CN1265588A - 中枢神经细胞的保护及存活促进剂 - Google Patents
中枢神经细胞的保护及存活促进剂 Download PDFInfo
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- CN1265588A CN1265588A CN98807701A CN98807701A CN1265588A CN 1265588 A CN1265588 A CN 1265588A CN 98807701 A CN98807701 A CN 98807701A CN 98807701 A CN98807701 A CN 98807701A CN 1265588 A CN1265588 A CN 1265588A
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Abstract
含有选自L-丝氨酸、甘氨酸以及其脂肪酸衍生物的物质,并以该物质为有效成分的中枢神经细胞存活促进剂;以及含有该物质并以该物质为有效成分的脑功能低下预防和/或治疗剂。具有保护脑细胞、抑制细胞死亡,同时延长细胞寿命的作用,例如可以抑制由脑出血、脑梗塞及头部外伤等伴有的脑水肿和脑内温度上升所引起的脑细胞死亡。
Description
技术领域
本发明涉及中枢神经细胞的保护及存活促进剂。
背景技术
海马神经元作为可以在实验室中培养的中枢神经细胞,在神经生理学的领域内被广泛应用,但众所周知的是,如果将这种细胞单独进行原代培养的话,培养开始一周后会出现大量神经元死亡。长期培养海马神经元的方法,已知的有和神经胶质细胞(神经元与其突起之间的神经胶质细胞)共存培养的方法,但是,由于这种培养系不是单一的培养系,所以并不适宜用作研究单纯海马神经元的营养要求条件以及神经元对蛋白性因子的应答。没有神经胶质细胞共存的条件下培养海马神经元的方法,已知的有在全部非必需氨基酸存在的条件下进行培养的方法,但是细胞的存活时间和存活数目与神经胶质细胞共存条件下进行的培养相比显著降低。
已知在海马神经元的原代培养系中,加入神经胶质细胞的培养上清(ACM,星形胶质细胞条件培养基)后,可以使神经元长期存活,Goslin等确立了这种培养方法(Goslin,K.and Banker,G.,“Culturing NerveCells,”Ed.By Bander,G.et al.,p.251-278,The MTP Press,England)。但是在这种培养上清液中,究竟是什么物质促进了神经元的存活尚未完全明了。
另一方面,已知L-丝氨酸在末稍神经细胞鸡胚神经节的形态分化中作为重要因子发挥作用(Savoca,r.,Ziegler,U.and Sonderegger,P.,Journal of Neuroscience Methods,61,pp.159-167,1995)。但是此文献中所公布的L-丝氨酸的作用只与神经元的形态形成相关,L-丝氨酸对神经元的存活是否具有一定的作用,对此完全没有揭示或作出提示。而且,在这个实验中所使用的细胞是末稍神经细胞,与海马神经元等中枢神经细胞在发生和机能方面存在显著差异,因此从此文献中无法明了L-丝氨酸对中枢神细胞的作用。
发明描述
本发明的目的,在于提供促进神经元存活的物质。本发明的另一目的,在于提供脑功能改善剂。
本发明者们为解决上述问题进行了深入细致地研究,结果发现神经胶质细胞分泌一种对海马神经元起到细胞存活促进作用的物质,这种物质海马神经元并不能产生,并且发现这种物质就是L-丝氨酸。此外,本发明人发现L-丝氨酸以及甘氨酸不仅对海马神经元,而且对小脑粒细胞和蒲肯野细胞等中枢神经细胞也具有细胞存活促进作用。本发明是在上述发现的基础上完成的。
也就是说,本发明提供含有选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质,优选的是L-丝氨酸和/或甘氨酸、更优选L-丝氨酸,并以该物质为有效成分的中枢神经细胞的细胞存活促进剂。根据本发明的优选方式,可以提供含有L-丝氨酸并以其为有效成分的中枢神经细胞的细胞存活促进剂。
从另一角度来看,依据本发明可以提供含有选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质,优选的是L-丝氨酸和/或甘氨酸、更优选L-丝氨酸,并以该物质为有效成分的脑功能低下的预防和/或治疗剂。此外还可以提供在上述预防和/或治疗剂制备中,选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质,优选的是L-丝氨酸和/或甘氨酸、更优选L-丝氨酸的应用,以及脑功能低下的预防和/或治疗方法。包括向患者提供所需的,选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质,优选的是L-丝氨酸和/或甘氨酸、更优选L-丝氨酸的有效预防和/或治疗量的方法。
从另一角度来看,本发明可以提供含有中枢神经细胞的细胞存活促进剂L-丝氨酸或甘氨酸,用于中枢神经细胞培养的培养基组合物。
附图简述
图1表示的是海马神经胶质细胞和神经元的培养上清液中的氨基酸浓度。
图2表示的是非必需氨基酸浓度对海马神经元存活的影响。
图3表示的是神经胶质细胞培养上清液中非必需氨基酸浓度随时间的变化。
图4表示的是在小脑蒲肯野细胞的原代培养中丝氨酸的效果。空心柱和实心柱分别表示在不含有丝氨酸及含有200μM丝氨酸的条件下的结果。缩写如下:Ser:L-丝氨酸;TNF:肿瘤坏死因子α;BDNF:脑源性神经营养因子;NT-3:神经营养蛋白-3;NGF:神经生长因子;GDNF:胶质细胞源性神经营养因子。
实施本发明的最佳方式
本发明中的细胞存活促进剂的特征是含有选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质,优选的是L-丝氨酸和/或甘氨酸、更优选是L-丝氨酸,并以该物质为有效成分。也可以使用L-丝氨酸和甘氨酸的酸加成盐和碱加成盐。L-丝氨酸或甘氨酸的脂肪酸衍生物并没有特殊的限定,比如可以适当选用十四烷基化的L-丝氨酸和甘氨酸。本发明的细胞存活促进剂具有抑制海马神经元、小脑粒细胞以及蒲肯野细胞等中枢细胞的细胞死亡,保护这些细胞,同时可使它们长期存活的作用。本发明中细胞存活促进剂的适用对象并无特殊限定,只要是中枢神经细胞均可适用。
本发明中的细胞存活促进剂可以添加到培养基中,用于中枢神经细胞的培养。在这种培养系中不需与神经胶质细胞共存,可以将单一系的中枢神经细胞长期培养。本发明所提供的中枢神经细胞培养用培养基组成物可举例如下:比如在伊格尔(Eagle’s)MEM培养基(25mM HEPES,含有最终浓度为30nM的***钠,500μM的丙酮酸钠,3.9mM的谷氨酸,16.7mM的葡萄糖,100μM的腐胺,10μg/ml的硫酸庆大霉素,0.1mg/ml的牛血清白蛋白)中,添加10-200μM,最好是50-100μM L-丝氨酸或甘氨酸(优选的是L-丝氨酸)制成的培养基组合物。但是,本发明中的培养基组合物,并不局限于以上举例,只要是在本领域技术人员可以利用的适宜培养基中加入上述浓度的细胞存活促进剂L-丝氨酸或甘氨酸(优选的是L-丝氨酸),所制成的培养基组合物不必说均属本发明的范畴。
依据本发明所提供的细胞存活促进剂,可以用作脑功能低下的预防和/或治疗剂的有效成分。本发明的药物,具有保护脑细胞、抑制细胞死亡,同时延长细胞寿命的作用。因此本发明的药物可以防止脑功能的低下和/或改善脑细胞活性降低所引起的脑功能低下。本发明的药物的适用对象并无特殊限定,可以用于预防和/或治疗伴有脑功能低下的各种疾病。比如可以抑制由脑出血、脑梗塞以及头部外伤等伴有的脑水肿和脑内温度上升所引起的脑细胞死亡。此外,还可以抑制伴随老化而出现的脑细胞数目减少,预防老年性痴呆的发病。还可用于预防和/或治疗阿尔茨海默病、帕金森氏病及亨庭顿舞蹈病等脑细胞变性引起的疾病。
作为本发明的药物,可以从L-丝氨酸、甘氨酸以及其脂肪酸衍生物中选取1种或者2两种以上的物质。作为本发明的药物,也可以使用上述物质在生理学上所允许的盐类,以及游离状态的物质或在生理学上所允许的盐类物质的水合物或者是溶剂合物。
作为本发明的药物,也可以单纯使用上述物质,但通常最好利用本领域技术人员可以应用的制剂用添加剂,制成含有上述物质,并以该物质为有效成分的药物组合物。作为药理学和制剂学上允许使用的制剂添加剂,可以使用的有赋形剂、崩解剂以及崩解辅助剂、结合剂、润滑剂、包衣剂、色素、稀释剂、基质、溶解剂以溶解辅助剂、等渗剂、pH调节剂、稳定剂、喷雾剂以及粘合剂。作为适用于口服的制剂,可以举例的有片剂、胶囊、细粒剂、颗粒剂、液体制剂或糖浆剂。此外作为非口服制剂,可以举例的有注射剂、输液剂、栓剂、吸入剂、经粘膜吸收剂、经皮吸收剂、滴鼻剂、滴耳剂、贴剂。
作为药理学和制剂学上所允许的,适用于口服或经皮或经粘膜给药制剂中的制剂添加剂,可以使用葡萄糖等赋形剂;羧甲基纤维素等崩解剂以及崩解辅助剂;羟甲基纤维素等结合剂;硬脂酸镁等润滑剂;羟丙基纤维素等包衣剂;凡士林等基质。此外制剂用添加剂也可以使用压缩气体等喷雾剂;聚丙烯酸钠等粘合剂;木棉等敷料。作为适用于注射和点滴给药制剂的添加剂,可以使用注射用蒸馏水等水性溶剂;组成即时溶解型注射剂的溶解剂以及溶解辅助剂;葡萄糖等渗剂;无机酸、有机酸、无机碱以及有机碱等pH稠节剂。
本发明中药物的给予量,可以根据所针对疾病的种类、患者的症状以及年龄、预防和治疗的目的等不同的条件进行适当增减,用量由本领域技术人员参考以上因素进行适宜选择。本发明人确认使用10-200μM最好是50-100μM的L-丝氨酸和甘氨酸(优选是L-丝氨酸)时,最适合于中枢神经细胞的存活,由此推断生物体内正常生理条件下可以从神经胶质细胞中得到这个浓度的L-丝氨酸。因此,为了抑制脑细胞的细胞死亡,最好适当调节用药量,使脑细胞与上述浓度的L-丝氨酸和甘氨酸(优选是L-丝氨酸)接触。此外,也可以把本发明中的脑功能低下的预防和/或治疗剂作为食品添加剂使用,也可以用作健康食品和饮料的成分。
实施例
以下举例对本发明进行具体说明,但本发明的范围并不局限于以下所举的实施例。
(1)材料及方法
(a)材料
由日本SLC公司购入确定妊娠18天的Wistar/ST大鼠。伊格尔MEM培养基、胎牛血清由Gibco公司购入,培养板由住友电木公司和BectonDickinson公司购入。所有的氨基酸都使用L-氨基酸。
(b)大鼠海马神经元和神经胶质细胞的原代培养
大鼠海马神经元的原代培养采用已经报道的方法(Enokido,Y.,andHatanaka,H.,Neuroscience,57,pp.965-972,1993)。培养基使用伊格尔MEM培养息25mM HEPES,含有最终浓度为30nM的***钠,500μM的丙酮酸钠,3.9mM的谷氨酸,16.7mM的葡萄糖,100μM的腐胺,10μg/ml的硫酸庆大霉素,0.1mg/ml的牛血清蛋白)。此外培养板预先用聚乙烯亚胺包被。
进行细胞计数和细胞形态观察时,直径35mm的6孔培养板每孔,将2×105细胞混悬于200μl 10%灭活胎牛血清的培养基中进行平皿接种。进行脂质抽提时,每直径100mm的培养板,将2×106的细胞混悬于2ml培养基中进行平皿接种。在5%CO2培养箱中保温2小时后,转移到加入非必氨基酸等添加物的1ml(35mm板)或6ml(100mm板)的无血清培养基(以10μg/ml胰岛素,100μg/ml转铁蛋白,20nM***代替灭活胎牛血清)中培养过夜。第二天,为防止神经胶质细胞的增殖,转移到含有1μM阿糖胞苷的无血清培养基中继续培养。
用上述相同的方法将神经胶质细胞分散混悬到含有10%灭活胎牛血清的伊格尔MEM培养基中,接种到未包衣的板上,培养至汇合。然后用胰蛋白酶-EDTA消化细胞1-2次,用同一培养基传代培养。再次汇合时,转移到上述无血清培养基中。
(c)小脑神经元的原代培养
小脑粒细胞和蒲肯野细胞的原代培养分别采用Kubo等的方法(Kubo,T.,Nonomura,T.,Enokido,Y.,and Hatanaka,H.,Dev.BrainRes.,85,pp.249-258,1995)和Furuya等的方法(Furuya,S.,Ono,K.,and Hirabayashi,Y.,J.Nurochem.,65,pp.1551-1561,1995)。
(d)免疫染色
采用抗MAP2(微管相关蛋白2)单克隆抗体(Boehringer Mannheim公司)和抗钙结合蛋白单克隆抗体(Sigma公司)进行神经元染色,分别采用Enokido等的方法(Enokido,Y.,and Hatanaka,H.,Neuroscinece,57,pp.965-972,1993),和Furuya等的方法(Furuya,S.,Ono,K.,and Hirabayashi,Y.,J.Nurochem.,65,pp.1551-1561,1995)。
(e)脂质分析
回收培养细胞,用氯仿/甲醇(1/2体积比)抽提出总脂质,用Folch分配法(Floch,J.,Lees,M.,and Sloane-Stanley,G.H.,J.Biol.Chem.,226,pp.495-509,1957)分配成两层之后,用HPLC板(Silica Gel 60 HPLC板,Merck公司)进行薄层层析。作为展开溶剂,磷脂质(氯仿层)的展开使用氯仿/甲醇/甲酸/乙酸/1M氯化镁(60/30/6.5/4.5/0.1体积比),糖脂质(水层)的展开用氯仿/甲醇/12mM氯化镁(5/4/1体积比)。用樱草黄试剂(非特异性)、碘蒸气(非特异性)、茚三酮(氨基)、Ryu-MacCoss试剂(磷酸基)等检测条带(井上圭三,永井克孝,脊山洋右,新生化学实验讲义,4,pp.37-47,1991)。
(f)氨基酸分析和质量测定
培养上清中的氨基酸是以溶液经5%三氯乙酸,或5%高氯酸处理后离心的。上清液作为氨基酸分析样品。脂质中的氨基酸是以脂质经薄层层析展开后抽提出脂质,加入6N HCl进行水解后的物质作为氨基酸分析用样品。氨基酸分析采用日立S-8500A型氨基酸分析仪。经TLC方法进行的脂质纯化以及质量分析(SIMS分析)采用已经报道的方法(Taki,T.,Kasama,T.,Handa,S.,and Ishikawa,D.,Anal.Biochem.,223,pp.232-238,1994;Kasama,T.,Hisano,Y.,Naksjima,M,Handa,S.,and Taki,T.,Glycoconj.J.,13,pp.461-469,1996),此外,蛋白质定量以牛血清白蛋白为标准,采用Pierce公司的BCA蛋白定量试剂盒。
(2)结果
(a)伊格尔MEM(基本必需培养基)中不含有非必需氨基酸(丙氨酸、天冬氨酸、谷氨酸、甘氨酸、天冬酰胺、脯氨酸、丝氨酸)。在这个培养基中海马神经元不能长期培养,由此提示:在生命体内这些非必需氨基酸可能是由与神经元共存的细胞,也就是神经胶质细胞所提供的。因此,首先研究了从神经元和神经胶质细胞中释放出了何种氨基酸。将大鼠海马神经元和神经胶质细胞分别用伊格尔MEM进行培养,一周后回收培养上清液,测定酸可溶性部分的非必需氨基酸浓度。结果可见:神经胶质细胞的培养上清液(星形胶质细胞条件培养基,ACM)中,丝氨酸、甘氨酸和天冬氨酸与其他氨基酸相比处于高浓度(图1)。
将海马神经元分别在不同非必需氨酸存在和不存在的条件下培养6天后,用抗MAP2抗体进行细胞染色,观察神经元的形态时,发现加入丝氨酸和甘氨酸的情况下可见到树突的延长以及存活率的增高,天冬氨酸、天冬酰胺、脯氨酸、丙氨酸、谷氨酸均未发现存活促进活性。为了研究非必需氨基酸浓度对海马神经元存活的影响,将海马神经元在不同非必需氨基酸存在的条件下培养6天,用抗MAP2抗体进行细胞染色后计数细胞数。结果如图2所示。图中表示的是3次实验结果的均值和±标准差。结果显示丝氨酸、甘氨酸大约在50-100μM时存活促进活性最高。
为研究神经胶质细胞的培养上清液中非必需氨基酸浓度的随时间的变化,经时收取神经胶质细胞的培养上清,测定酸可溶性部分的非必需氨基酸浓度,结果如图3所示。图中表示的是3次实验结果的均值±标准差。结果可见:与其他氨基酸相比丝氨酸最早释放到细胞外,培养两天后大约在70μM达到平衡。这一浓度正是能够维持神经元存活的浓度,因此认为这一现象也具有生理学上意义。另一方面,甘氨酸没有释放出维持神经元存活所必需的量。由以上结果可见:丝氨酸对于海马神经元的存活是必需的,由神经胶质细胞释放出必需的量。
(b)从以上结果可以推测,丝氨酸传递着促进神经元存活的信号,或者是神经元不能合成丝氨酸,因而造成细胞死亡。前一推测因丝氨酸添加前后蛋白质磷酸化未发生变化,故予以否定。为验证神经元由于不能合成丝氨酸从而造成细胞死亡的可能性,对神经元细胞膜中存在的含有丝氨酸的脂质进行研究。
将海马神经元在100μM丝氨酸或甘氨酸存在及不存在的条件下,分别培养6天后,抽提出脂质进行Folch分配。用TLC将氯仿层和水层展开后,分别用茚三酮和樱草黄试剂进行显色反应,检出脂质。结果可见:在氯仿层的脂质成分中,丝氨酸非存在条件下培养的细胞中磷脂酰丝氨酸的含量很少,与之相比生成了含量稍大的新脂质(X-3)。此外,在水层中丝氨酸存在条件下所见的GT1b神经节苷脂和未鉴定脂质,在丝氨酸非存在的条件下消失。
因此,对丝氨酸非存在条件下出现的脂质X-3进行结构分析。这种脂质对Ryu-MacCoss反应和茚三酮反应均呈阳性,由此提示该脂质是含有氨基的磷脂质。此外,用碘蒸气检出TLC上的脂质(X-3)带,进行纯化水解后,以TLC板上没有碘染的部分作为对照,进行氨基酸分析时,确认有苏氨酸的存在。此外,用TLC层析法进行脂质(X-3)纯化后,经SIMS分析的结果是,这种脂质作为结合有2个钠离子的脂肪酸长度不同的两个峰(846,18∶1/18∶0、870,20∶3/18∶0)被检出,确认它为磷脂酰苏氨酸。
以上的结果提示,在不供给丝氨酸的情况下,苏氨酸被作为替代物,用以合成磷脂酰苏氨酸。因此,神经元本身不具备合成丝氨酸的能力,在磷脂酰丝氨酸的合成中所必需的丝氨酸,极大地依赖于神经胶质细胞的供给。磷脂酰丝氨酸是神经元存活所必需的蛋白激酶C活性化中的必需脂质(Kaibuchi,K.,Takai,Y.,and Nishizuka,Y.,J.Biol.Chem.,256,pp.7146-7149,1981),这种脂质的缺乏成为威胁细胞存活的重要原因之一。此外,以往尚无关于磷脂酰苏氨酸存在于神经中的报道。
丝氨酸一般是从葡萄糖降解体系的代谢中间产物3-磷酸丝氨酸,经过3个阶段的酶促反应生成,即由3-磷酸丝氨酸→(磷酸丝氨酸脱氢酶)→3-磷酸羟基丙酮酸→(磷酸丝氨酸转氨酶)→磷酸丝氨酸→(磷酸丝氨酸磷脂酶)→丝氨酸。上述结果可以证明,神经胶质细胞将这个合成途径中合成的丝氨酸快速分泌到细胞外,推测海马神经元由于某种原因丝氨酸的合成非常少,依赖于神经胶质细胞的供给。
此外,甘氨酸也可抑制神经元细胞的死亡,这可以用甘氨酸摄入细胞内后,利用丝氨酸羟甲基转移酶很容易合成丝氨酸来说明。当然丝氨酸也可以作为丝氨酸羟甲基转移酶催化的甘氨酸从头合成的前体加以利用。由此丝氨酸的枯竭带来甘氨酸的枯竭。因此认为在这两种氨基酸不存在的情况下,蛋白质的合成自然会受到影响。
(c)丝氨酸存活促进作用不仅在海马神经元,在小脑粒细胞中也可见到。小脑粒细胞培养一周后转移到不含丝氨酸的无血清培养基中继续培养,在第7天几乎全部细胞出现死亡;但在转移到加入200μM丝氨酸的培养基中继续培养7天的情况下,可以完全抑制细胞死亡,并可维持细胞形态。同样在小脑蒲肯野细胞中,也可确认丝氨酸具有促进存活作用。由此可以推论中枢神经细胞一般不能合成足以维持存活的丝氨酸。
为了在小脑蒲肯野细胞的原代培养中确认丝氨酸的作用,将小脑的蒲肯野细胞如图4所示分别进行处理,培养6天(A)和12天(B)后,用抗钙结合蛋白抗体进行蒲肯野细胞染色,计数细胞数,结果如图4所示。图中空心柱表示丝氨酸非存在条件下的结果,实心柱表示含有200μM丝氨酸的培养基中的结果。可以证实丝氨酸具有增强肿瘤坏死因子-α(TNF-α)和脑源性神经营养因子(BDNF:brain-derived nerotroficfactor)等蛋白性营养因子活性的作用。
工业实用性
本发明的药物,具有保护脑细胞、抑制细胞死亡,同时延长细胞寿命的作用。因此本发明的药物可以防止脑功能低下和/或改善脑细胞存活性降低所引起的脑功能低下。
Claims (6)
1.中枢神经细胞存活促进剂,含有选自L-丝氨酸、甘氨酸以及其脂肪酸衍生物的物质为有效成分。
2.权利要求1中所述的中枢神经细胞存活促进剂,含有L-丝氨酸为有效成分。
3.脑机能低下的预防和/或治疗剂,含有选自L-丝氨酸、甘氨酸以及其脂肪酸衍生物的物质作为有效成分。
4.选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质在制备权利要求3的脑机能低下预防和/或治疗剂中的应用。
5.脑功能低下的预防和/或治疗方法,包括向患者提供有效量的选自L-丝氨酸、甘氨酸及其脂肪酸衍生物的物质。
6.用于中枢神经细胞培养的培养基组合物,含有细胞存活促进剂L-丝氨酸或甘氨酸。
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