CN116672341A - 雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用 - Google Patents
雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用 Download PDFInfo
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Abstract
本发明提供了雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用。具体地,本发明提供了雷帕霉素或其药学上可接受的盐或酯的用途,它被用于制备治疗或预防渐冻人症和/或额颞叶痴呆的药物组合物。雷帕霉素可进入脑内中枢神经系统,清除神经细胞中的多聚蛋白poly‑GA聚集,缓解神经细胞损伤和毒性。同时也能够抑制poly‑GA导致的星形胶质细胞和小胶质细胞激活,继而抑制神经炎症,并显著改善C9orf72突变所导致的ALS和FTD等神经退行性病变。
Description
技术领域
本发明涉及药物治疗领域,具体地涉及雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用,包括雷帕霉素及其组合物在制备用于治疗渐冻症和额颞叶痴呆中的药物中的用途。
背景技术
肌萎缩侧索硬化症(ALS),也被称为渐冻人症,是一种常见的神经退行性疾病,患者的脊髓、大脑皮层、小脑等神经元出现损伤,临床症状主要表现为肌肉萎缩,运动功能障碍以及麻痹。ALS患病率致死率极高,通常在症状出现后2-3年内即死亡,属于危害性极大的一类神经退行性疾病。
额颞叶痴呆(FTD)是排名第二的早老性痴呆,主要是因为大脑额叶和颞叶病变退化,导致病人出现语言、人格、认知功能障碍。
研究报道,FTD和ALS在病理上存在着重叠,即部分FTD病人呈现出运动功能障碍,而多达50%的ALS患者也出现行为和认知功能方面的障碍。
然而,无论是ALS还是FTD,目前尚无令人满意的有效治疗药物和方法。
目前,本领域迫切需要开发新的有效治疗或缓解ALS和FTD的药物和方法。
发明内容
本发明的一个目的是提供雷帕霉素的新的医药用途,即其在制备用于治疗渐冻人症和额颞叶痴呆中的药物中的用途。
本发明的第一方面,提供了一种雷帕霉素或其药学上可接受的盐或酯的用途,它用于制备一药物组合物或制剂,所述药物组合物或制剂用于:
(a)治疗或预防渐冻人症、额颞叶痴呆或组合;
(b)清除促进脑部poly-GA蛋白的聚集;和/或
(c)抑制小胶质细胞和/或星形胶质细胞的激活。
在另一优选例中,所述的渐冻人症为C9orf72突变型渐冻人症。
在另一优选例中,所述的C9orf72突变型为GGGGCC重复序列及其反义链CCCCGG突变型。
在另一优选例中,所述的C9orf72突变型为产生poly-GA的突变型。
在另一优选例中,所述的渐冻人症包括家族性渐冻人症或散发性渐冻人症。
在另一优选例中,所述的额颞叶痴呆为C9orf72突变型额颞叶痴呆。
在另一优选例中,所述的C9orf72突变型为GGGGCC重复序列及其反义链CCCCGG突变型。
在另一优选例中,所述的C9orf72突变型为产生poly-GA的突变型。
在另一优选例中,所述的额颞叶痴呆包括家族性渐冻人症或散发性额颞叶痴呆。
在另一优选例中,所述的药物用于清除促进脑部poly-GA蛋白的聚集。
在另一优选例中,所述的药物用于清除促进脑部选自下组区域的poly-GA蛋白的聚集:运动皮层、海马体、小脑、或其组合。
在另一优选例中,所述药物用于抑制小胶质细胞和/或星形胶质细胞的激活。
在另一优选例中,所述药物为含有以下成分的药物组合物:(a)第一活性成分,所述第一活性成分为雷帕霉素;和(b)药学上可接受的载体。
在另一优选例中,所述组合物或制剂被施用于人和非人哺乳动物。
在另一优选例中,所述的组合物还含有额外的治疗药物。
在另一优选例中,所述的额外的治疗药物为治疗渐冻人症和/或额颞叶痴呆的药物。
在另一优选例中,所述的额外的治疗药物选自下组:白藜芦醇,地高辛等。
在另一优选例中,所述的药物组合物为固态或液态制剂。
在另一优选例中,所述的药物组合物的剂型选自下组:口服制剂、注射剂、冻干剂。
在另一优选例中,所述的药物组合物的剂型为口服制剂,较佳地为片剂、胶囊剂、颗粒剂。
本发明的第二方面,提供了一种试剂盒,所述的试剂盒包括:
(U1)检测试剂,所述的检测试剂用于检测C9orf72突变;和
(U2)第一活性成分,所述的第一活性成分选自下组:雷帕霉素、或其药学上可接受的盐或酯。
在另一优选例中,所述的试剂盒用于治疗渐冻人症和/或额颞叶痴呆。
在另一优选例中,所述的试剂盒还含有:
(U3)第二活性成分,所述的第二活性成分为额外的治疗渐冻人症和/或额颞叶痴呆的药物。
在另一优选例中,所述的检测试剂包括:用于检测C9orf72是否存在GGGGCC重复突变的试剂,用于检测是否存在poly-GA多聚重复蛋白的试剂、或其组合。
在另一优选例中,所述的检测用检测选自下组的样本:脑细胞、神经细胞、外周血、或其组合。
在另一优选例中,所述检测试剂包括:引物、探针、芯片或其组合。
本发明的第三方面,提供了一种体外非治疗性地减少神经细胞中poly-GA蛋白聚集的方法,包括步骤:
在雷帕霉素存在下,培养神经细胞,从而减少所述神经细胞中poly-GA蛋白聚集。
在另一优选例中,所述的神经细胞是具有C9orf72突变型的神经细胞。
在另一优选例中,所述的C9orf72突变型为GGGGCC重复序列及其反义链CCCCGG突变型。
在另一优选例中,所述的C9orf72突变型为产生poly-GA的突变型。
在另一优选例中,所述的神经细胞包括:N2a细胞、SH-sy5y细胞。
本发明的第四方面,提供了一种治疗治疗或预防渐冻人症和/或额颞叶痴呆的方法,包括步骤:给需要的对象施用安全有效量的雷帕霉素或其药学上可接受的盐或酯。
在另一优选例中,所述的对象包括人和非人哺乳动物。
在另一优选例中,所述的非人哺乳动物包括啮齿动物(如小鼠、大鼠)。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了雷帕霉素可显著改善ALS小鼠行为学退变。A:造模及给药方案;B:小鼠转棒实验;C和D:爬杆试验;E、F和G:旷场实验;
图2显示了雷帕霉素可显著改善ALS小鼠的脑内炎症和细胞凋亡。A:小鼠脑中GFAPmRNA丰度检测;B:小鼠脑中Iba1 mRNA丰度检测;C:小鼠脑切片,免疫荧光检测GFAP;D:小鼠脑切片免疫荧光观察Iba1;E:Tunel染色检测小鼠脑中细胞死亡;
图3显示了雷帕霉素可显著降低小鼠脑内poly-GA蛋白聚集。
具体实施方式
本发明人经过广泛而深入的研究,首次意外地发现,雷帕霉素可有效地治疗渐冻症和额颞叶痴呆,尤其是C9orf72突变相关的渐冻症和额颞叶痴呆。雷帕霉素可以进入脑内中枢神经系统产生作用,清除神经细胞中的多聚蛋白poly-GA聚集,缓解神经细胞损伤和毒性。此外,雷帕霉素可抑制poly-GA导致的星形胶质细胞和小胶质细胞激活继而抑制神经炎症,最终显著改善C9orf72突变导致的神经退行性病变,从而可用于治疗渐冻症和额颞叶痴呆。在此基础上完成了本发明。
具体地,动物行为学的实验结果表明,在C57BL/6J小鼠ALS模型中,行为学出现显著退化,主要表现为运动能力出现显著下降,同时其平衡能力和学习记忆能力也出现显著降低。而给予雷帕霉素后,可以显著改善模型小鼠的运动,平衡和学习记忆能力,最终缓解致病蛋白的神经退行性病变。针对脑内细胞的进一步实验表明,给予雷帕霉素后,能够显著降低模型小鼠脑内Iba1和GFAP mRNA水平和炎症,并显著降低模型小鼠脑内不同脑区的细胞损伤。此外,雷帕霉素可显著降低模型小鼠脑内poly-GA蛋白的聚集。
术语和缩写
ALS指肌萎缩性侧索硬化症,英文为Amyotrophic lateral sclerosis,又称渐冻人症。
FTD指额颞叶痴呆,英文为Frontotemporal dementia。
GFP-(GA)150指GFP和150个甘氨酸(Glycine,缩写G)丙氨酸(Alanine,缩写为A)重复形成的融合蛋白,用于模拟多聚重复二肽poly-GA。由于该融合蛋白含有GFP元件,因此可在激发条件下发出荧光。
活性成分
雷帕霉素(Rapamycin)是一种新型大环内酯类免疫抑制剂,为白色固体结晶。早在20世纪70年代起初被作为低毒性的抗真菌药物,随后发现具有免疫抑制作用。1989年开始作为治疗器官移植的排斥反应的新药进行试用。根据现有的临床前及临床数据看,是一种疗效好,低毒,无肾毒性的新型免疫抑制剂。
目前,雷帕霉素已经作为维持移植器官免疫能力的常用药物(特别是肾移植),以减缓器官移植手术后的免疫排斥反应。
雷帕霉素的结构式如下:
mTOR通路
mTOR通过两种大型分子复合物产生生物学功能,分别是是mTOR复合物1(mTORC1)和mTOR复合物2(mTORC2)。
雷帕霉素是mTORC1抑制剂,可抑制mTOR活性。
C9orf72基因和蛋白
C9orf72基因,即9号染色体上开放阅读框72基因,英文为chromosome 9openreading frame 72。
位于C9orf72基因第一个内含子上的GGGGCC六碱基高度重复序列突变是导致ALS/FTD重要原因。正常人体内的GGGGCC重复序列一般是2-8个,而在携带C9orf72基因突变的病人中重复数目是几百甚至可达上千。
药物组合物和施用方式
本发明的雷帕霉素或其药学上可接受的盐或酯,用于制备治疗或预防渐冻人症和/或额颞叶痴呆的药物组合物。
本发明的药物组合物包括安全有效量范围内的雷帕霉素或其药理上可接受的盐或酯。其中“安全有效量”指的是:化合物的量足以明显改善病情,而不至于产生严重的副作用。通常,药物组合物含有1-10000mg雷帕霉素化合物/剂,更佳地,含有100-8000mg雷帕霉素类化合物/剂,最佳地含有200-6000mg癸酸甘油酯类化合物/剂。较佳地,所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是:一种或多种相容性固体或液体填料或凝胶物质,它们适合于人使用,而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和,而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如 )、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制,代表性的施用方式包括(但并不限于):口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药,优选的给药方式为口服给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中,活性化合物与至少一种常规惰性赋形剂(或载体)混合,如柠檬酸钠或磷酸二钙,或与下述成分混合:(a)填料或增容剂,例如,淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(b)粘合剂,例如,羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶;(c)保湿剂,例如,甘油;(d)崩解剂,例如,琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠;(e)缓溶剂,例如石蜡;(f)吸收加速剂,例如,季胺化合物;(g)润湿剂,例如鲸蜡醇和单硬脂酸甘油酯;(h)吸附剂,例如,高岭土;和(i)润滑剂,例如,滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠,或其混合物。胶囊剂、片剂和丸剂中,剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备,如肠衣和其它本领域公知的材料。它们可包含不透明剂,并且,这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时,活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外,液体剂型可包含本领域中常规采用的惰性稀释剂,如水或其它溶剂,增溶剂和乳化剂,例知,乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油,特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外,组合物也可包含助剂,如润湿剂、乳化剂和悬浮剂、甜味剂、娇味剂和香料。
除了活性化合物外,悬浮液可包含悬浮剂,例如,乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂,或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药,或者与其他药学上可接受的化合物联合给药(如治疗渐冻人症的其他药物)。
使用药物组合物时,是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人),其中施用时剂量为药学上认为的有效给药剂量,对于60kg体重的人而言,日给药剂量通常为1~10000mg,优选100~8000mg,更优选为200-6000mg。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
本发明的主要优点包括:
(a)雷帕霉素可以显著缓解该ALS模型小鼠的神经行为学退化,改善小鼠的运动能力和平衡能力以及学习记忆能力。
(b)本发明人意外地发现了雷帕霉素的新的药效,其促进细胞内多聚蛋白降解,最终缓解细胞损伤,恢复小鼠运动功能。因此,雷帕霉素可以用于治疗c9orf72突变的ALS和FTD等疾病。
(c)雷帕霉素能够显著改善小鼠的神经退行性症状,以及减少脑内神经元的死亡。
下面结合具体实施例,进一步阐述本发明。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。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例1:雷帕霉素缓解ALS模型小鼠的神经行为学退化
在本实施例中,采用图1A所示的实验方案,在C9orf72突变翻译的poly-GA的ALS模型小鼠上,测试了雷帕霉素对该模型小鼠的行为学退化的治疗效应。
1、实验原理
利用腺相关病毒递送突变的基因来建造ALS小鼠模型是一种经典的造模方法。当小鼠神经感染病毒并表达突变蛋白后可以很好的模拟临床上ALS各种行为学及病理学特征。本发明以该递送技术在小鼠脑内过表达目的基因继而构建ALS小鼠模型,并评价雷帕霉素的治疗效应。
2、实验材料与方法
1)动物来源:C57BL/6J小鼠购置于北京维通利华实验动物技术有限公司。
2)动物培养条件:SPF级动物房饲养;温度:22-24℃;湿度:45-80%;光照:150-300Lx,12小时昼夜交替。其饲养,给药和处死均严格按照动物实验和福利的指导(参照上海市实验动物管理条例)
3)ALS小鼠模型的建立:构建AAV-GFP-GA150,血清型为PHP.eB的病毒,尾静脉注射五个月后对小鼠进行行为学及病理学分析。病毒注射五个月后,行为学上小鼠出现明显的行为缺陷,病理学上小鼠脑皮层能够显著激活胶质细胞,引发神经炎症。
4)动物分组与给药:C57BL/6J小鼠饲养于SPF级动物房中,适应性驯养一周后,在30天时,尾静脉注射腺相关病毒,剂量为100ul(病毒量MOI:1010)。
动物分为4组(n=6):
过表达空载GFP为对照组,包括GFP+对照组(给予4mg/kg 5%PEG-400和5%Tween80的PBS溶液),以及GFP+雷帕霉素(施用4mg/kg剂量的雷帕霉素);
过表达GFP-GA150为ALS造模组,第90天开始隔天通过腹腔注射雷帕霉素给药或对照药物。其中GFP-GA150+对照为施用5%Tween 80的PBS溶液,而GFP-GA150+雷帕霉素(施用4mg/kg剂量的雷帕霉素)。
于180天时进行行为学测试。行为学测试分转棒实验评价其运动能力,爬杆实验评价其四肢肌肉力量及平衡力量,旷场实验评价其学习记忆能力。
5)观察指标
行为学测试分转棒实验评价其运动能力,爬杆实验评价其四肢肌肉力量及平衡力量,旷场实验评价其学习记忆能力。
转棒实验:连续四天测试对照组和造模组,以及给药对照组和给药造模组的转棒实验。记录每只小鼠的持续运动时间。
爬杆实验:测试对照组和造模组,评价给药对照组和给药造模组的四肢力量和平衡能力。
旷场实验:测试对照组和造模组,评价给药对照组和给药造模组的学习记忆能力。
数据处理和统计分析:数据以均值±标准差(mean±sem)表示,采用t检验对数据进行统计学分析。
3、实验结果
结果如图1B-1G所示。
小鼠转棒实验的结果如图1B和表1所示,其中,GFP+对照组和GFP+雷帕霉素组无统计学差异。然而,GFP-GA150+对照组和GFP-GA150+雷帕霉素组从第3天和第4天起存在统计学的显著差异。施用雷帕霉素可以显著提高小鼠的掉落潜伏期(latency to fall)。
表1小鼠转棒实验的掉落潜伏期(s)
*给予雷帕霉素后,导致的掉落潜伏期增减幅度。
旷场实验的结果如图1的C、D和E所示,与表达GFP+Control,GFP+Rapamycin或GFP-GA150+Rapamycin的小鼠相比,GFP-GA150小鼠后肢站立的时间更少,表明GFP-GA150小鼠运动协调能力的缺陷,雷帕霉素治疗后,能明显缓解这一缺陷(图1C);GFP-GA150小鼠在中央区域的时间与距离也显著降低,表明了其探索能力下降产生焦虑行为。同样,雷帕霉素治疗后,GFP-GA150小鼠探索能力得到明显改善(图1D,E)。
尤其是图1E的结果表明,小鼠在中央区域的时间表明了其探索能力,雷帕霉素治疗后,GFP-GA150小鼠探索能力得到明显加强,小鼠ALS模型组给予雷帕霉素后,中央区的停留时间高于正常对照(即GFP+对照组),这表明雷帕霉素治疗GFP-GA150小鼠后其探索能力增强,焦虑行为降低。
爬杆试验的结果图1的F和G所示,爬杆实验用于检测小鼠运动协调能力,小鼠完全转身向下并面向地板的时间记为潜伏期(T-turn),小鼠爬下杆子四肢到达杆底的时间记作下杆时间(T-LA)。GFP-GA150小鼠出现明显的运动协调能力缺陷,它表现出更长时间的T-turn及T-LA,然而雷帕霉素治疗后,GFP-GA150小鼠的T-turn及T-LA时间显著减少(图1F,G)。
上述结果表明,C57BL/6J小鼠ALS模型组经造模后,与对照组相比其行为学出现显著退化,主要表现为运动能力出现显著下降,同时其平衡能力和学习记忆能力也出现显著降低。
当给予雷帕霉素后,可以显著改善模型小鼠的运动,平衡和学习记忆能力,最终缓解致病蛋白的神经退行性病变。
实施例2:雷帕霉素对ALS模型小鼠的脑内细胞的影响
本实施例采用组织免疫荧光以及组织病理学切片的实验技术,观察雷帕霉素对ALS模型小鼠脑内细胞的细胞影响,并考察雷帕霉素对模型小鼠尤其是脑内神经细胞的损伤以及其他影响,比如对于神经免疫炎症的影响。
1、实验原理:
针对雷帕霉素很好的缓解了小鼠神经行为学退变,可以推测是雷帕霉素通过缓解致病蛋白所诱导的细胞损伤发挥保护作用。因此通过TUNEL染色来考察雷帕霉素对细胞毒性的缓解作用,同时通过qPCR和免疫荧光的方法来考察其对小胶质细胞和星形胶质细胞的影响来判断其对脑内神经炎症的影响。
2、实验材料与方法
(1)样品处理:在实施例1的行为学评测完成后,即用PBS灌流后取小鼠脑组织,一半脑置于液氮用来做生化实验如qPCR,另一半脑先置入蔗糖梯度脱水,然后PFA固定后用OCT(optimal cutting temperature compound,是一种市售或常规的聚乙二醇和聚乙烯醇的水溶性混合物)包埋,最后制成20um的冰冻切片。
(2)TUNEL染色:按照说明书配置成1:的稀释液,加到冰冻脑切片,孵育x小时后,PBS清洗,加入抗淬灭剂盖玻片封片,于显微镜观察并拍照。
(3)qPCR检测小鼠脑内神经炎症:精密称取小鼠脑匀浆10mg,并加入1ml Trizol,匀浆机匀浆;4℃,12000rpm离心5min,取上清移入新的1.5ml离心管中;加入200μL氯仿,颠倒混匀2min,静置5min,4℃,12000rpm离心10min;取400μL上层水相至干净的离心管中,加入200μL无水乙醇,混匀后,静置10min;将RNA吸附柱放入收集管中,将上层水相和无水乙醇混合液移入RNA吸附柱中,静置5min,4℃,10000rpm离心5min,倒去收集管中的废液;配制RPE混合液(RPE:无水乙醇=1:4),向吸附柱中加入500μL RPE混合液,静置2min,4℃,10000rpm离心30s,倒去废液;将吸附柱放入干净的1.5mL离心管中,向吸附柱中加入50μLDEPC水溶解RNA,静置5min,4℃,12000rpm离心2min,提取到的RNA溶液置于-80℃保存。
(2)实时定量荧光PCR:A、利用酶标仪检测抽提的RNA浓度;B、将RNA反转录为cDNA,反转录体系(20μL),见下表。反转录过程:①37℃,15min;②85℃,5s;③保持10℃。
表2反转录体系
C、定量PCR:反应体系(每孔)见下表。
表3定量PCR反应体系
定量PCR过程:①50℃,2min;②95℃,20s;③95℃,5s;④60℃,30s;③-④循环40次。
表4引物序列表
基因 | 引物序列(5'-3') | SEQ ID No: |
mGAPDH正向引物 | CATGGCCTTCCGTGTTCCTA | 1 |
mGAPDH反向引物 | CCTGCTTCACCACCTTCTTGAT | 2 |
mIba1正向引物 | GGATTTGCAGGGAGGAAAAG | 3 |
mIba1反向引物 | TGGGATCATCGAGGAATTG | 4 |
GFAP正向引物 | GGAGAGGGACAACTTTGCAC | 5 |
GFAP反向引物 | AGCCTCAGGTTGGTTTCATC | 6 |
(4)免疫荧光检测小鼠脑内神经炎症:
冰冻切片用0.5%tritonX-100,处理30分钟后,加入含1%山羊血清的PBST封闭1小时,用漂洗液漂洗(0.5%tritonX-100的PBS)10分钟,并重复漂洗2次,加入一抗(小胶质细胞标识蛋白Iba1抗体,星形胶质细胞标识蛋白GFAP抗体)4℃孵育过夜,用漂洗液漂洗(0.5%tritonX-100的PBS)10分钟并重复2次,加入配好的荧光二抗(ALEXA-594),室温孵育2小时,用漂洗液漂洗(0.5%tritonX-100的PBS)10分钟并重复2次,DAPI染细胞核5分钟后,PBS漂洗5分钟,再封片。于激光共聚焦显微镜下观察。
(5)数据处理和统计分析:数据以均值表示,采用t检验对数据进行统计学分析。对于神经免疫炎症统计共9张切片的荧光强度。
D、数据处理:采用2^-△△Ct法。①△Ct=Ct(目的基因)-Ct(内参基因);②-△△Ct=-(△Ct(给药组)-△Ct(溶剂组));③对-△△Ct进行2的幂运算,即2^-△△Ct就得出给药组与溶剂组相之间的倍数关系。数据以均值±标准差(mean±sem)表示,采用t检验对数据进行统计学分析。
3、实验结果
结果如图2所示。
神经炎症通常与星形胶质细胞和小胶质细胞的激活有关。图2A显示,对于ALS模型小鼠,在给予雷帕霉素后,小鼠脑中GFAP mRNA丰度明显降低。这表明,ALS模型小鼠星形胶质细胞激活被雷帕霉素抑制。
图2B显示,对于ALS模型小鼠,在给予雷帕霉素后,小鼠脑中Iba1 mRNA丰度也降低,这提示ALS模型小鼠小胶质细胞的激活被雷帕霉素抑制所抑制。
图2C显示,对于ALS模型小鼠,在给予雷帕霉素后,小鼠脑切片的免疫荧光检测出的GFAP阳性细胞降低。这表明ALS模型小鼠激活的星形胶质细胞被雷帕霉素抑制。
图2D显示,对于ALS模型小鼠,在给予雷帕霉素后,小鼠脑切片免疫荧光观察Iba1阳性细胞数目降低,这表明ALS模型小鼠激活的小胶质细胞被雷帕霉素抑制。
图2E显示,对于ALS模型小鼠,在给予雷帕霉素后,Tunel染色检测小鼠脑中细胞死亡显著减少。
上述结果表明,对于ALS模型小鼠,给予雷帕霉素后,能够显著降低模型小鼠脑内Iba1和GFAP mRNA水平和蛋白水平,表明雷帕霉素有效地减少了poly-GA聚集诱导的神经炎症。Tunel染色结果显示雷帕霉素能够显著降低模型小鼠脑内不同脑区的细胞损伤。
实施例3:组织水平测试雷帕霉素对模型小鼠脑内蛋白聚集的影响
在本实施例中,采用激光共聚焦荧光显微镜观察小鼠不同脑区蛋白聚集的变化,通过显微镜拍照并统计不同小鼠脑区的荧光强度,从而分析不同脑区的蛋白聚集情况。
GFP-(GA)150为GFP和(GA)150形成的融合蛋白,用于模拟poly-GA。由于该融合蛋白含有GFP元件,因此可在激发条件下发出荧光。
1、实验材料与方法:
(1)脑组织中冰冻切片制备:小鼠PBS灌流后,取脑先置入蔗糖梯度脱水,然后PFA固定后用OCT包埋,最后制成20um厚度的冰冻切片。
(2)DAPI稀释液孵育脑切片5分钟后,漂洗液洗三次,然后置于载玻片,脱水后,加入封闭液封闭,加上盖拨片,激光共聚焦显微镜观察并拍照。
(3)数据处理:采用t检验对数据进行统计学分析。对于神经免疫炎症则统计共9张切片的荧光强度。
2、实验结果
结果如图3和表5所示。
表5多聚蛋白聚集(归一化的GFP荧光信号强度)
运动皮层 | 海马体 | 小脑 | |
GFP-GA150+对照组 | 1 | 1 | 1 |
GFP-GA150+雷帕霉素组 | 0.09 | 0.12 | 0.19 |
对于ALS模型小鼠,雷帕霉素给药组中,运动皮层、海马体、小脑等不同脑内区域的多聚蛋白聚集明显减少。这表明,雷帕霉素可以有效降低ALS模型小鼠脑区中poly-GA等疾病相关蛋白的聚集,从而有效治疗或缓解ALS和FTD。
讨论
ALS和FTD是一类系统性疾病,目前尚无有效治疗药物和方法。
自从C9orf72基因突变被发现与ALS/FTD有关后,其致病机理迅速成为了研究的热点,无论是家族性ALS/FTD,还是散发性ALS/FTD,其所占比例均远远超过其他基因突变:在家族性ALS/FTD,C9orf72基因突变比例可达40%,在散发性ALS/FTD也高达6%。
针对C9orf72基因突变致病机制,目前研究最多最受关注的致病假说即非ATG依赖翻译的多聚重复蛋白毒性假说(Repeat-associated non-ATG,RAN)。研究发现,位于内含子区域的GGGGCC重复序列及其反义链CCCCGG即使在无ATG起始密码子的情况下依然能够编码出五种多聚重复蛋白:poly-GA,poly-GP,poly-GR,poly-PA和poly-PR。
一系列的临床研究表明,携带C9orf72突变体内poly-PR和poly-GR丰度远远低于poly-GA,而随着重复数目的增加poly-GA的毒性也随之增加,同时研究进一步表明在携带C9orf72突变的临床病人组织中poly-GA才是丰度最高的。Poly-GA在C9orf72突变的病人中高表达,这表明其极有可能是诱发发病的重要因素。因此,降低病人体内的poly-GA很可能是一种潜在的治疗方法。
虽然,用poly-GA抗体处理可明显抑制poly-GA聚集并显著抑制病人脑组织提取物中的蛋白聚集。然而,抗体类药物不易通过血脑屏障,因此在临床应用的难度较大。
本发明的研究表明,高度重复poly-GA的ALS小鼠表现出显著的行为学缺陷和运动障碍以及脑内神经炎症即细胞死亡。通过雷帕霉素治疗,能够显著的消除脑内神经炎症并改善神经细胞死亡。雷帕霉素可有效进入脑部,促进脑部细胞内的蛋白聚集降解,降低ALS小鼠脑内poly-GA聚集,继而发挥治疗效应,本发明为开发治疗ALS提供了有效的充分的实验证据。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
1.一种雷帕霉素或其药学上可接受的盐或酯的用途,其特征在于,用于制备一药物组合物或制剂,所述药物组合物或制剂用于:
(a)治疗或预防渐冻人症、额颞叶痴呆或组合;
(b)清除促进脑部poly-GA蛋白的聚集;和/或
(c)抑制小胶质细胞和/或星形胶质细胞的激活。
2.如权利要求1所述的用途,其特征在于,所述的渐冻人症为C9orf72突变型渐冻人症。
3.如权利要求1所述的用途,其特征在于,所述的额颞叶痴呆为C9orf72突变型额颞叶痴呆。
4.如权利要求1所述的用途,其特征在于,所述的药物用于清除促进脑部poly-GA蛋白的聚集。
5.如权利要求4所述的用途,其特征在于,所述的药物用于清除促进脑部选自下组区域的poly-GA蛋白的聚集:运动皮层、海马体、小脑、或其组合。
6.如权利要求1所述的用途,其特征在于,所述药物用于抑制小胶质细胞和/或星形胶质细胞的激活。
7.如权利要求1所述的用途,其特征在于,所述药物为含有以下成分的药物组合物:(a)第一活性成分,所述第一活性成分为雷帕霉素;和(b)药学上可接受的载体。
8.一种试剂盒,其特征在于,所述的试剂盒包括:
(U1)检测试剂,所述的检测试剂用于检测C9orf72突变;和
(U2)第一活性成分,所述的第一活性成分选自下组:雷帕霉素、或其药学上可接受的盐或酯。
9.一种体外非治疗性地减少神经细胞中poly-GA蛋白聚集的方法,其特征在于,包括步骤:
在雷帕霉素存在下,培养神经细胞,从而减少所述神经细胞中poly-GA蛋白聚集。
10.如权利要求9所述的方法,其特征在于,所述的神经细胞是具有C9orf72突变型的神经细胞。
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