CN100560718C - 原儿茶酸在促进神经干细胞体外增殖及诱导分化方面的用途 - Google Patents
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Abstract
本发明属于细胞生物学和药理学技术领域,涉及一种中药单体化合物的用途,特别涉及原儿茶酸在促进神经干细胞体外增殖及诱导分化方面的新用途。其特征在于:制备神经干细胞移植治疗神经退行性疾病药物中的应用;制备修复损伤神经组织及功能重建的神经营养素和细胞分化剂中的应用;构建高效率的药物筛选模型,并利用该模型进行药物药效的初步筛选与评价中的应用。本发明确定了原儿茶酸的新的药理活性,为中药的防治作用提供物质基础,同时也为开发拥有自主知识产权的新药提供借鉴。
Description
技术领域
本发明属于细胞生物学和药理学技术领域,涉及一种中药单体化合物的用途,特别涉及原儿茶酸在促进神经干细胞体外增殖及诱导分化方面的新用途。
背景技术
益智(Alpinia oxyphylla MIQUEL)为姜科山姜属(Alpinia Roxb.)植物,自古以来就是中医临床上的常用药材,有温脾止泻,摄唾涎,固精缩尿的功效。现代研究表明,益智中含有特殊的化学成分和显著的生物活性,具有强心、抗癌、抗过敏及神经保护等广泛的药理活性。发明人通过建立的体外神经细胞损伤模型,首次从中药益智中筛选并证明了原儿茶酸是益智中的主要神经活性成分(An LJ et al.,Food Chem Toxicol,2006,44(3):436-443.)。原儿茶酸,即3,4-二羟基苯甲酸,化学结构式如下:
其制备过程为:益智仁粉碎成适当大小颗粒,95%乙醇室温浸渍24小时,布氏漏斗过滤,滤渣重复提取三次。合并滤液,减压蒸馏浓缩至浸膏,加水分散后用氯仿、乙酸乙酯、正丁醇依次萃取,收集各部分,减压蒸干后,乙酸乙酯组分采用100~140目硅胶柱层析,干法上样,氯仿-甲醇梯度洗脱,薄层层析(TLC)检测收集相同馏分。取氯仿-甲醇为50∶1的馏分重新溶解于甲醇中,用0.45μm的滤膜过滤后进行Sephadex LH-20凝胶层析,甲醇洗脱。重结晶后即得原儿茶酸。
原儿茶酸是天然存在于许多蔬菜和果实中的一种酚酸类物质,也是除益智以外其它很多中药(如四季青、丹参、芙蓉等)的有效活性成分。早期的研究已经证明,原儿茶酸具有抗血小板凝集、降低心肌耗氧量、抑菌、镇痛等多方面药理活性,是一种重要的天然活性物质。近年来的研究发现了原儿茶酸许多新的药理作用。如Ueda等的研究发现原儿茶酸具有显著的抗氧化作用,其抗氧化能力是α-生育酚的10倍以上(Ueda et al.,Arch Biochem Biophys,1996,333(2):377-384.)。Tseng小组从中药芙蓉中分离得到了原儿茶酸,证明了原儿茶酸抗氧化及抗肿瘤作用,并且初步探讨了原儿茶酸的作用机制(Tseng TH et al.,ChemBiol Interact,1996,101(2):137-148;Tseng TH et al.,Cancer Lett,1998,126(2):199-207;Tseng TH et al.,Biochem Pharmacol,2000,60(3):307-315.)。更多的研究表明,原儿茶酸可作为许多化学致癌物的有效抑制剂,例如二乙基亚硝基胺所致的肝癌(Tanaka T et al.,Cancer Res,1993,53(12):2775-2779.)、4-硝基喹啉-1-氧化物导致的口腔癌(Tanaka T et al.,Cancer Res,1994,54(9):2359-2365.)、azoxymethane所致的结肠癌(Kawamori T et al.,Jpn J Cancer Res,1994,85(7):686-691.)、N-甲基-N-亚硝基脲所致的胃癌(Tanaka T et al.,Cancer,1995,75(6Suppl):1433-1439.)、N-丁基-N-(4-羟基丁基)亚硝胺导致的膀胱癌(Hirose Y et al.,Carcinogenesis,1995,16(10):2337-2342.)等等。这些数据提示了原儿茶酸有可能成为一种天然的抗癌保护剂。到目前为止,有关原儿茶酸神经活性的研究报道较少。本研究对益智中原儿茶酸的神经保护活性进行了大量研究,结果发现原儿茶酸对氧化应激损伤的PC12细胞具有明显的抗氧化及抗凋亡的保护作用。原儿茶酸能够保护质膜的完整性,提高细胞内谷胱甘肽的含量,增强超氧化物歧化酶和过氧化氢酶的活性,从而保持细胞内的抗氧化防御体系处于正常的水平(Guan S et al.,Eur J Pharmacol,2006,538(1-3):73-79.);而且原儿茶酸能够抑制细胞凋亡,调节线粒体的通透性,阻止膜电位的丧失,抑制活性氧的产生,激发内源性抗氧化机制及拮抗caspase-3的激活和上调Bcl-2蛋白的表达,从而保持线粒体功能的完整性(Guan et al.,Food Chem Toxicol,2006,44(10):1659-1666.)。
然而迄今未见国内外对原儿茶酸在促进神经干细胞体外增殖及诱导分化方面的研究报道。
发明内容
本发明公开了原儿茶酸的一种新用途,是发现原儿茶酸具有显著促进体外培养的神经干细胞增殖的作用,使其持续保持增殖状态,并在适宜条件下能分化为有功能的神经元,用于神经干细胞移植治疗神经退行性疾病和神经再生及功能重建的神经营养素和细胞分化剂。
本发明的一个目的是利用原儿茶酸能诱导神经干细胞体外定向分化获得的有功能的神经元,用于构建高效率的药物筛选模型,并利用该模型进行药物药效的初步筛选与评价。
本发明的技术方案包括以下内容:
(1)神经干细胞的体外分离培养及鉴定
选用胚胎14天(E14d)小鼠引颈处死,无菌操作取出胚胎中脑组织,制成分离细胞悬液。在无血清DMEM/F12培养基中采用悬浮培养方式培养,培养液中添加EGF和bFGF。7~10天后将原代培养所形成的“神经干细胞球”分离,进行传代培养。取第二代细胞培养7~10天后进行鉴定。采用免疫细胞化学方法鉴定神经干细胞:Nestin染色检测神经干细胞、GFAP染色检测星形胶质细胞、TuJ1染色检测神经元、GalC染色检测少突胶质细胞以及BrdU染色标记增殖的细胞。
(2)原儿茶酸对体外培养的神经干细胞的促增殖作用
将原儿茶酸直接作用于原代或传代的神经干细胞,实验分为五组:对照组、6×10-6mol/L原儿茶酸组、6×10-5mol/L原儿茶酸组、6×10-4mol/L原儿茶酸组及1.2×10-3mol/L原儿茶酸组。于体外分别培养4天和7天后显微镜下观察,在添加原儿茶酸条件下培养的神经干细胞球明显增多,经统计分析,当原儿茶酸的浓度超过6×10-5mol/L以上时,神经球的形成数目明显高于对照组。CCK-8结果也显示,原儿茶酸组的细胞存活率均明显高于对照组。
(3)原儿茶酸诱导体外培养的神经干细胞分化为有功能的神经元
实验采用以下两种条件培养的神经球:正常培养组(无任何处理的神经球)、原儿茶酸增殖组(经原儿茶酸处理后的神经球)。将不同条件下培养的神经干细胞球分别在分化培养基(DMEM/F12+10%胎牛血清)中吹打成单细胞悬液,接种于多聚左旋赖氨酸处理过圆玻片的24孔板内,24小时后可见细胞均贴壁生长,添加原儿茶酸处理后,继续培养7天,用免疫细胞化学方法检测神经元的特异性标记物、星型胶质细胞的特异性标记物和少突胶质细胞的特异性标记物的表达,并计数分化为神经元、星型胶质细胞和少突胶质细胞的百分率。结果显示,添加原儿茶酸的神经干细胞分化后形成的神经元生长状态良好,神经元的数目多于对照组。而且对于已由原儿茶酸处理后增殖的神经球而言,其分化为有功能的神经元的比例明显高于正常培养的神经球。表明经原儿茶酸处理后增殖的神经干细胞的分化能力并没有受到明显的影响,而且更有利于向神经元方向分化。
本发明的效果和益处是在细胞层面上对原儿茶酸的药效进行了严格论证,对其中蕴含的大量生物学信息进行了深入探讨,优点显著:
①本发明开拓了原儿茶酸的新用途,即原儿茶酸具有显著促神经干细胞增殖的作用,使其持续保持增殖状态,并在适宜条件下能分化为有功能的神经元。
②原儿茶酸可用于神经干细胞移植治疗神经退行性疾病和神经再生及功能重建的神经营养素和细胞分化剂。干细胞移植治疗神经退行性疾病目前正处于实验研究阶段,原儿茶酸的研究将有助于该领域的深入发展。
③本发明采用一种中药单体成分来诱导神经干细胞的增殖及定向分化为有功能的神经元,替代受损伤的细胞或死亡的细胞,为中药的防治作用提供物质基础,另一方面药效机制的阐明为中药现代化开发拥有自主知识产权的新药提供借鉴。
④本发明利用原儿茶酸能诱导神经干细胞体外定向分化获得的有功能的神经元,用于构建高效率的药物筛选模型,可为治疗神经退行性疾病的新药开发提供检测平台。
附图说明
图1是本发明所述的原儿茶酸体外培养的“神经球”增殖的影响,为原代培养的第二代神经干细胞培养4天后的显微照片图。图中A对照组;B 6×10-5mol/L原儿茶酸组。
图2是本发明所述的原儿茶酸对体外培养的神经干细胞生存能力的影响,为原儿茶酸促进神经干细胞增殖的量效图。图中横坐标轴的数字表示各实验组(其中0,对照组;1,6×10-6mol/L原儿茶酸组;2,6×10-5mol/L原儿茶酸组;3,6×10-4mol/L原儿茶酸组;4,1.2×10-3mol/L原儿茶酸组),纵坐标轴表示神经干细胞的细胞增殖百分率(*P<0.05,表示同对照组之间的显著性差异)。
具体实施方式
以下结合技术方案和附图详细说明本发明的具体实施例。
实施例1体外神经干细胞的分离培养和鉴定
(1)中脑来源的神经干细胞的分离与培养
取E14d小鼠引颈处死,无菌操作取出胚胎,分离胚胎中脑组织,消化成单细胞后,台盼蓝计数,以2×105个/mL接种于T25培养瓶中37℃、5%CO2悬浮培养,干细胞培养基为DMEM/F12(1∶1)、2%B27、bFGF(10ng/mL)、EGF(20ng/mL)。每3~4天半量换液,培养一周后观察生长的悬浮细胞球的数目及大小。收集神经球机械吹打成单细胞悬液后,置于同样条件下继续培养,视生长情况决定是否传代。
(2)细胞亚克隆的连续传代及诱导分化
用巴斯德细管从培养瓶中取出单个较大的神经干细胞球(直径约为150μm左右),机械吹打成单细胞悬液,接种于干细胞培养基中,观察细胞生长情况及神经干细胞球的数目。七天后,再用巴斯德吸管从培养瓶中取出单个较大的干细胞球在分化培养基中(DMEM/F12+10%胎牛血清)轻柔机械吹打,尽可能分散神经球,并接种于多聚左旋赖氨酸处理过圆玻片的24孔板内,继续培养7天,3~4天半量换液。
(3)神经干细胞的鉴定
原代培养的神经干细胞球,经传代后接种在底部涂有多聚赖氨酸的35mm的培养皿中,培养3天后,用4%的多聚甲醛(以0.1MPBS制备,pH7.2~7.4)固定30min,0.01MPBS冲洗3次后,进行神经干细胞标记物Nestin和BrdU抗体免疫细胞化学染色。
实验结果:在原代培养神经干细胞7~9天后,可获得大量大小不一、呈悬浮生长的克隆球。原代单个的克隆球机械分离后进行传代培养,可形成新的细胞克隆(亚克隆)。这些克隆球经Nestin和BrdU抗体鉴定呈阳型染色,表明它们是神经干细胞,具有***增殖能力。
实施例2原儿茶酸对神经干细胞的促增殖作用
(1)实验设计及药物处理
实验分为五组,分别为对照组、6×10-6mol/L原儿茶酸组、6×10-5mol/L原儿茶酸组、6×10-4mol/L原儿茶酸组及1.2×10-3mol/L原儿茶酸组。将原代培养的第二代神经干细胞收集,以2×105个/mL的浓度接种于24孔板,每孔500μL,每组处理设6个复孔,分别培养4天和7天后观察神经球的数目及细胞的存活率。
(2)神经干细胞增殖的检测方法
①神经球计数法:原代培养的第二代神经干细胞分别在不同浓度的原儿茶酸作用下培养4天和7天后,双盲法计数神经球的形成个数,在10×20荧光显微镜下每孔随机计数10个视野,每组6孔,计算其平均值。组间比较用非配对Student’s T-test。
②CCK-8法:细胞的增殖率采用CCK-8比色法检测。对照组为正常对照和空白对照(正常对照为不加药物组,空白对照为与实验孔平行的不含细胞外其它条件相同的对照孔)。CCK-8比色法基本原理为:该试剂中含有WST-8[其化学名称为:2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐],它在电子载体1-甲氧基-5-甲基吩嗪硫酸二甲酯的作用下被细胞线粒体中的脱氢酶还原为具有高度水溶性的黄色甲臢染料。生成的甲臢物的数量与活细胞的数量成正比,在酶标仪上进行比色,所检测OD值的大小可反映细胞代谢活性的强弱。检测时每孔加50μL CCK-8(终浓度约为0.5mg/mL)于5%CO2,37℃培养3小时,用酶标仪测定450nm,参考波长为630nm的OD值,计算细胞的增殖率。
细胞的增殖率=(OD加药孔-OD加药孔空白)/(OD对照孔-OD对照孔空白)×100%。
实验结果:神经干细胞在原儿茶酸作用下培养4天和7天后,显微镜下观察,与对照相比,神经球的数量明显增多。经统计分析,原儿茶酸在6×10-5~1.2×10-3mol/L的浓度范围内,可明显促进神经球的增殖。6×10-5mol/L的原儿茶酸作用4天后,增殖的神经球数目为正常对照的2.9倍(如图1),同样细胞的生存能力也得到了显著提高,细胞的增殖率达到了对照的140.32±9.17%(如图2)。
实施例3原儿茶酸诱导神经干细胞分化为有功能的神经元的能力
(1)原儿茶酸对神经干细胞的诱导分化
将原代培养的第二代神经干细胞球,和经6×10-5mol/L的原儿茶酸作用7天后的神经干细胞球收集,分别在分化培养基(DMEM/F12+10%胎牛血清)中吹打成单细胞悬液,以2×105个/ml的浓度接种于多聚左旋赖氨酸处理过玻片的24孔板内,每孔500μL,每组处理设六个复孔。24小时后可见细胞均贴壁生长,添加不同浓度原儿茶酸(6×10-5、6×10-4、1.2×10-3mol/L)处理后,将培养板置于饱和湿度,5%CO2、37℃培养箱继续培养7天。
(2)免疫荧光细胞化学染色
培养结束后,取出24孔培养板各孔中的盖玻片。用4%的多聚甲醛(以0.1MPBS制备,pH7.2~7.4)固定30min,0.01M PBS冲洗3次后,各组进行免疫荧光细胞化学染色,检测神经元的特异性标记物(TuJ1)、星型胶质细胞的特异性标记物(GFAP)和少突胶质细胞的特异性标记物(GalC)的表达。
(3)细胞计数
在10×20荧光显微镜下,对免疫荧光细胞化学染色TuJ1阳性的细胞进行计数(以0.25mm2区域作为计数单位面积)。每组有6个培养神经干细胞的盖玻片,每个盖玻片上随机选取4个计数单位面积,计数细胞总数和染色阳性细胞数及染色阳性细胞的百分率。同样方法计数GFAP和GalC染色阳性细胞数及染色阳性细胞的百分率。
实验结果:原儿茶酸能有效促进神经干细胞分化为有功能的神经元。添加原儿茶酸的神经干细胞分化后形成的神经元生长状态良好,不同浓度的原儿茶酸实验组,神经干细胞分化为神经元的百分率都高于对照组(P<0.05),但不同浓度的原儿茶酸实验组之间没有显著差异。而且对于已由原儿茶酸处理后增殖的神经球而言,其分化为有功能的神经元的比例明显高于正常培养的神经球。表明经原儿茶酸处理后增殖的神经干细胞的分化能力并没有受到明显的影响,而且更有利于向神经元方向分化。
尽管本发明是以原儿茶酸在促进神经干细胞体外增殖及诱导分化方面的用途为例来描述的,但这种描述并不意味着对本发明构成限制。参照本发明的描述,其它种类的细胞以及实施例的其它变形,对于本领域技术人员都是可以预料的。因此,这样的变形不会脱离所属权利要求限定的范围及精神。
Claims (1)
1.一种利用原儿茶酸促进神经干细胞体外增殖及诱导分化的方法,是将原儿茶酸作用体外培养的神经干细胞,诱导其增殖并分化为有功能的神经元,其特征在于:原儿茶酸诱导体外培养的神经干细胞分化为有功能的神经元:对于由原儿茶酸处理后增殖的神经球而言,其分化为有功能的神经元的比例明显高于正常培养的神经球;其中原儿茶酸浓度为6×10-5~1.2×10-3mol/L。
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