CN101180068B - 萃取自石斛的多糖在制备用于治疗哺乳动物自体免疫葡萄膜炎的药物中的用途 - Google Patents
萃取自石斛的多糖在制备用于治疗哺乳动物自体免疫葡萄膜炎的药物中的用途 Download PDFInfo
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Abstract
一种包含由石斛属植物制备而得的多糖的组合物。所述组合物通过诱导口服耐受性与促进先天性免疫力以对自体免疫疾病与黏膜疾病产生作用。
Description
技术领域
本发明是关于对自体免疫疾病与黏膜疾病有效用的组合物、制备方法及其用途,更特别的是所述组合物包含得自于石斛的多糖,且所述组合物的使用方法包含服用取自于石斛的多糖,透过诱导口服耐受性(oral tolerance)与促进先天性免疫力(innate immunity)以对自体免疫疾病与黏膜疾病产生作用。
背景技术
免疫***的基本性质是免疫耐受性,可分辨自身与非自身分子,因而免疫***可以保护宿主不受外来病原的侵袭且不对自身攻击。当自身受到免疫***的攻击时,即形成自体免疫疾病。(Annu.Rev.Med.48:341-351,1997)
目前已证实口服耐受性可应用于治疗自体免疫疾病,包含葡萄膜炎(uveitis)、胶原蛋白所引起的关节炎(collagen-induced arthritis)、佐剂性关节炎(adjuvant arthritis)、***性红斑性狼疮(Systemic Lupus Erythematosus,SLE)、多发性硬化症(multiple sclerosis)、甲状腺炎、重症肌无力、发炎性肠道疾病(inflammatory bowel disease,IBD)以及第一型糖尿病。(Annals of the New YorkAcademy of Sciences 778(1):217-227,1996)
口服抗原经由食道到了肠相关淋巴组织(gut-associated lymphoid tissue,GALT),所述肠相关淋巴组织是一种非常完整的免疫网状***。GALT包含绒毛(villi)、固有层(lamina propria)、上皮细胞间淋巴球(intraepitheliallymphocytes)与皮尔氏斑(Peyer’s Patch),其中皮尔氏斑是指散布于绒毛上的淋巴小结。肠道的上皮细胞包含M细胞,负责传送外来的抗原与微生物。M细胞是负责将病原传送至其下免疫细胞,抗原与病原仅能透过M细胞而穿过肠道障蔽。而后,辨识到抗原的上皮间淋巴球会移至皮尔氏斑,以引发免疫反应。在皮尔氏斑,抗原呈现细胞将抗原呈现给淋巴球,而被抗原刺激过的淋巴球细胞经由***及血液循环,回到远离肠道的全身黏膜组织(如呼吸道等)固有层。
肠道免疫***中抵御经口服而感染疾病的机制,可分为先天性免疫(innateimmunity)和后天性免疫(adaptive immunity)。病原侵犯人体时,被感染的部位分泌化学激素(chemokine)促进巨噬细胞、单核球(monocytes)、颗粒球和自然杀手细胞进行非特异性的先天性免疫反应。巨噬细胞和颗粒球直接吞噬病原。自然杀手细胞是病毒感染和其他细胞内致病菌感染时最先反应的免疫细胞,自然杀手细胞不仅对标的细胞进行毒杀作用,而且分泌多种细胞激素调节免疫反应,如:IFN-γ、TGF-β、TNF-α、IL-5和IL-10等。巨噬细胞及其他抗原呈现细胞(antigenpresenting cell,APC),如树突状细胞(dendritic cells),利用细胞表面的第二类MHC分子及共刺激分子(co-stimulators),将抗原呈现给T细胞,以进一步活化免疫作用,也就是由T细胞与B细胞分别执行特异性的细胞免疫(cell-mediatedimmunity)和抗体免疫(humoral immunity)反应。黏膜免疫主要受CD4+T细胞分泌细胞激素的调控。第二型辅助T细胞(Th2)所分泌的IL-4、IL-5、IL-6及IL-10参与刺激B细胞产生抗体,第三型辅助T细胞(Th3)主要分泌TGF-β,引起B细胞产生黏膜中最主要的抗体IgA。(Trends Immunol.22:244-247,2004;J.Glin.Invest.106:935-937,2000)
值得注意的是肠道黏膜免疫反应不局限在肠道,而是引起全身黏膜***的免疫反应。一旦有抗原在小肠引起抗体产生,淋巴细胞经由淋巴液与血液直接作用于直肠、乳汁、肺、生殖道等处,而使得受到作用的组织也都侦测得到抗体,此现象称为共同黏膜免疫***(common mucosal immune system)。由于共同免疫***的存在,经由口服途径引起的抗体可遍布全身。经口服途径进入的抗原,若能诱导***耐受性,或使黏膜表现IgA,就称为口服耐受性(oral tolerance)。口服耐受性包含***反应(systemic unresponsiveness)和黏膜反应(mucosal responsiveness),前者避免身体发生自体免疫疾病,后者针对特定病原诱发主动的抑制。因此,黏膜免疫***倾向于抑制***内发炎的免疫反应,而促进黏膜局部非发炎性IgA的表现(Immunol.Today 18:335-343,1997)。
肠道上皮组织由单层上皮细胞所组成,形成肠道与外来物质(食物、细菌)的屏障,上皮细胞无法允许大分子或微生物通透,且在上皮细胞之间有非常紧密的连结(tight junction),用以阻止小分子进入。***中包埋的腺窝(crypts)含有干细胞(stem cells),这些干细胞可再生肠上皮细胞。除了再生肠上皮细胞之外,肠干细胞也可以分化为负责分泌肠道黏液(mucin)的杯状细胞(Gobelt cells)与肠内分泌细胞(enteroendocrine cells),以及分泌抗微生物肽(antimicrobialpeptides)。当这些细胞分化后,会往绒毛顶端移动,只有潘氏细胞(Paneth cells)停留在干细胞的区域(J.Clin.Invest.105:1493-1499,2000;Science294:2115-2116,2001)。
可合理的推测:由于肠道上皮细胞特殊的位置,因此对于肠腔中的各式抗原,它可能扮演一个影响免疫***走向的重要角色。肠道上皮细胞可以像抗原呈现细胞(APCs)一样表现抗原呈现分子(antigen-presenting molecules),以调节肠黏膜中的T细胞反应(Immunol.Today 21:123-128,2000)。研究发现肠道上皮细胞中的抗原呈现分子包括第一类MHC、第二类MHC、以及类似第一类MHC的CD1d(Gastroenterology 124:1420-1431,2003)。MHC class I负责与CD8+T细胞结合,MHC class II负责与CD4+T细胞结合,这两类的抗原呈现分子主要表现于上皮细胞的底侧边细胞膜(basolateral membrane)。CD1d可呈现糖脂质(glycolipid)而活化自然杀手T细胞。这些证据都显示了肠道上皮细胞在黏膜免疫***中,具有十分重要的地位。
肠道上皮细胞除了担任物理性的屏障(physical barrier),防止病源进入体内,还可以分泌对抗微生物的抗微生物肽(antimicrobial peptides),并且提供讯息给其他细胞(Immunol.Today21:123-128,2000;J.Clin.Invest.95:55-65,1995)。已知肠道中含有一种可对抗微生物感染的肽(antimicrobial peptides),称为防卫素(defensins)。防卫素(defensins)是分子量为3-5kD的蛋白质,可分为阿法-防卫素(α-defensin)家族与贝塔-防卫素(β-defesin)家族,共有八种肽。这些防卫素遍布于人体的上皮细胞,包含口腔、肺脏、肠道等,在不同器官有不同的表现型态(Eur.J.Gastroenterol.Hepatol.13:771-776,2001;Nat.Rev.Immunol.3:710-720,2003)。当肠道受感染时,上皮细胞会表现防卫素(defensins),而防卫素(defensins)具有类似化学激素(chemokine)的功能,吸引自然杀手细胞(NKcells)及树突状细胞(Dendritic cells)到达感染的区域,快速发挥防御作用,这样的作用可视为一种先天性免疫反应(Innate immunity)。防卫素(defensins)不只具有先天性免疫反应的特性,还可以藉由Toll样受体4(toll-like receptor 4)使树突状细胞(Dendritic cells)表现共刺激素(B7.2),促进T细胞增生。因此防卫素与先天性免疫反应及后天性免疫反应之间的连结有关。
另一种重要的抗微生物肽-血管生成素(angiogenins),原本被认为与癌细胞血管新生有关,但后来Hooper在2003年发现,在正常的生理状况下,血管生成素主要由肠道当中的潘氏细胞(Paneth cell)所分泌,平时存在细胞的颗粒(granule)中,可受到脂多糖類(lipopolysaccharide,LPS)的刺激而释放到肠道中。若以无菌鼠(germ-free mice)与带有一般肠内共生菌的老鼠相比,结果显示它受到肠内菌的影响而大量表现。血管生成素与防卫素一样具有增强细胞表面防御功能,及对抗微生物感染的效果(Nat.Immunol.4:269-273,2003)。
除了分泌抗微生物肽抵抗病原的感染之外,肠道上皮细胞也提供许多讯息来调节肠道免疫反应。肠道上皮细胞为了能抵抗微生物的侵犯,必须透过防御机制认出病原并做快速的反击。所述防御机制即为先天性免疫反应***,它是以型态识别接受器(Pattern-recognition receptor,PRR)来识别微生物,它可以识别大多数微生物共有且必要的大分子,如革兰氏阴性菌(Gram-negative)细胞壁的成份多糖脂(Lipopolysaccharide,LPS)。近年来已知型态识别受器如何启动(活化)细胞。已知在果蝇细胞中,当受到感染之后,Toll蛋白质诱导抗真菌与抗细菌的肽。Medzhitov及Janeways等人发现在哺乳动物中,Toll样受体(Toll-like receptor,TLR)会诱导活化后天免疫反应所需的细胞激素与共刺激分子(Cell 91:295-298,1997)。由于TLR分子的细胞内段与IL-1受器相似(Toll-IL-1R homology),因此细胞内讯号传导的路径被认为是类似的。当TLR与内毒素结合时,会使得NF-kB移转至细胞核(Nuclear translocation)对其他免疫调节基因进行转录控制(transcription regulation)。结果就是启动前发炎细胞激素(proimflammatorycytokine)基因的表现、抗微生物肽的分泌,以及如IL-8,MIP,MCP-1等化学激素(chemokines)的分泌。这些化学激素(chemokines)负责活化巨噬细胞及自然杀手细胞(NK cell),到达被感染的区域,消灭受到感染的细胞(Cell 91:295-298,1997;Gurr.Opin.Immunol.14:103-110,2002)。Toll样受体除了活化先天性免疫力之外,也因为启动了NF-kB,造成前发炎细胞激素的分泌,间接活化了后天性免疫力。
人类细胞中至少有十种已知的TLR,而在TLR家族中以TLR-2、TLR-4及TLR-9的研究最多。已知TLR参与辨识多种病原,包括细菌与病毒。TLR-4是革兰氏阴性菌LPS的受器,TLR-2则是革兰氏阳性菌肽聚糖(peptidoglycan)的受器,同时也是结核杆菌脂肽(lipopeptide)的受器,而TLR-9则是含非甲基化磷化CG寡核苷酸(Unmethylated & phosphorylated cytosine-guanine oligonucleotide,CpG)的受器。(Curr.Opin.Immunol.14:103-110,2002)TLR-3可辨识双股RNA病毒(Nature 433:887-892,2005),而TLR-7可辨识单股RNA病毒(Proc Natl Acad SciU S A.101(15):5598-5603,2004)。当活化TLR讯息传递路径时,则诱导先天性免疫力或是抗病毒反应以保护自身。
在肠道上皮细胞中,正常情况下,TLR-3与TLR-5会持续表现,而TLR-2与TLR-4的表现量极少。(Infection and Immunity 68:7010-7017,2000)在肠道上皮细胞中,细菌的CpG寡核苷酸是TLR-9的配体(ligand),当受到细菌DNA的刺激时,上皮细胞会产生IL-8(FASEB J.17:1319-1321,2003)。再者,肠道表皮细胞并不会对所有的外来物质而进行诱导发炎,亦即肠道上皮细胞对于共生的微生物具有耐受性,而在潜在的病原条件或是自体免疫疾病下,肠道上皮细胞提供危险信号至抗原呈现细胞,因而藉由抗原呈现细胞诱发T细胞所参与的发炎反应(Cell118:229-241,2004)。
在正常稳定的情况下,TLR所辨识的共生微生物对于保护肠道免于受到损害扮演相当重要的角色(Cell 118:229-241,2004)。然而,在人类发炎性肠道疾病(Inflammatory bowel disease,IBD)中,由Th1所引起的病理现象是由于对微生物的异常黏膜免疫反应(Gastroenterol.Clin.North Am.31:41-62,2002)。这些发现显示TLR调控宿主与共生微生物之间的黏膜平衡。
已知口服中草药萃取物可调节免疫力,极可能是因为中草药萃取物透过肠道黏膜免疫而调节免疫力的发展。石斛(Dendrobium)属于兰科植物,性甘味甜微咸,为一种非常珍贵的中药材。有些中药药典揭露石斛(Dendrobium)物种可有效治疗黏膜疾病、胃病与眼疾。根据我们之前的研究显示Herba Dendrobii对于免疫***疾病以及黏膜疾病具有治疗效果。
本发明是提供一种用于自石斛(Dendrobium)制备多糖的方法,且更提供由石斛(Dendrobium)所得多糖的医药使用。
发明内容
本发明的一目的是提供自植物制备多糖的方法。所述方法包含的步骤为a)以第一醇类萃取该植物,以获得一第一醇类萃取物以及剩余植材,b)以一溶剂萃取所述剩余植材,以获得一第二萃取物,以及c)以第二醇类沉淀所述第二萃取物,以获得所述多糖。
所述植物较佳是属于石斛(Dendrobium)属。
所述第一醇类较佳是甲醇。
所述溶剂较佳是水。
所述第二醇类较佳是乙醇。
根据本发明,在步骤b)中以所述溶剂萃取所述剩余植材后,所述方法更包含离心与过滤,以获得所述第二萃取物。
本发明的另一目的是提供一种用于治疗自体免疫疾病与黏膜疾病的组成物。所述组成物包含由一植物所制备的多糖,其中所述植物属于石斛(Dendrobium)属,以及与诱发所述自体免疫疾病相关的抗原。
所述多糖较佳是由本发明所提供的方法所制备而得。
根据本发明,所述自体免疫疾病是葡萄膜炎(uveitis)。
本发明的另一目的是提供一种治疗哺乳动物自体免疫疾病与黏膜疾病的方法。所述方法包含将一抗原与一多糖投予所述哺乳动物,其中所述抗原是关于所述自体免疫疾病的诱发,以及所述多糖是自石斛(Dendrobium)属的植物中制备而得。
根据本发明,以口服的方式投予所述多糖。
根据本发明,所述自体免疫疾病是葡萄膜炎(uveitis)。
本发明的另一目的是提供一种用于治疗哺乳动物自体免疫疾病与黏膜疾病的方法。所述方法包含将一多糖投予所述哺乳动物,其中所述多糖是自石斛(Dendrobium)属的植物中制备而得。
根据本发明,以口服的方式投予所述多糖。
根据本发明,所述自体免疫疾病是葡萄膜炎(uveitis)。
通过以下的详细说明与附随图式,可使本领域技术人员更清楚了解本发明的目的与优点。
附图概述
图1是说明IEC-6细胞以DeCaPS处理后,PCR分析细胞激素与抗微生物肽的mRNA表现。
图2是说明IEC-6细胞以DeCaPS处理后,PCR分析Toll样受体(Toll-likereceptor,TLR)的mRNA表现。
图3是说明C3H老鼠口服DeCaPS之后,以ConA刺激脾脏细胞行细胞***的效应。
图4是说明C3H老鼠口服DeCaPS之后,以GM-CSF共刺激骨髓细胞行细胞***的效应。
图5是说明C3H老鼠口服DeCaPS之后,PCR分析所取得的皮尔氏斑(Peyer’sPatch)与脾脏细胞中特定细胞激素的mRNA表现。
图6是说明C57BL/6j老鼠在有无口服DeCaPS下,口服白蛋白(ovalbumin)之后,分析肠液中针对白蛋白的IgA含量。
图7是说明C57BL/6j老鼠在有无口服DeCaPS下,口服白蛋白(ovalbumin)之后,分析血清中针对白蛋白的IgM含量。
图8是说明C57BL/6j老鼠在有无口服DeCaPS下,口服白蛋白(ovalbumin)之后,分析血清中针对白蛋白的IgG含量。
图9是说明以PCR分析C57BL/6j老鼠皮尔氏斑(Peyer’s Patch)中淋巴细胞的细胞激素表现。
图10是说明C57BL/6j老鼠的DTH反应。
图11A是一视网膜电流图,说明正常组老鼠闪动强度范围。
图11B是正常组老鼠的眼睛组织切片图。
图12A是一视网膜电流图,说明对照组老鼠闪动强度范围。
图12B是对照组老鼠的眼睛组织切片图。
图13A是一视网膜电流图,说明DC-10组老鼠闪动强度范围。
图13B是DC-10组老鼠的眼睛组织切片图。
图14A是一视网膜电流图,说明DC-40组老鼠闪动强度范围。
图14B是DC-40组老鼠的眼睛组织切片图。
图15A是一视网膜电流图,说明DC-160组老鼠闪动强度范围。
图15B是DC-160组老鼠的眼睛组织切片图。
图16A是说明图11A、12A、13A、14A与15A中a波的振幅。
图16B是说明图11B、12B、13B、14B与15B中b波的振幅。
本发明的最佳实施方案
请参阅以下实施例,以更进一步了解本发明。须注意的是以下本发明较佳实施例的说明仅作为阐示用途,而并不限制本发明的揭露形式。
本发明提供一种自植物制备多糖的方法,所包含的步骤为a)以第一醇类萃取该植物,以获得一第一萃取物以及剩余植材,b)以一溶剂萃取所述剩余植材,以获得一第二萃取物,以及c)以第二醇类沉淀所述第二萃取物,以获得所述多糖。
范例I:自石斛(Dendrobium)制备多糖
将10公斤的新鲜Herba Dendrobii干燥成为2.92公斤的干燥材料,而后以73.3公升的甲醇浸泡且萃取所述的干燥材料,而得到136.79公克的粗抽物以及剩余植材。将所述的剩余植材进行去甲醇制程,接着以水浸泡过夜。以55℃至60℃的热水萃取反应液30分钟,而后于10℃以离心机(ER-RC13C-124,HITACHI)转速5000转进行离心。离心后,以6微米的滤纸过滤上清液。以乙醇沉淀滤液,因而获得20.684克的多糖(而后称为DeCaPS)。
范例II:以DeCaPS处理的IEC-6细胞的免疫反应
a.培养IEC-6细胞
在37℃下,于含有5%CO2的培养箱中,将源自于正常老鼠小肠的IEC细胞培养于含有5%胎牛血清、4.5g/L葡萄糖、5μg/ml胎牛胰岛素与2mML-谷氨酰胺(Glutamine)的DMEM培养液中。
b.以DeCaPS处理IEC-6细胞
当6孔盘中长满IEC-6细胞时,分别以0μg/ml、1μg/ml、10μg/ml与100μg/ml浓度的DeCaPS处理IEC细胞6小时。
c.自以DeCaPS处理的IEC细胞分离总RNA
在以DeCaPS处理之后,收取IEC细胞并将其悬浮于1毫升的Ultraspec TMRNA分离试剂(美国Biotex laboratories公司),并以所述试剂中的标准程序取得总RNA并定量。
d.反转录并PCR分析细胞激素与Toll样受体
反应体积为26.5μl(微升),包含0.1微克的寡dT、5微克的所取得总RNA以及DEPC处理过的无菌水,于70℃反应10分钟。而后加入4微升的10mM dNTP、0.5微升的rRNAsin、1微升的AMV(禽类成髓细胞性白血病病毒(Avian myeloblastosisvirus,AMV)反转录酵素(10单位)以及8微升的5x RT缓冲液,使得总体积为40微升,并在42℃下反应60分钟,而后在90℃反应5分钟,藉此自反转录反应得到cDNA产物。接着,将0.5微升的10mM dNTP、0.5微升10x Prozyme缓冲液、引物(0.8微升的5μM sense DNA与0.8微升的5μM antisense DNA)以及无菌水加入2.5微升的所述cDNA产物,形成总体积25微升,并放置于DNA热循环器(Perkin-Elmer-Cetus)中培养,进行35循环的PCR反应。表一是说明包含目标基因的正股(sense)与反股(antisense)引物(序列编号1-32)的所述引物序列,以及对应的接合(annealing)温度。
表一
序列编号 | 目标基因(PCR产物大小) | 接合温度(℃) |
1,2 | β-actin(肌动蛋白)(510bps) | 57 |
3,4 | IL-1β563bps | 61 |
5,6 | IL-2(502bps) | 61 |
7,8 | IL-4(399bps) | 61 |
9,10 | I1-6(638bps) | 61 |
11,12 | IL-10(455bps) | 61 |
序列编号 | 目标基因(PCR产物大小) | 接合温度(℃) |
13,14 | TNF-α(308bps) | 57 |
15,16 | IFN-γ(460bps) | 61 |
17,18 | TGF-β(525bps) | 61 |
19,20 | 老鼠α-防卫素(900bps) | 59 |
21,22 | 老鼠血管生成素(900bps) | 59(热起始) |
23,24 | TLR2(495bps) | 55 |
25,26 | TLR4(508bps) | 57 |
27,28 | TLR5(737bps) | 59 |
29,30 | TLR7(729bps) | 59 |
31,32 | TLR9(725bps) | 60 |
请参阅图1,其是说明IEC-6细胞以DeCaPS处理后,以PCR分析细胞激素与抗微生物肽的mRNA表现。分别以浓度1μg/ml、10μg/ml与100μg/ml的DeCaPS处理6小时的IEC-6细胞,IEC-6细胞中TNF-α(序列编号:33)的mRNA表现量大约分别被抑制29%、20%与30%;然而,IEC-6细胞中IL-10(序列编号:34)的mRNA表现量大约分别上升9.89倍、6.55倍与1.77倍。这表示DeCaPS可诱导肠道上皮细胞提供耐受信号至免疫***中的其他细胞,进而诱发TH2/3路径。此外,在以浓度1μg/ml、10μg/ml与100μg/ml的DeCaPS处理6小时后,IEC-6细胞中α-防卫素(序列编号:35)的mRNA表现量大约分别被抑制42%、33%与51%;然而,IEC-6细胞中血管生成素(序列编号:36)的mRNA表现量大约分别上升1.96倍、1.42倍与1.70倍。
请参阅图2,说明IEC-6细胞以DeCaPS处理后,以PCR分析Toll样受体(Toll-like receptor,TLR)的mRNA表现。在未处理DeCaPS的IEC-6细胞中,所表现的Toll样受体为TLR2(序列编号:37)、TLR4(序列编号:38)、TLR7(序列编号:39)以及TLR9(序列编号:40)。在以浓度1μg/ml、10μg/ml与100μg/ml的DeCaPS处理6小时后,IEC-6细胞中TLR4(序列编号:38)的mRNA表现量大约分别被抑制6%、16%与13%,IEC-6细胞中TLR5(序列编号:41)的mRNA表现量大约分别被抑制13%、37%与35%,IEC-6细胞中TLR7(序列编号:39)的mRNA表现量大约分别被抑制11%、7%与48%;然而,IEC-6细胞中TLR9(序列编号:40)的mRNA表现量大约分别上升2.21倍、1.09倍与3.56倍。这表示自石斛取得的多糖不仅抑制TNF-α(序列编号:33)的表现,也抑制TLR4(序列编号:38)、TLR5(序列编号:41)与TLR7(序列编号:39)的表现。
范例III:动物模式口服DeCaPS对于免疫***的效应
将由范例I制备的DeCaPS,透过饮用水,以10毫克/公斤/日、50毫克/公斤/日以及250毫克/公斤/日的剂量喂食C3H老鼠(13周大)5天,而后牺牲老鼠以取得脾脏与骨髓。
a.以刀豆蛋白A(Concanavalin A,Con A)刺激自C3H老鼠所取得的脾脏细胞并进行MTT分析
自以DeCaPS处理的C3H老鼠所取得的脾脏细胞(4×105细胞/槽)培养于含有10%胎牛血清与浓度为1或5微克/毫升的刀豆蛋白A(Con A)的RPMI-1640培养液中,处理72小时,而后以1毫克/毫升的MTT处理3小时。而后,将含有50%DMF与20%SDS的溶解(lysis)缓冲液加入细胞培养中,进行反应16小时。分析反应在570纳米的吸光值,作为生长指标,如图3所示。
请参阅图3,其说明C3H老鼠口服DeCaPS之后,以ConA刺激来自经处理C3H老鼠的脾脏细胞行细胞***的效应。分别以口服剂量为10毫克/公斤/日、50毫克/公斤/日以及250毫克/公斤/日的DeCaPS处理C3H老鼠5天后,脾脏细胞对于1微克/毫升的ConA刺激反应大约被抑制28%、49%与30%。
b.GMCSF刺激自C3H老鼠所取得骨髓细胞的MTT分析
自口服DeCaPS的C3H老鼠的腿部,取得骨髓细胞。将所述骨髓细胞培养于含有2%FCS与4ng/ml GMCSF的α-MEM培养液中,而正对照组的骨髓细胞于含有2%FCS与20ng/ml GMCSF的α-MEM培养液中培养72小时。接着将培养液换为含有1mg/ml MTT与2%FCS的RPMI-1640处理骨髓细胞3小时,而后细胞以MTT溶解缓冲液(pH4.5,20%SDS与50%DMF)处理培养过夜。反应于570钠米分析吸光值,以代表生长指标,如图4所示。
请参阅图4,说明C3H老鼠口服DeCaPS处理后,以GM-CSF刺激得自C3H老鼠的骨髓细胞行细胞***的效应。分别以口服剂量为10毫克/公斤/日、50毫克/公斤/日以及250毫克/公斤/日的DeCaPS处理C3H老鼠5天后,骨髓细胞对于GM-CSF刺激的细胞***反应大约被提升1.71倍、1.62倍与1.76倍。
c.得自C3H老鼠的皮尔氏斑与脾脏细胞中,细胞激素的mRNA表现
自口服DeCaPS处理的C3H老鼠,取得皮尔氏斑与脾脏细胞。以范例II中的方法,用PCR分析皮尔氏斑与脾脏细胞中特定细胞激素的mRNA,而PCR的结果如图5所示。
请参阅图5,说明C3H老鼠口服DeCaPS处理后,PCR分析自C3H老鼠所取得的皮尔氏斑(Peyer’s Patch)与脾脏细胞中特定细胞激素的mRNA表现,其中1-4分别代表口服DeCaPS剂量为0毫克/公斤/日、10毫克/公斤/日、50毫克/公斤/日以及250毫克/公斤/日的C3H老鼠。
在C3H老鼠口服DeCaPS 5天后,皮尔氏斑中IL-4(序列编号:42)、IL-6(序列编号:43)、IL-1β(序列编号:44)、IFN-γ(序列编号:45)与TGF-β(序列编号:46)的mRNA表现量增加,如图5所示。在C3H老鼠口服剂量为10毫克/公斤/日或50毫克/公斤/日的DeCaPS达5天后,IL-4(序列编号:42)的mRNA表现量分别增加为2.64倍与2.46倍。在C3H老鼠口服剂量为10毫克/公斤/日、50毫克/公斤/日或250毫克/公斤/日的DeCaPS达5天后,IL-6(序列编号:43)的mRNA表现量分别增加为2.61倍、3.99倍与5.35倍,IL-1β(序列编号:44)的mRNA表现量分别增加为1.83倍、2.00倍与1.11倍,IFN-γ(序列编号:45)的mRNA表现量分别增加为3.47倍、5.47倍与5.57倍,以及TGF-β(序列编号:46)的mRNA表现量分别增加为2.38倍、2.54倍与1.89倍。由于皮尔氏斑是决定对于抗原的免疫反应的主要位置,所以对于IL-4(序列编号:42)与IL-6(序列编号:43)的增加,会诱发Th2路径,透过IL-1β(序列编号:44)增加,可活化T辅助细胞分泌细胞激素,例如TGF-β(序列编号:46),以及TGF-β(序列编号:46)诱导IgA类转换的B细胞分泌IgA,以抑制某些免疫反应。
在C3H老鼠口服DeCaPS处理达5天后,脾脏细胞中IL-1β(序列编号:44)、IL-4(序列编号:42)与TGF-β(序列编号:46)的mRNA表现量受到抑制,如图5所示。在C3H老鼠以口服剂量为10毫克/公斤/日、50毫克/公斤/日或250毫克/公斤/日的DeCaPS处理达5天后,IL-1β(序列编号:44)的mRNA表现量大约抑制32%、43%与79%,且IL-4(序列编号:42)的mRNA表现量大约抑制7%、67%与14%。在C3H老鼠口服剂量为50毫克/公斤/日或250毫克/公斤/日的DeCaPS达5天后,TGF-β(序列编号:46)的mRNA表现量大约抑制27%与44%,以及IFN-γ(序列编号:45)的mRNA表现量大约抑制33%与97%。然而,在C3H老鼠以口服剂量为10毫克/公斤/日、50毫克/公斤/日或250毫克/公斤/日的DeCaPS处理达5天后,IL-6(序列编号:43)的mRNA表现量增加为1.84倍、1.4倍与1.23倍。值得注意的是对于DeCaPS处理并没有明显的细胞反应,且此现象是与对于ConA刺激的反应一致。
范例IV:以DeCaPS于动物模式中增加口服耐受性
由于已知动物受到卵白蛋白(OVA)免疫化可建立自体免疫疾病的动物模式(TheJournal of Pharmacology and Experimental Therapeutics 288:849-857,1999),所以本发明建立以OVA诱导自体免疫疾病的老鼠,并且以DeCaPS在OVA诱导自体免疫疾病的老鼠上促进口服耐受性,如以下的说明。在第4天与第5天,将C57/BL6j老鼠(65周大)透过饮用水,口服喂食0.5毫克/毫升卵白蛋白(OVA),以及在第3-7天,将DeCaPS投予C57/BL6j老鼠作为佐剂(adjuvant),剂量分别为10毫克/公斤/日、40毫克/公斤/日与160毫克/公斤/日。在第8天时,以CFA为佐剂的50微克OVA,以i.p.注射投予老鼠作为抗原,在第22天时,于血液收集中侦测到针对OVA的IgG与IgM,在第24天时,将50微克的OVA以i.p.注射投予老鼠,在第30天时,侦测到针对OVA的抗体;在第32天,将老鼠牺牲,以获得肠黏膜的肠液与肺黏膜,而后侦测到针对OVA的IgA。详细的实验过程如表II中所述。
表二、在动物模式中口服含/不含DeCaPS的OVA
在第32天取得老鼠肠黏膜肠液,其中测得肠液中针对卵白蛋白的IgA量,如图6所示。请参阅图6,以OVA与DeCaPS(40毫克/公斤/日)处理的C57/BL6j老鼠中由肠黏膜所分泌的IgA增加为1.9倍(p<0.01),以及以OVA与DeCaPS(160毫克/公斤/日)处理的C57/BL6j老鼠中由肠黏膜所分泌的IgA增加为4.35倍(p<0.001)
在第22天与第30天,检测血清中的IgM抗体,如图7所示。相较于第22天对照组所表现的IgM抗体量,正常组所表现的IgM抗体量大约被抑制49%(p<0.05),正对照组所表现的IgM抗体量大约被抑制87%(p<0.05),DC-1组所表现的IgM抗体量大约被抑制46%(p<0.02),DC-2组所表现的IgM抗体量大约被抑制88%(p<0.001),以及DC-3组所表现的IgM抗体量大约被抑制86%(p<0.001)。再者,第30天时,相较于对照组所表现的IgM抗体量,正常组所表现的IgM抗体量大约被抑制47%(p<0.01),正对照组所表现的IgM抗体量大约被抑制72%(p<0.001),DC-1组所表现的IgM抗体量大约被抑制38%(p<0.01),DC-2组所表现的IgM抗体量大约被抑制58%(p<0.01),以及DC-3组所表现的IgM抗体量大约被抑制71%(p<0.001)。
在第22天与第30天,检测血清中的IgG抗体,如图8所示。相较于第22天对照组所表现的IgG抗体量,正对照组所表现的IgG抗体量大约被抑制94%(p<0.01),DC-1组所表现的IgG抗体量大约被抑制67%(p<0.01),DC-2组所表现的IgG抗体量大约被抑制76%(p<0.01),以及DC-3组所表现的IgG抗体量大约被抑制88%(p<0.001)。再者,相较于第30天对照组所表现的IgG抗体量,正对照组所表现的IgG抗体量大约被抑制75%(p<0.001),DC-2组所表现的IgG抗体量大约被抑制62%(p<0.001),以及DC-3组所表现的IgG抗体量大约被抑制63%(p<0.001)。
此外,在第32天由老鼠取得皮尔氏斑,萃取皮尔氏斑中淋巴球的RNA并分析细胞激素。皮尔氏斑中淋巴球的细胞激素表现,如图9所示,其中A行代表正常组,B行代表对照组,C行代表正对照组,D行代表DC-1组,E行代表DC-2组,以及F行代表DC-3组。PCR放大程序为在94℃变性(denature)45秒,在61℃接合(annealing)45秒以及在72℃延长(extension)1分钟。在所述PCR程序中有37循环。以2%的洋菜胶体,电泳分离PCR产物,以及将胶体以溴化乙锭(ethidium bromide)染色并以UV灯照射。
值得注意的是相较于对照组,在DC-3组中TGF-β(序列编号:46)表现量大约被抑制30%,DC-3组中IL-4(序列编号:42)的表现量大约增加为2.2倍,以及DC-3组中IL-10(序列编号:34)的表现量大约增加为1.3倍。相较于对照组,DC-2组中IL-2(序列编号:47)的表现量大约增加为1.4倍,以及DC-2组中IL-4(序列编号:42)的表现量大约增加为1.6倍。再者,相较于对照组,DC-1组中IL-4(序列编号:42)的表现量大约增加为1.4倍,以及DC-1组中IL-6(序列编号:43)的表现量大约增加为1.3倍。
皮尔氏斑中淋巴球的IL-4(序列编号:42)与IL-6(序列编号:43)mRNA表现量上升。必须要强调的是当抗原诱发T细胞或是B细胞透过机制回到小肠时,皮尔氏斑是决定对于抗原的免疫反应的位置,因应IL-4(序列编号:42)与IL-6(序列编号:43)的增加,活化Th2路径以产生抗体,血清中的IgG与IgM表现量受到抑制,以抑制过敏反应,并且达到口服耐受性。
承上所述,得自于石斛(Dendrobium)的多糖可抑制胰脏中T细胞的细胞***反应(mitogenic response);然而,骨髓中颗粒细胞(granulocyte)与巨噬细胞(macrophage)则是会受到石斛(Dendrobium)多糖的的活化。换句话说,可藉由口服石斛多糖而抑制自免疫疾病中被活化的T细胞;然而,藉由口服石斛多糖而活化颗粒细胞系(granulocyte lineage),进以藉由促进先天性免疫力(innateimmunity),而防止病原入侵。
关于肠道免疫,IL-4(序列编号:42)、IL-6(序列编号:43)、IL-1β(序列编号:44)、IFN-γ(序列编号:45)与TGF-β(序列编号:46)的mRNA表现量增加,而诱发肠道内的Th2/Th3路径,因而形成口服耐受性。根据以石斛多糖所处理的IEC-6细胞中TNF-α(序列编号:33)、TLR2(序列编号:37)、TLR4(序列编号:38)、TLR5(序列编号:41)与TLR7(序列编号:39)的表现,显示藉由石斛多糖,Toll样受体(toll-like receptor)所传达的讯息可被调节为免疫抑制型反应或是抗发炎反应,以促进口服耐受性的相关讯息,亦即Th2/3路径,并且在感染病原状态下,例如自体免疫疾病与黏膜疾病,保持体内黏膜的平衡(mucosal homeostasis)。有趣的是如范例II所述,口服石斛多糖可诱导具有抗微生物活性的血管生成素(angiogenin)的表现。因此,口服石斛多糖不仅促进口服耐受性,更可诱发先天性免疫力(innate immunity)以防止病原入侵。根据这些发现,石斛多糖可有效治疗自体免疫疾病与黏膜疾病,例如发炎性肠道疾病(inflammatory bowel disease,IBD),以减缓长期发炎与伤害。
已知血管生成素(angiogenin)具有的活性不仅可以对抗肠道微生物,例如乳酸球菌(Enterococcus faecalis),更可以对抗引起人类***感染的微生物,例如白色念珠菌(Candida albicans)与肺炎链球菌(Streptococcus pneumoniae)。(Nat.Immunol.4:269-273,2003)在临床研究中,女性的白带(leucorrhea)是多种微生物的表浅***感染(polymicrobial,superficial vaginal infection),通常是受到细菌感染,例如***伽氏杆菌(G.vaginalis)、念珠菌(Candida spp.)、白色念珠菌(C.albicans)、***毛滴虫(T.vaginalis)、链球菌族群D(Streptococcusgroup D)、b型溶血性链球菌(Streptococcus b hemolytic)、大肠杆菌与克雷白式菌(Klebsiella spp.)等。(Salud publica Mex vol.45 suppl.5,pS694-S697,2003)很明显地,由微生物如白色念珠菌(Candida albicans)所引起的女性白带可藉由口服石斛多糖,诱导血管生成素(angiogenin)的活性而得到舒缓。
范例IV:以DeCaPS治疗自体免疫疾病
实验用的自体免疫葡萄膜炎(Uveitis)是T细胞所参与的自体免疫疾病,作为一些眼睛自体免疫疾病的模式。已经确认实验用的自体免疫葡萄膜炎是以140-kD的糖脂蛋白“光受体间维生素A酸结合蛋白质(interphotoreceptor retinoidbinding protein,IRBP)”免疫诱导而形成。亦为已经确定的是可藉由IRBP肽1-20(序列编号:48:GPTHLFQPSLVLDMAKVLLD),此为IRBP的第1至20个胺基酸,进以诱导自体免疫葡萄膜炎。此外,IRBP肽1-20在老鼠与人类皆相同(conserved)。(Investigative Ophthalmology & Visual Science 41(1):127-131,2000)所以,在本发明中使C57BL/6j老鼠口服IRBP肽以建立自体免疫模式。
a.合成IRBP肽
以Fmoc化学肽合成器(PS3)合成IRBP肽1-20(序列编号:48:GPTHLFQPSLVLDMAKVLLD),以Agilent HPLC纯化并以Brukeer esquire 2000 MS进行纯化。
b.投予IRBP与DeCaPS
将无特定病原(specific-pathogen-free,SPF)的C57BL/6j老鼠随机分组,分为正常组、对照组、DC-10组、DC-40组以及DC-160组,其中各组有6只老鼠。以i.p.注射的方式,将含有人类IRBP肽(序列编号:48)(150微克/鼠)与CFA(1∶1体积/体积)的0.2毫升乳化液(emulsion)中投予除正常组外的各组SPF鼠。透过饮用水,以10毫克/公斤/天剂量的DeCaPS喂食DC-10组的老鼠28天,以40毫克/公斤/天剂量的DeCaPS喂食DC-40组的老鼠28天,以160毫克/公斤/天剂量的DeCaPS喂食DC-160组的老鼠28天。以视网膜电流图(electro-retinogram,ERG)分析老鼠的视网膜功能,并且在第28天将老鼠牺牲,以获得其眼睛进行生物分析与组织病理鉴定。所述实验过程如以下表三所述。
表三、在动物模式中投予/不投予DeCaPS治疗自体免疫疾病
组别 | 老鼠数 目 | 第0天的 ERG鉴 定 | 第1天以 IRBP免疫 (immunizatio n) | DeCaPS (第1-28 天)毫克/ 公斤/天 | DTH分析 (第21与23天) | 第28天ERG & 组织病理 |
正常 | 6 | + | - | - | + | + |
控制 | 6 | + | + | - | + | + |
DC-10 | 6 | + | + | 10 | + | + |
DC-40 | 6 | + | + | 40 | + | + |
DC-160 | 6 | + | + | 160 | + | + |
c.迟发型过敏免疫反应(Delayed type hypersensitivity,DTH)测试
在以IRBP肽免疫后的第21天,以IFA(20微升)中乳化的20微克IRBP肽皮下注射于老鼠左脚掌。以IFA注射右脚掌。在48小时后,以尺测量脚掌的厚度。
请参阅第10图,显示老鼠的DTH反应。相对于注射磷酸盐缓冲溶液(PBS)的老鼠的脚掌厚度增加量,以脚掌厚度增加量呈现DTH反应。值得注意的是投予DeCaPS(160毫克/公斤/天)的老鼠的脚掌增加量是明显降低。
d.视网膜电流图分析
将老鼠置于暗室2小时,而后以戊巴比妥酸钠(sodium pentobarbital)麻醉。老鼠的角膜被麻醉且瞳孔放大。以闪光刺激视网膜,并且将视网膜对于闪光的反应记录成为视网膜电流图。所述视网膜电流图显示光受体的作用与视网膜近端,如两极细胞与Müller细胞的功能。视网膜电流图用以反应整个视网膜的状态。在视网膜电流图中,典型为负向a波而后为正向b波。A波前缘是测量光受体层锥细胞与杆细胞的活性,而b波则是反应内核层中两极细胞的作用。
在第28天随机自正常组选取的2只老鼠,其视网膜电流图的结果分别显示于图11A的上部与下部,其中闪光是起始于时间100毫秒且闪光期间约为600毫秒。在第28天随机自正常组选取的2只老鼠,其眼睛的组织病理鉴定分别显示于图11B的上部与下部(400倍放大),其中光受体层标示为P、外核层标示为ONL且内核层标示为INL。
在第28天随机自对照组选取的2只老鼠,其视网膜电流图的结果分别显示于图12A的上部与下部,其中闪光是起始于时间100毫秒且闪光期间约为600毫秒。在第28天随机自对照组选取的2只老鼠,其眼睛的组织病理鉴定分别显示于图12B的上部与下部(400倍放大)。显示由于以IRBP肽免疫(immunization),对照组老鼠的光受体层(P)、外核层(ONL)与内核层(INL)受到严重的伤害。
在第28天随机自DC-10组选取的2只老鼠,其视网膜电流图的结果分别显示于图13A的上部与下部,其中闪光是起始于时间100毫秒且闪光期间约为600毫秒。在第28天随机自DC-10组选取的2只老鼠,其眼睛的组织病理鉴定分别显示于图13B的上部与下部(400倍放大)。显示由于以IRBP肽免疫(immunization)后并口服DeCaPS(10毫克/公斤/天)28天,DC-10组老鼠的光受体层(P)、外核层(ONL)与内核层(INL)受到严重的伤害。
在第28天随机自DC-40组选取的2只老鼠,其视网膜电流图的结果分别显示于图14A的上部与下部,其中闪光是起始于时间100毫秒且闪光期间约为600毫秒。在第28天随机自DC-40组选取的2只老鼠,其眼睛的组织病理鉴定分别显示于图14B的上部与下部(400倍放大)。显示由于以IRBP肽免疫(immunization)后并口服DeCaPS(40毫克/公斤/天)28天,DC-40组老鼠的光受体层(P)、外核层(ONL)与内核层(INL)仅受到轻微的伤害。
在第28天随机自DC-160组选取的2只老鼠,其视网膜电流图的结果分别显示于图15A的上部与下部,其中闪光是起始于时间100毫秒且闪光期间约为600毫秒。在第28天随机自DC-160组选取的2只老鼠,其眼睛的组织病理鉴定分别显示于图15B的上部与下部(400倍放大)。显示由于以IRBP肽免疫(immunization)后并口服DeCaPS(160毫克/公斤/天)28天,DC-160组老鼠的光受体层(P)、外核层(ONL)与内核层(INL)并未受到伤害。
据此,在以IRBP肽免疫(immunization)后,发现内核层有更多的细胞死亡,而死亡的细胞并不会下陷至外核层。亦发现IRBP肽处理会伤害内核层中的细胞,然而以160毫克/公斤/天剂量的处理可明显防止细胞自内核层脱落。
此外,在图16A中比较图11A、12A、13A、14A与15A中所示的a波(统计p<0.02),以及在图16B中比较图11A、12A、13A、14A与15A中所示的b波(统计p<0.02)。要强调的是以IRBP肽免疫的对照组老鼠的a波明显低于正常组老鼠的a波;然而,相较于正常组,以DeCaPS(40毫克/公斤/天)处理的老鼠,明显可防止细胞自内核层脱落。再者,以DeCaPS(160毫克/公斤/天)处理的老鼠,其a波与b波的振幅几乎等于正常组老鼠的a波与b波振幅。更要进一步强调的是以IRBP肽免疫所造成的伤害可由自石斛(Dendrobium)所制备的多糖(160毫克/公斤/天)处理而挽救。换句话说,口服由石斛所制备的多糖可使视网膜免于受到由IRBP所诱导的发炎。
根据上述实验,本发明是提供一种组合物,用于治疗自体免疫疾病与黏膜疾病,其中所述组合物包含由石斛(Dendrobium)所制备的多糖以及诱导所述自体免疫疾病的抗原,这是由于自石斛(Dendrobium)所制备的多糖可促进口服耐受性的诱发且促进先天性免疫力。再者,本发明是提供仅包含自石斛(Dendrobium)所制备的多糖的组合物,用于治疗自体免疫疾病与黏膜疾病,这是由于自石斛(Dendrobium)所制备的多糖可单独诱导口服耐受性且调节先天性免疫力。
虽然本发明已说明于上述较佳实施例中,然而可以理解的是本发明并不会因为所揭露的实施例而受到限制。相反地,本发明可包含不同的修饰与相似的配置,却仍不脱本发明权利要求的精神与范围,根据对于本发明权利要求最宽广的解释,可包含所有的修饰与类似的架构。
Claims (3)
1.一种萃取自石斛(Dendrobii Herba)的多糖在制备用于治疗哺乳动物自体免疫葡萄膜炎的药物中的用途。
2.如权利要求1的用途,其中所述多糖通过如下方法制备,所述方法包含的步骤为:
(a)以甲醇萃取石斛,以获得一第一萃取物;
(b)以水萃取所述第一萃取物,以获得一第二萃取物;以及
(c)以乙醇沉淀所述第二萃取物,以获得所述多糖。
3.如权利要求1的用途,其中所述药物是一口服药物。
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