前囟，调节位置并进行定位(前囟后0.2mm，旁开1.0mm，深度2.5mm)。用微量注射器将1.5μl生理盐水、1.5μl sg-NC或1μlsgALPK1-2慢病毒注射到侧脑室，注射速度为0.5μl/min，留针5min后，缓慢取出进样针，缝合伤口。14天后对小鼠进行2VO造模。
2)建立2VO模型
按照文献方法建立小鼠2VO模型。
使用气体麻醉系统对C57BL/6小鼠进行麻醉，小鼠仰卧于体温维持垫上，小鼠肛温维持在36.5℃～37.5℃。暴露小鼠的双侧颈总动脉，剥离迷走神经，颈动脉夹闭20min后松开，并确保再灌注成功，假手术组仅暴露双侧颈总动脉，不进行夹闭。
3)于再灌72h后对小鼠进行神经行为学功能进行评分，评分方法见表1。
表1：神经行为学评分表
4)ALPK1敲减对脑缺血小鼠神经功能的影响
小鼠再灌注72h后，采用神经行为学评分对小鼠神经行为功能进行评分。如图所示(图1-A)，2VO术后72h，2VO组小鼠神经行为学评分显著高于Sham组(P<0.01)，Sham组的神经行为学评分都为0，而Saline/2VO、sgNC/2VO和sgALPK1-2/2VO的神经行为学评分分别为6.7±0.5、6.5±0.5和4.1±0.4，表明2VO给小鼠造成了严重的神经行为学障碍。而ALPK1敲减小鼠可以显著改善2VO术后的神经行为学障碍：与sgNC/2VO组相比，sgALPK1-2/2VO组小鼠神经行为学评分明显降低(P<0.01)。
神经元是神经系统的基本结构和功能单位，通过突触形成复杂的反射弧以对机体功能做出调控。脑缺血发生后，释放的炎性因子对神经元造成损害，使得神经元的数目和形态都发生改变。
采用免疫荧光法考察了小鼠海马CA1区神经元丢失情况，如图所示(图1-B)。与Sham组相比，2VO组小鼠海马CA1区神经元数目显著减少(P<0.01)，表明2VO造成了严重的神经元损伤。而ALPK1敲减小鼠可以显著改善神经元丢失情况：与sgNC/2VO组相比，sgALPK1-2/2VO组小鼠海马CA1区神经元存活得到明显改善(P<0.01)
实施例2：ALPK1基因敲除对短暂局灶性脑缺血小鼠(tMCAO)的神经保护作用
1)小鼠脑缺血后ALPK1蛋白及mRNA水平变化情况
将雄性C57BL/6小鼠分为Sham组和tMCAO组，采用线栓法制备短暂性脑缺血小鼠模型，具体步骤如下：雄性C57BL/6小鼠(～27g)吸入混有氮气与氧气的异氟烷(浓度为2％)。气体麻醉后，将其仰卧位固定在手术台上。剪开小鼠颈部正中间的皮肤，轻柔分离出左侧CCA、ECA和ICA，尽量避开迷走神经，以免引发小鼠深呼吸甚至死亡。用活结结扎CCA和ECA的远心端。用显微手术剪在ECA剪一斜形切口，插入尼龙线栓，翻转ECA直至与ICA位于同一直线位置，线栓插入ICA约10mm，感觉到微小阻力时停止继续插入。将线栓另一端用手术缝线固定于ECA，打开CCA的活结，缝合手术切口。1小时后再次麻醉小鼠，解除原来的缝线，打开颈部切口，将线栓拔出以实现阻塞的大脑中动脉血液再灌注。整个手术过程中用实验动物体温维持仪将小鼠体温维持在37±0.5℃。手术过程中用激光散斑血流成像仪检测脑血流改变，小鼠局部流血流下降不低于75％，再灌后血流量恢复者视为模型成功，用于后续实验。正常对照组除了不插入线栓外，其他操作均与上述相同。缺血24h后麻醉小鼠，用预冷的生理盐水从小鼠心脏灌流至肝脏变白，在冰上取下脑组织立即冻存到-20℃，用于ALPK1mRNA和蛋白水平的检测。
图2显示，与Sham组相比，tMCAO组小鼠脑缺血24h后，缺血脑组织中的ALPK1 mRNA和蛋白水平显著上调(P<0.01)，提示ALPK1可能在小鼠脑缺血后发挥了重要的调节作用。
2)ALPK1敲除对脑缺血小鼠神经行为功能及脑水肿的影响
针对上述实验结果，采用ALPK1基因敲除小鼠进行验证实验。将小鼠分为WT/Sham组、WT/tMCAO组和KO/tMCAO组。采用上述线栓法制备短暂性脑缺血小鼠模型。缺血24h后，采用mNSS评分对小鼠整体神经功能障碍程度进行评价。具体评分标准如表2所示。mNSS总分为18分,0分正常,1-6分为轻度损伤，7-12分为中度损伤，13-18分为重度损伤。
表2：改良神经功能缺损评分(mNSS)量表
图3显示，与WT/tMCAO组相比，KO/tMCAO组小鼠神经功能障碍有显著改善(P<0.01)，提示ALPK1敲除可改善小鼠脑缺血后神经功能障碍。
3)干湿重法测量脑水肿
实验小鼠缺血24h后，大脑被收集起来，分为缺血半脑、非缺血半脑和小脑。每个部分称量以得到湿重(WW)，然后在摄氏110度的环境下烘干24h达到恒重，测定其干燥时的干重，脑组织含水量为-用以下公式计算:(WW-DW)/WW×100％。图3显示，与WT/tMCAO组相比，KO/tMCAO组小鼠缺血半脑的脑水肿程度明显减轻(P<0.01)，提示ALPK1敲除可改善小鼠脑缺血后脑水肿。综上所述，ALPK1在小鼠脑缺血后神经功能损伤中发挥了关键的调节作用。
Claims (9)
1.α-激酶1基因作为中枢神经系统疾病预防或治疗靶点的应用。
2.如权利要求1所述的应用，其特征在于，所述的中枢神经系统疾病为脑缺血导致的相关疾病。
3.如权利要求2所述的应用，其特征在于，所述的中枢神经系统疾病为缺血性脑卒中。
4.如权利要求3所述的应用，其特征在于，所述的缺血性脑卒中为脑缺血后神经功能障碍。
5.α-激酶1的抑制剂或拮抗剂在制备用于预防或治疗脑缺血导致的中枢神经系统疾病的制品中的应用。
6.用于敲减或敲除α-激酶1基因的制剂在制备用于预防或治疗脑缺血导致的中枢神经系统疾病的制品中应用。
7.如权利要求6所述的应用，其特征在于，所述的制剂为RNAi相关的制剂。
8.如权利要求6所述的应用，其特征在于，所述的制剂为基因编辑的制剂。
9.一种筛选用于预防或治疗脑缺血导致的中枢神经系统疾病的药物的方法，所述的方法是使用构建的动物模型进行筛选；所述的动物模型，是通过上调α-激酶1基因表达量构建的。
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