CN110548143A - 大麻素受体cb1在视网膜感光细胞退行性疾病中的应用 - Google Patents
大麻素受体cb1在视网膜感光细胞退行性疾病中的应用 Download PDFInfo
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Abstract
本发明涉及一种大麻素受体CB1在视网膜感光细胞退行性疾病中的应用，属于基因功能与应用领域。本发明确定了CB1在RP、AMD等视网膜感光细胞退行性疾病中的作用，同时确定了CB1的抑制剂SR1能有效缓解视网膜光感受器的凋亡，保护该细胞的功能作用，因此，CB1基因可作为药物靶标，用于在筛选保护视网膜光感受器细胞功能和/或预防、缓解和/治疗RP、AMD等视网膜感光细胞退行性疾病的药物；CB1基因可作为基因治疗中的靶基因，用于在设计和制备在保护视网膜光感受器细胞功能和/或预防、缓解和/治疗RP、AMD等疾病的药物和/或生物学试剂，为视网膜感光细胞退行性疾病的治疗提供了一条有效的新途径。
Description
技术领域
本发明属于基因的功能与应用领域，涉及大麻素受体CB1及其特异拮抗剂SR141716A在治疗视网膜感光细胞退行性疾病中的功能及应用，具体是在缓解或治疗视网膜感光细胞退行性疾病的药物中应用。
背景技术
视网膜感光细胞退行性疾病，是一组以进行性感光细胞及色素上皮功能丧失为共同表现的退行性疾病。该类疾病主要是由基因突变或RPE细胞功能障碍引起的，常见的典型例子是视网膜色素变性(RP)和老年黄斑变性(AMD)，也是两种重要的棘手的致盲眼病。由遗传性原因所导致的感光细胞退行性改变的患病率约为1/3500-1/4000，中国目前有视网膜色素变性的病人40万人，全世界的患者在150万人以上。而后天药物和疾病原因所引起的继发性视网膜感光细胞退行性疾病也日益增高。
视锥细胞和视杆细胞上的基因缺失或突变可以直接导致光感受器细胞缺失，RPE细胞是为光感受器细胞提供能量的细胞，因此RPE细胞功能障碍也会导致光感受器细胞的凋亡和缺失，视网膜外层的光感受器细胞功能障碍，其下游的神经元无法接收到光视觉信号，是这类疾病视力丧失的最终原因。由于视杆细胞和视锥细胞是相互支持的，所以一旦一方受累，另一种细胞也会受到影响；一旦视杆细胞受累，RP患者就会出现夜盲，然后周边视野逐渐丢失，最终导致视力完全丧失。干性AMD患者也就有类似的病理过程，黄斑部凋亡的RPE细胞累及到光感受细胞，从而导致中心视力不可逆的损害，最后的结果是不可逆的双目失明。因为视网膜感光细胞退行性疾病中感光细胞的凋亡不可逆且有大多数疾病具有高度的遗传异质性，相关疾病的治疗也变得十分困难。虽然有传统的药物保守治疗，及近年来的基因治疗、干细胞治疗、视网膜假体等方法，但目前仍未有权威且普适的治疗方案。因此寻找能有效缓解疾病中光感受器细胞的凋亡和缺失的特异性分子，对进一步阐明视网膜感光细胞退行性疾病的发生发展机制，具有重要的理论意义，可为临床防治RP和AMD疾病提供新靶点和新策略。
大麻素受体是中枢神经系统分布最广泛的G蛋白偶联受体，主要包括大麻素受体1(Cannabinoid receptor,CBl)、大麻素受体2(Cannabinoid receptor,CB2)、G蛋白偶联受体119(GPR119)、蛋白偶联受体55(GPR55)、瞬时受体电位香草酸亚型1(TRPV1)。大麻素受体可以通过影响细胞间突触连接，调节神经元细胞的功能，从而对神经系统发挥调节作用。其中SR141716A(SR1)是CB1特异拮抗剂，能显著抑制CB1受体发挥功能作用。
多项研究表明，大麻素对中枢神经系统具有保护作用，大麻素受体可以通过激活其下游的信号通路对神经系统损伤发挥保护作用，其主要的通路包括三类：1)G蛋白偶联受体信号通路；2)CB1受体介导的离子通道；3)丝裂原活化蛋白激酶(MAPK)信号通路。
视网膜是中枢神经系统的延伸。视网膜中的光感受器细胞、双极细胞、神经节细胞都属于神经元细胞。视网膜神经节细胞的轴突形成视神经，因此视神经纤维实际上是中枢神经系统的轴突。视网膜独特的物理结构，局部排列的表面分子和细胞因子能产生特定的免疫反应，这与中枢神经系统相类似，这两个系统在细胞免疫、突触传递、神经再生等方面有许多共同之处，同时中枢神经退变性疾病和视网膜变性疾病有共同之处。已知大麻素在中枢神经外伤、神经炎和神经退行性疾病中有神经保护作用，但在视网膜慢性变性疾病比如RP和AMD疾病中，大麻素及其受体的作用尚不明确。目前的研究表明，大麻素受体1(CB1)介导的神经保护作用主要包括：1)抑制兴奋性突触间神经递质的分泌；2)抑制胞内韩离子浓度的上升；3)激活细胞内信号途径，比如PDK/Akt、ERK、BDNF、NF-kB等信号通路，发挥神经保护作用。
发明内容
本发明的目的是确定CB1在视网膜感光细胞退行性疾病中对光感受器细胞凋亡的调控作用，提供一种CB1基因靶点及其抑制剂SR1作为药物靶标在筛选预防、缓解和/治疗RP和AMD疾病的药物中的应用，提供一个用于预防、缓解和/或治疗视网膜感光细胞退行性疾病的靶基因CB1的新途径。
本发明解决上述技术问题所采用的方案是：
大麻素受体CB1作为药物靶标在预防、缓解和/治疗视网膜感光细胞退行性疾病的药物中的应用。
大麻素受体CB1特异拮抗剂SR141716A在制备预防、缓解和/治疗视网膜感光细胞退行性疾病的药物中的应用，所述视网膜感光细胞退行性疾病包括视网膜色素变性和老年黄斑变性。
本发明还提出一种预防、缓解和/治疗视网膜感光细胞退行性疾病的药物，包含大麻素受体CB1及其特异拮抗剂SR141716A。
本发明还提出一种保护视网膜光感受器细胞功能的方法，采用大麻素受体CB1作为基因靶点，采用大麻素受体CB1特异拮抗剂SR141716A作为靶向药物。
本发明还提出一种保护视网膜光感受器细胞功能的药物，包含大麻素受体CB1及其特异拮抗剂SR141716A。
本发明相对于现有技术具有如下优点：
(1)本发明发现大麻素受体CB1在光感受器细胞中的表达，并提出其可以作为药物靶标，用于筛选保护视网膜光感受器细胞功能和/或预防、缓解和/治疗RP、AMD等视网膜感光细胞退行性疾病的药物。
(2)本发明发现CB1抑制剂SR1能有效缓解视网膜光感受器的凋亡，保护该细胞的功能作用，因此可以作为保护视网膜光感受器细胞功能和/或预防、缓解和/治疗RP、AMD等疾病的药物和/或生物学试剂，为视网膜感光细胞退行性疾病的治疗提供了一条有效的新途径。
附图说明
图1为CB1在视网膜中分布和表达情况，其中A：没有加一抗的对照，B：CB1抗体免疫荧光染色的视网膜切片，C：用Western blot方法，在全视网膜组织匀浆中检测到CB1蛋白，条带位于60kd的位置，IF×400；
图2为CB1在视网膜双极细胞上的表达，其中A：微分干涉差(differentialinterference contrast microscope，DIC)模式白光照明下的急性分离得到的单个双极细胞，B：用CB1抗体对单个双极细胞进行免疫荧光染色，C：用双极细胞标记抗体PKC对单个双极细胞进行免疫荧光染色，D：用CB1抗体对单个双极细胞进行免疫荧光染色，E：用双极细胞标记抗体PKC对单个双极细胞进行免疫荧光染色，F：图D和图E两图重叠后的图像，IF×400；
图3为CB1在视网膜在各细胞层的表达和分布，其中A从左至右：CB1、视锥细胞、DAPI、合并图，B：CB1、双极细胞、DAPI、合并图，C：CB1、神经节细胞、DAPI、合并图，D：CB1、Müller细胞、DAPI、合并图，IF×400；
图4为不同给药组的视网膜外核层厚度；
图5为DAPI染色显示不同给药组的三个核层厚度，其中A：CON组外核层厚度大概是内核层的2倍；B：给MNU5天后，外核层变薄，比内核层厚度小；C：给MNU前给以SR1干预，外核层厚度丢失减轻；D：给MNU前给以WIN干预，外核层厚度的丢失仍然明显；E：给MNU前给以WIN和SR1干预，外核层厚度的丢失仍然明显，IF×400；
图6为不同给药组外核层与内核层的厚度比及外核层与三个核层的厚度比，其中A：不同给药组外核层与内核层的厚度比(ONL/INL)，B：外核层与三个核层的厚度比(ONL/TL)；
图7为不同给药组Müller细胞的增生情况，其中A：对照组(CON)Müller细胞的荧光信号主要集中于外界膜基底部，没有胶原增生情况；B：MNU组Müller细胞增生明显，增生的胶原丝遍布整个视网膜；C：MNU+SR1组视网膜胶原增生不明显；D：MNU+WIN组胶原增生明显；E：MNU+WIN+SR1组胶原增生明显，右下图为用Image J软件分析得到的不同组间Müller细胞的免疫荧光染色情况，与GFAP免疫荧光染色结果一致，IF×400；
图8为SR1对双极细胞的影响作用，其中：A-D依次为MNU后5天，GFAP、PKC、DAPI的标记情况以及合并图；E-H依次为MNU加SR1干预后5天，GFAP、PKC、DAPI的标记情况以及合并图；I-L依次为MNU加SR1干预后5天，GFAP、Brn-3a、DAPI的标记情况以及合并图，IF×400；
图9为SR1对视网膜血管的保护作用，其中A：对照组呈现正常的视网膜血管；B：给MNU后视网膜血管结构破坏，末梢出现异常血管丛样结构；C：MNU造模前给予SR1，大血管结构正常，见少量末梢血管丛；D：MNU造模前给予WIN，大血管结构损伤严重，大量末梢血管丛；E：MNU造模前后给予WIN和SR1，大血管结构损伤严重，大量末梢血管丛；F：统计分析单位面积内视网膜血管丛的个数，对照组无异常血管丛样结构，给MNU组见大量异常视网膜血管丛，MNU造模后给予SR1组见少量末梢血管丛，MNU造模后给予WIN组见大量末梢血管丛，MNU造模后给予WIN和SR1组见大量末梢血管丛，IF×200；
图10为MNU对视网膜双极细胞的损伤，其中A为对ON细胞的损伤情况，对照组给药物LY341495，可以在ON型双极细胞上观察到一个ON型波，给MNU后第7天，ON型双极细胞对LY341495的反应完全消失；B为OFF细胞的损伤情况，对照组给药物AMPA，可以在OFF型双极细胞上观察到一个OFF型波，给MNU后第7天，OFF型双极细胞对AMPA的反应仅部分消失；
图11为SR1对MNU诱导的双极细胞电活动损伤的缓解效果，其中A为CNQX处理前后AMPA药物的反应情况，对照组给药物AMPA后，可以在OFF型双极细胞上观察到一个OFF型波；预先CNQX药物灌流孵育视网膜组织片，AMPA反应被完全阻断；将CNQX洗脱掉后，再给AMPA药物，AMPA受体介导的OFF反应又重新出现；B为MNU处理前后AMPA药物的反应情况，对照组给给药物AMPA后，可以在OFF型双极细胞上观察到一个OFF型波；给MNU后第7天，OFF型双极细胞对AMPA的反应部分仍保留；给药SR1药物干预后，OFF型双极细胞对AMPA的反应被部分挽回。
具体实施方式
为更好的理解本发明，下面的实施例是对本发明的进一步说明，但本发明的内容不仅仅局限于下面的实施例。
实施例1
(1)揭示CB1在视网膜组织上广泛表达。
利用免疫荧光和Western blot对CB1在视网膜中分布和表达进行检测，发现CB1在在光感受器细胞、OPL、ONL、IPL、GCL有表达，OPL和IPL的表达最显著(图1)。为了能观察单个视网膜细胞中CB1的表达情况，对急性分离的成年小鼠视网膜的双极细胞，进行免疫荧光染色，用ON型双极细胞的标志物PKC抗体做标记，观察到在双极细胞上，CB1和PKC有共染色的情况，说明小鼠ON型双极上有CB1的表达(图2)。为了更好的了解大麻素受体在视网膜的分布情况，采用多个细胞的标志抗体和CB1抗体进行共标。发现CB1在视锥细胞、ON型双极细胞、神经节细胞上有表达，在Müller细胞上没有CB1的表达(图3)。
(2)明确CB1的抑制剂SR1能缓解视网膜外核层损伤，减少视网膜感光细胞的凋亡。
利用MNU腹腔注射建立视网膜感光细胞退行性疾病模型，给予3种大麻素相关的药物WIN 55，212-2(WIN，CB1非特异性激动剂)、SR141716A(SR1，CB1特异性抑制剂)和SR144528(SR2，大麻素受体2的特异性抑制剂)，利用免疫荧光和Western blot检测视网膜组织结构的变化。结果发现，注射MNU前给SR1干预，能缓解MNU导致的视网膜外层损伤；单独给WIN或WIN+SR1或WIN+SR2，没有观察到外核层的改善。在注射MNU之前给予SR1干预，发现DAPI标记的外核层厚度比其他组更厚(图4-6)，GFAP标记的胶原反应也变轻(图7)，PKC标记的双极细胞树突丢失减轻(图8)。用视网膜血管染色评估给予SR1干预后MNU诱导的视网膜外层的损伤程度，发现视网膜血管的损伤减轻(图9)。说明CB1的抑制剂SR1能有效缓解视网膜感光细胞退行性疾病中视网膜外核层损伤，减少视网膜光感受器细胞凋亡。
(3)揭示CB1的抑制剂SR1对视网膜感光细胞退行性疾病中光感受器视锥细胞和OFF型双极细胞有保护作用。
采用视网膜薄片膜片钳技术，观察给各种大麻素相关药物和MNU后，ON型和OFF型双极细胞上，代谢型谷氨酸受体(mGLUR6，ON型双极细胞上特征性受体)和离子型谷氨酸受体(AMPA，OFF型双极细胞特征性受体)的电生理反应。结果显示，给MNU后第7天，ON型双极细胞反应完全消失，OFF型双极细胞的反应仅部分消失(图10)，同时，给予SR1药物干预后，OFF型双极细胞对AMPA的反应被部分挽回(图11)。说明MNU对视网膜ON型双极细胞的损伤更大，同时SR1能缓解MNU诱导的双极细胞电活动损伤。
由以上述结果可知CB1的抑制剂SR1能显著改善MNU诱导的双极细胞的上游的神经元即光感受器细胞的功能，提示CB1在视网膜感光细胞退行性疾病中能促进光感受器细胞的凋亡，导致细胞功能的丧失，促进疾病的进展和加重。因此，CB1及其抑制剂SR1在视网膜感光细胞退行性疾病的细胞功能调控中起到重要的作用。
以上所述是本发明的优选实施方式而已，当然不能以此来限定本发明之权利范围，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和变动，这些改进和变动也视为本发明的保护范围。
Claims (6)
1.大麻素受体CB1作为药物靶标在预防、缓解和/治疗视网膜感光细胞退行性疾病的药物中的应用。
2.大麻素受体CB1特异拮抗剂SR141716A在制备预防、缓解和/治疗视网膜感光细胞退行性疾病的药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述视网膜感光细胞退行性疾病包括视网膜色素变性和老年黄斑变性。
4.一种预防、缓解和/治疗视网膜感光细胞退行性疾病的药物，其特征在于，包含大麻素受体CB1及其特异拮抗剂SR141716A。
5.一种保护视网膜光感受器细胞功能的方法，其特征在于，采用大麻素受体CB1作为基因靶点，采用大麻素受体CB1特异拮抗剂SR141716A作为靶向药物。
6.一种保护视网膜光感受器细胞功能的药物，其特征在于，包含大麻素受体CB1及其特异拮抗剂SR141716A。
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