CN101273983B - 异补骨脂甲素诱导小鼠干细胞定向分化为神经细胞的用途 - Google Patents
异补骨脂甲素诱导小鼠干细胞定向分化为神经细胞的用途 Download PDFInfo
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Abstract
本发明提供异补骨脂甲素诱导干细胞(包括胚胎干细胞、神经干细胞、骨髓间充质干细胞)体外定向分化为单一类型神经细胞在制备干细胞移植治疗神经退行性疾病药物中的应用,可在制备修复重建损伤神经组织的促细胞分化剂中应用,也可在构建药效筛选与评价模型中应用。本发明开拓了异补骨脂甲素的新用途,为异补骨脂甲素中药防治作用提供物质基础,也为药物调控再生医学或组织工程提供依据。
Description
技术领域
本发明属细胞生物学和药理学领域,涉及一种异戊烯基中药单体化合物的用途,具体涉及异补骨脂甲素在诱导干细胞体外定向分化方面的用途,可用于药物治疗神经退行性疾病和修复重建损伤神经组织的再生医学促细胞分化剂。
背景技术
补骨脂为豆科植物补骨脂(Psoralea corylifolial)的果实,具有补肾助阳、固精缩尿、温脾止泻、止血止汗、纳气平喘之功能。补骨脂的化学成分含香豆精类、黄酮类、单萜酚类以及挥发油、皂苷、多糖、类脂等成分。已经分离纯化的黄酮类化合物有异补骨脂甲素(isobavachin),新补骨脂异黄酮(neobavaisoflavone),异补骨脂查耳酮(isobavachalcone),补骨脂次素(psoralidin)。现代药理研究表明补骨脂具有多种生理活性,包括以下几个方面:
1.心血管***作用:动物实验显示补骨脂有扩张冠状动脉作用,可兴奋心脏、提高心脏作功率,但心肌耗氧量增加不明显。
2.抗菌作用:对金黄色葡萄球菌有抑制作用。
3.泌尿生殖***作用:能壮阳固精,是治阳痿及早泄的常用药,具有固涩之功效。
4.对平滑肌的作用:能使离体和在位肠管兴奋,对豚鼠子宫则呈松弛,舒张支气管平滑肌作用。
5.升高白细胞作用:动物实验表明对粒细胞的生长有促进作用,并能保护动物在注射环磷酰胺后引起的白细胞下降。
6.免疫调节作用:对体液免疫有一定调节作用。尚能调节神经和血液***,促进骨髓造血,促进吞噬细胞功能,增强免疫和内分泌功能,从而发挥抗衰老延寿作用。
7.护肝作用:对用氢化可的松造成小鼠肝脏损害有良好的治疗作用。
干细胞体外定向分化为神经细胞模型,已用于胚胎神经发育学和再生医学研究。药物调控细胞微环境可用于研究干细胞定向分化神经元和胶质细胞表型 及分化信号通路机制的研究,也可用以筛选促神经分化“药样”小分子化合物。本发明申请者研究小组的前期工作发现,异戊烯基化合物淫羊藿素具有显著诱导小鼠胚胎干细胞体外定向分化为神经细胞表型作用。此外未见其它关于药物诱导干细胞分化为神经细胞的报道。鉴于异补骨脂甲素和淫羊藿素的化学结构式相似,均属异戊烯基黄酮类化合物,亟待进一步探讨异戊烯基黄酮结构类似物在发挥神经分化和神经保护方面的活性和机制,旨在为新型结构化合物防治神经退行性改变,药物调控再生医学或组织工程提供依据。
迄今未见国内外对补骨脂提取物的单体化合物,尤其是异补骨脂甲素诱导干细胞定向分化为神经细胞效应的研究报道。
补骨脂分离的异戊烯基化合物结构式为:
异补骨脂甲素 新补骨脂异黄酮
补骨脂次素 异补骨脂查耳酮
发明内容
本发明的一个目的是提供异补骨脂甲素诱导干细胞(包括胚胎干细胞、神经干细胞、骨髓间充质干细胞)体外定向分化为单一类型神经细胞在制备干细胞移植治疗神经退行性疾病药物中的应用。
本发明的另一个目的是提供异补骨脂甲素能诱导干细胞(包括胚胎干细胞、神经干细胞、骨髓间充质干细胞)体外定向分化获得的神经细胞在制备修复重建损伤神经组织的促细胞分化剂中应用。
本发明的再一个目的是提供异补骨脂甲素能诱导干细胞(包括胚胎干细 胞、神经干细胞、骨髓间充质干细胞)体外定向分化获得神经细胞在构建药效筛选与评价模型中应用。
本发明的异补骨脂甲素能诱导干细胞(包括胚胎干细胞、神经干细胞、骨髓间充质干细胞)体外定向分化获得神经细胞通过以下步骤实现:
主要采用经典的4-/4+法进行诱导分化培养。胚胎干细胞(ES细胞)按约900个/30μl细胞接种于培养皿盖内表面,翻转后形成悬滴,置于培养箱中3天。将已形成拟胚体(embryoid bodies,EBs)的悬滴转入盛有10ml分化培养液的培养皿内,继续悬浮培养1天。第二天加入受试药物异补骨脂甲素与ES细胞拟胚体(Ebs)悬浮共培养4天。再将EBs转入预铺多聚鸟氨酸的细胞培养板内。评价神经样细胞的指标是出现细胞突起长度为胞体直径3-5倍以上。
本发明在细胞层面上对异补骨脂甲素的药效进行了严格论证,对其中蕴藏的大量生物学信息进行了初步探讨,具体有以下有益之处:
1.本发明开拓了异补骨脂甲素的新用途,即诱导干细胞(包括胚胎干细胞、神经干细胞、骨髓间充质干细胞)定向分化为单一类型神经细胞。
2.异补骨脂甲素可用作干细胞移植治疗神经退行性疾病和修复重建损伤神经组织的促细胞分化剂。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(含临床实验研究),预计促细胞分化剂异补骨脂甲素的介入将有助于该领域的研究。
3.本发明一方面明确了具备药理活性的成分异补骨脂甲素,为中药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。
附图说明
图1为异戊烯基黄酮类化合物异补骨脂甲素促神经分化作用。
图2为流式细胞术定量检测nestin阳性神经前体细胞比例。
图3为异补骨脂甲素诱导ES细胞分化成的细胞阳性表达神经细胞亚型特异性蛋白。
图4为异补骨脂甲素诱导ES细胞分化的神经细胞β-tubulinIII和神经胶质细胞特异性胶质纤维酸性蛋白(GFAP)mRNA表达。
具体实施方式
本发明结合附图和实施例作进一步的说明。此外应理解,下面的优选具体实施方案仅仅是举例说明,而非以任何方式限制本发明的范围。
实施例1:异补骨脂甲素诱导胚胎干细胞(embryonic stem cell,ES细胞)体外定向分化为神经细胞的研究
1.ES细胞支持培养:
小鼠ES细胞D3细胞株(ES-D3细胞)在基础培养基(含高糖DMEM、非必需氨基酸、10%胎牛血清、白血病抑制因子)中,按1×106个/ml的密度接种于饲养层细胞(小鼠成纤维细胞)上,2~3天进行传代培养。
2.异补骨脂甲素诱导ES细胞定向分化为神经细胞的培养:
ES-D3细胞用胰酶消化,用分化培养基(含高糖DMEM、非必需氨基酸、10%胎牛血清、不含白血病抑制因子)将其制成单细胞悬液,按约600个/30μL接种于培养皿盖子内表面上,将盖子翻转形成悬滴,置培养箱中培养3天。此后将已形成拟胚体(embryoid bodies,EBs)的悬滴转入盛有10mL分化培养基的培养皿内,继续悬浮培养1天。第二天加入异补骨脂甲素与ES细胞拟胚体悬浮共培养4天。之后,再将拟胚体转移到预铺了多聚鸟氨酸及层粘连蛋白的96孔板内,每孔加入100μL ES细胞分化培养基(DMEM/F12+N2/B27+1%胎牛血清)及异补骨脂甲素。此时开始用倒置显微镜每天观察细胞,以捕获出现神经样细胞(细胞突起的长度为胞体直径的5倍以上)的最初时间。
参见图1,通过以上实验获得的拟胚体,若能出现神经样细胞(细胞突起的长度为胞体直径的5倍以上),则以此作为定向分化成神经细胞的指标。通过相差显微镜观察,结果显示异补骨脂甲素诱导ES细胞定向分化为神经细胞作用非常明显,与溶剂对照组相比,其与ES细胞拟胚体共孵育8-10天时,就有80%以上拟胚体出现神经样细胞,数量众多,神经细胞突起较密,相互连接成网状,在拟胚体周围铺展分布,缓慢向远端延伸。图1中A:溶剂对照(低倍),B:溶剂对照(高倍),C:异补骨脂黄酮0.1mol/L(低倍),D:异补骨脂黄酮0.1mol/L(高倍)。
实施例2:免疫荧光实验鉴定异补骨脂甲素诱导ES细胞体外定向分化为神经前体细胞
ES细胞分化早期,三胚层结构处在不断分化中,其中神经前体细胞的形成是细胞分化为神经细胞的关键步骤。巢蛋白(nestin)是神经前体细胞的标志蛋白,以此为标志我们对分化早期异补骨脂甲素诱导作用下神经前体细胞的差异进行了考察。结果显示,在8+0天,经异补骨脂甲素诱导后nestin免疫染色阳性细胞有较明显的差异,流式细胞术定量检测结果显示,异补骨脂甲素能显著增加ES细胞分化而来的nestin阳性神经前体细胞比例,参见图2。图2 是异补骨脂甲素能显著增加ES细胞分化而来的nestin阳性神经前体细胞比例图,(维A酸RA为阳性对照)其中图2A是流式检测结果图;图2B是A图的定量分析柱形图。
实施例3:免疫荧光实验鉴定异补骨脂甲素诱导ES细胞体外定向分化为神经细胞
收集分化8+6天的异补骨脂甲素诱导组细胞样本,用纯冷甲醇固定10min,再用牛血清封闭处理30min,然后加入一抗:(1)神经胶质细胞特异性蛋白monoclonal mouse anti-GFAP,1∶200稀释,(2)神经元细胞特异性蛋白monoclonal mouse anti-β-tubulin III,1∶100稀释,4℃孵育过夜。第二天加入二抗:FITC-Conjugated goat anti-Mouse IgG或Rhodamine-Conjugated goatanti-Rabbit IgG,1∶1000稀释,37℃孵育1.5h。无荧光甘油封片后用荧光显微镜观察,拍照记录。
参见图3,结果显示,异补骨脂甲素诱导ES细胞分化8+6天,出现有神经胶质细胞特异性胶质纤维酸性蛋白(GFAP)阳性的神经胶质细胞及神经元细胞特异性微管蛋白(β-TubulinIII)阳性的神经元细胞,其中以β-TubulinIII阳性细胞最为多见,呈现红色荧光,多分布于克隆边缘,胞体为圆形、椭圆形,突起为一个至数个,长度大小不一,其细长突起彼此相连联交织成复杂的网状,它们可能是不同发育阶段的神经元细胞;少数细胞表达神经胶质细胞标志物GFAP,呈现绿色荧光。图3中A:β-tubulin III阳性细胞(红色),B:GFAP阳性细胞(绿色)。
实施例4:RT-PCR实验鉴定异补骨脂甲素诱导ES细胞体外定向分化为神经细胞
收集分化d 8+6由异补骨脂甲素诱导组和溶剂对照组细胞,按照Trizolreagent说明书提取总mRNA。引物序列与实验条件如下(表1):
表1.引物序列和PCR反应条件
PCR反应体系:94℃变性30s,72℃延伸60s,β-tubulinIII、GFAP、β-actin退火温度分别是58℃、58℃、55℃,分别循环40、40、30轮,末次循环后72℃再延伸10min,4℃冷却保存。PCR产物1.5%琼脂糖凝胶电泳,用凝胶成像***观察及拍照分析。
参见图4,实验结果显示,经异补骨脂甲素诱导ES细胞分化8+6天时,β-tubulin III和GFAP基因有表达,且表达量与溶剂对照组比较明显增加。
实施例5:异补骨脂甲素诱导骨髓间充质干细胞(bone mesenchymal stemcells,BMSCs)定向分化为神经细胞的研究
BMSCs具有干细胞的自我复制和多向分化潜能两个特性,可以向多种***和部分来源于外胚层和中胚层的组织分化,其中包括神经细胞。
1.BMSCs的分离培养:取大鼠股骨、胫骨骨髓置于离心管中,微吸管反复吸吹骨髓,形成分散的单细胞悬液,加入配制的PERCOLL分离液,密度为11073g/mL,离心2000r/min,20min后可见中间有一层约1~2mm厚的白色层,其成分主要为MSCs,仔细用吸管吸取这一层,再用PBS离心后,按1×104个/mL细胞密度接种至含10%FBS的DMEM培养基中培养。
2.将传至第3,5,7代的BMSCs按照0.4×106/ml接种于事先放置有消毒盖玻片的六孔板内制备细胞爬片。当细胞达到70%~80%融合时,弃培养液,加入含bFGF和20%FCS的L-DMEM培养液预诱导24h后,更换培养液,PBS洗两次,再分别加入不同浓度异补骨脂甲素对BMSCs进行诱导分化,采用免疫细胞化学检测神经细胞特异性表达蛋白。
实施例6:异补骨脂甲素诱导神经干细胞定向分化为神经细胞的研究
神经干细胞(neural stem cells,NSCs)是神经***终生保持增殖能力和分化潜能的细胞,可通过对称或不对称***,生成新的干细胞和分化潜能逐渐降低的子细胞,最终生成神经元、星形胶质细胞及少突胶质细胞。
取孕13天胎鼠大脑,在无菌条件下用机械法将其制备成单细胞悬液,加入含B27,bFGF和insulin的DMEM/F12培养液培养。NSC培养7~10天后,形成神经球,呈悬浮状生长。收集NSCs集落,以800r/min离心5min后,计数后接种于预先用多聚赖氨酸处理过的6孔板内,加入含不同浓度异补骨脂甲素的DMEM/F12分化培养液进行诱导培养。采用免疫细胞化学检测神经细胞特异性表达蛋白。
异补骨脂甲素对神经干细胞分化为神经细胞这一过程具有显著诱导分化作用。异补骨脂甲素有望成为一种以神经为作用靶点的新型诱导分化剂。由于 在异补骨脂甲素诱导干细胞定向分化为神经细胞过程中富含发育依赖性生物信息,能提供很多靶点供药效评价用,因此可将神经发育依赖性特殊基因和蛋白表达等能够反映神经细胞功能的指标,借助PCR、免疫组化、Western Blot等技术对异补骨脂甲素的药效进行深入评价,以论证异补骨脂甲素对神经细胞生长和功能改善的促进作用。
无需进一步详细阐述,相信采用前面所公开的内容,本领域技术人员可最大限度地应用本发明。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。
本发明涉及的序列
<110>浙江大学
<120>异补骨脂甲素诱导干细胞定向分化为神经细胞的用途
<160>6
<210>1
<211>30
<212>DNA
<213>人工序列
<220>
<223>根据β-actin mRNA序列设计的PCR检测上游引物序列
<400>1
TGACGGGGTCACCCACACTGTGCCCATCTA 30
<210>2
<211>30
<212>DNA
<213>人工序列
<220>
<223>根据β-actin mRNA序列设计的PCR检测下游引物序列
<400>2
CTAGAAGCATTTGCGGTGGACGATGGAGGG 30
<210>3
<211>20
<212>DNA
<213>人工序列
<220>
<223>根据β-tubulinIII mRNA序列设计的PCR检测上游引物序列
<400>3
GGTGTCCGAGTACCAGCAGT 20
<210>4
<211>20
<212>DNA
<213>人工序列
<220>
<223>根据β-tubulinIII mRNA序列设计的PCR检测下游引物序列
<400>4
GAAGAGCACCAGAGACCCAG 20
<210>5
<211>25
<212>DNA
<213>人工序列
<220>
<223>根据GFAP mRNA序列设计的PCR检测上游引物序列
<400>5
AAGCTCCAAGATGAAACCAACCTGA 25
<210>6
<211>23
<212>DNA
<213>人工序列
<220>
<223>根据GFAP mRNA序列设计的PCR检测下游引物序列
<400>6
GCAAACTTAGACCGATACCACTC 23
Claims (4)
1.一种异补骨脂甲素在诱导干细胞体外定向分化方面的用途,其特征在于,在体外定向分化为单一类型神经细胞,在干细胞移植时作为制备治疗神经退行性疾病的促细胞分化剂中的应用,所述干细胞是指小鼠胚胎干细胞、神经干细胞或骨髓间充质干细胞。
2.一种异补骨脂甲素在诱导干细胞体外定向分化方面的用途,其特征在于,在干细胞移植时作为制备修复重建损伤神经组织的促细胞分化剂中的应用,所述干细胞是指小鼠胚胎干细胞、神经干细胞或骨髓间充质干细胞。
3.一种异补骨脂甲素在诱导干细胞体外定向分化方面的用途,其特征在于,在构建药效筛选与评价模型中应用,所述干细胞是指小鼠胚胎干细胞、神经干细胞或骨髓间充质干细胞。
4.根据权利要求1-3任一所述的异补骨脂甲素的应用,其特征是:所述的异补骨脂甲素能诱导干细胞体外定向分化获得单一类型神经细胞,通过以下步骤实现:
(1)采用经典的4-/4+法进行诱导分化培养,小鼠胚胎干细胞按900个/30μl细胞接种于培养皿盖内表面,翻转后形成悬滴,置于培养箱中3天,(2)将已形成拟胚体的悬滴转入盛有10m1分化培养液的培养皿内,继续悬浮培养1天,第二天加入受试药物异补骨脂甲素与拟胚体悬浮共培养4天,(3)再将已形成的拟胚体转入预铺多聚鸟氨酸的细胞培养板内,评价神经样细胞的指标是出现细胞突起长度为胞体直径3-5倍以上。
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