CN106497877A - 一种促进骨髓间充质干细胞向神经诱导分化的基质材料 - Google Patents
一种促进骨髓间充质干细胞向神经诱导分化的基质材料 Download PDFInfo
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Abstract
本发明涉及一种促进骨髓间充质干细胞向神经诱导分化的基质材料,具体提供了一种促进骨髓间充质干细胞分化的支架粒子,所述支架粒子为烟草花叶病毒与异亮氨酸‑赖氨酸‑缬氨酸‑丙氨酸‑缬氨酸五肽的缀合物。本发明还提供了包含上述支架粒子的培养基,所述培养基使骨髓间充质干细胞增殖加快,细胞密度增加,有利于更好的细胞粘附和增殖。
Description
技术领域
本发明涉及一种促进骨髓间充质干细胞向神经诱导分化的基质材料。
背景技术
最近的证据表明,骨髓间充质干细胞可以跨分化为神经外胚层细胞。这已经引起了科学界用骨髓间充质干细胞治疗神经系统退行性变和自身免疫性疾病巨大的兴趣。骨髓细胞容易获得,不仅克服利用胎儿组织相关的伦理和免疫问题而且克服从脑中获得脑神经干细胞的有关风险。尽管已知骨髓间充质干细胞有很多潜能,深入了解这些多能干细胞如何发育成神经细胞仍是一个核心问题。各国科学家在不断探索如何在体外将骨髓间充质干细胞向神经系统细胞高效诱导分化。除了多种化学品和细胞因子被用于尝试启动骨髓间充质干细胞向神经分化,细胞外微环境的特性越来越被认为是影响幼稚骨髓间充质干细胞增殖,活力和分化的相关细胞刺激。研究表明,细胞外基质的表面化学、刚度和纳米形貌都对控制骨髓间充质干细胞的命运起着重要的作用。能够容纳骨髓间充质干细胞生长并诱导其分化成特定谱系的优化支架在各种组织工程和细胞治疗应用中是非常可取的。在这里,我们报告如何通过烟草花叶病毒产生的纳米形貌结合多价异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽抗原决定簇显示与神经诱导剂维甲酸协同启动骨髓间充质干细胞向神经样细胞表型分化,从而在大鼠脊髓全横断后诱导强健的源自骨髓间充质干细胞神经形成。
发明内容
本发明所要解决的技术问题是提供一种促进骨髓间充质干细胞向神经诱导分化的基质材料。
本发明解决上述问题的技术方案如下所述:
利用烟草花叶病毒(TMV)的自然结构特征通过重氮偶联和Cu(I)催化叠氮-炔的1,3-偶极环加成(CuAAC)两步反应在其壳体Y139基团铆接IKVAV配体将其开发成一种MSC神经形成优化支架,称为TMV-IKVAV纳米管,如示意图1所示。
上述技术方案中,所述的IKVAV配体是异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽抗原决定簇;所述的烟草花叶病毒通过市售途径获得或可采用植物病毒学方法从感染烟草花叶病毒的烟草叶中提取得到,具体提取方法可参照Gooding GV Jr和Hebert TT所公开的方法实施(Gooding GV Jr,Hebert TT,A simple technique for purification oftobacco mosaic virus in large quantities,Phytopathology,1967,57(11):1285)。
上述TMV-IKVAV纳米管的制备方法由以下步骤组成:
(1)按植物病毒学方法从感染烟草花叶病毒的烟草叶中提取烟草花叶病毒,然后将其加入到缓冲液中,使烟草花叶病毒在稳定液中的重量浓度为15-30mg/mL,得到含烟草花叶病毒的储存液;
(2)取3-乙炔苯胺1原位生成的重氮盐;
(3)TMV与3-乙炔苯胺1原位生成的重氮盐在4℃pH 9的缓冲溶液中处理形成炔烃接枝TMV粒子2。
(4)溴-端基GIKVAV用NaN3在DMSO衍生化为叠氮-端基GIKVAV 3。
(5)2和3用来通过铜(I)催化叠氮-炔环加成反应(CuAAC))合成IKVAV接枝TMV粒子(TMV-IKVAV)。作为一个通用的CuAAC反应方案,2mg/ml 2和3mM 3加入含CuSO4(1mM)和抗坏血酸钠(2mM)的Tris缓冲(10mM,pH 7.8)溶液中。在4℃孵育18h后,TMV-IKVAV粒子通过蔗糖梯度离心纯化。
本发明所述的TMV-IKVAV纳米管可按常规的方法制成涂层覆在细胞培养基质表面,以促进骨髓间充质干细胞向神经诱导分化。
具体地,本发明一个方面提供了一种促进骨髓间充质干细胞分化的支架粒子,所述支架粒子为烟草花叶病毒与异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽的缀合物。
本发明的另一个方面提供了上述支架粒子的制备方法,其包括如下步骤:
1)将烟草花叶病毒将加入到缓冲液中,使烟草花叶病毒的重量浓度为15-30mg/ml,得到含烟草花叶病毒的储存液;
2)取3-乙炔苯胺1原位生成的重氮盐;
3)烟草花叶病毒的储存液与3-乙炔苯胺1原位生成的重氮盐在处理形成炔烃接枝烟草花叶病毒粒子;
4)将溴-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽用NaN3衍生化为叠氮-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽;5)将步骤3)所得炔烃接枝烟草花叶病毒粒子和步骤4)所得叠氮-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽加入含CuSO4和抗坏血酸钠的Tris缓冲溶液中,在2-10℃下孵育后纯化,获得异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽接枝烟草花叶病毒支架粒子。
本发明的再一个方面提供了一种促进骨髓间充质干细胞分化的培养基,其包含烟草花叶病毒或上述的一种促进骨髓间充质干细胞分化的支架粒子。
进一步地,是将包含烟草花叶病毒或权利要求1所述的一种促进骨髓间充质干细胞分化的支架粒子涂覆在氨基丙基三乙氧基甲硅烷涂层基板。
进一步地,其还含维甲酸,优选维甲酸的含量为10-50μM,更优选为15-30μM。
进一步地,其还含有胎牛血清、青霉素和/或链霉素。
本发明另一个方面提供了上述支架粒子在促进骨髓间充质干细胞向神经诱导分化的应用。
本发明另一个方面提供了上述的支架粒子在制备促进骨髓间充质干细胞向神经诱导分化的培养基的应用。
本发明另一个方面提供了上述培养基培养获得的神经细胞,以及该神经细胞在制备治疗神经损伤疾病的药物中的应用。
由于烟草花叶病毒为一种具有一定的刚性的自然分子支架,可近似为一个纳米杆,长300nm,直径18nm,其独特的形状类似于主要的细胞外基质成分的结构。此外,TMV其衣壳蛋白外表面提供2130个非常活跃的酪氨酸-139(Y139)基团,可以很容易地用各种各样的小分子修饰。因此它提供了一种理想的多价显示支架能够沿此纳米棒多重显示活性序列。每个TMV-IKVAV颗粒上有共约2130个IKVAV抗原决定簇。充分研究的TMV结构允许精确控制配体的间距和空间取向,其结果是,大多数的抗原决定簇可用于与受体结合;且IKVAV抗原决定簇在TMV-IKVAV表面上高浓度呈现,进一步促进有效和强大的细胞-配体相互作用和信号转导。
附图说明
图1为用IKVAV修饰TMV示意图。
图2为天然烟草花叶病毒(17528m/z),炔衍生物2(17656m/z)和TMV-IKVAV(18380m/z)亚基蛋白的基质辅助激光解吸离子飞行质谱(MALDI-TOF MS)。
图3a-f为骨髓间充质干细胞在TMV,TMV-IKVAV,laminin和APTES基板的行为结果。
图4a-f为骨髓间充质干细胞向神经分化结果。
图5a-b为动物实验五组后肢随着时间的推移BBB开放场行走评分以及术后三个月所有五组大鼠的典型表现。
具体实施方式
实施例1诱导分化的基质材料的制备
(1)烟草花叶病毒通过市售途径获得或按植物病毒学方法从感染烟草花叶病毒的烟草叶中提取,然后将其加入到10mM pH7.4磷酸盐缓冲液中,使烟草花叶病毒在稳定液中的重量浓度为15-30mg/mL,得到含烟草花叶病毒存储液;
(2)取3-乙炔苯胺原位生成的重氮盐;
(3)将步骤(1)所得的含烟草花叶病毒缓冲液与3-乙炔苯胺原位生成的重氮盐在4℃100mM pH 9的硼酸盐缓冲溶液中处理2小时形成炔烃接枝烟草花叶病毒粒子;
(4)将溴-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸(GIKVAV)用NaN3在DMSO中衍生化为叠氮-端基GIKVAV;
(5)将2mg/ml步骤(3)所得炔烃接枝烟草花叶病毒粒子和3mM步骤(4)所得叠氮-端基GIKVAV加入含CuSO4(1mM)和抗坏血酸钠(2mM)的Tris缓冲(10mM,pH 7.8)溶液中,在4℃下孵育18h后通过蔗糖梯度离心纯化,获得IKVAV接枝TMV粒子(TMV-IKVAV);其基质辅助激光解吸离子飞行质谱见图2。
(6)氨基丙基三乙氧基甲硅烷(APTES)涂层基板可由市售途径获得,将IKVAV接枝TMV粒子涂覆到细胞培养APTES基板表面形成致密层,获得APTES-TMV-IKVAV基板。同时,也将TMV粒子和层粘连蛋白(laminin)分别涂覆到APTES基板表面,分别得到APTES-TMV和APTES-laminin基板。
实施例2骨髓间充质干细胞培养,粘附和铺展研究
(1)实验方法
初级骨髓间充质干细胞是从80g Sprague-Dawley大鼠的骨髓中分离。该程序是按照南方医科大学动物护理和伦理委员会动物实验的指导方针进行。MSC在含10%胎牛血清,100U/ml青霉素,100mg/ml链霉素的完全DMEM培养基中,在37℃湿润的5%CO 2培养箱中培养。在生长阶段(约90%汇合)的骨髓间充质干细胞单层用0.25%胰蛋白酶-0.02%EDTA消化2分钟从培养板上剥离,并重新悬浮于完全培养基中用于随后的细胞粘附研究。
APTES-TMV,APTES-TMV-IKVAV,APTES-laminin和APTES基板(10×10mm2)放入6孔板;在完全DMEM溶液中2mL骨髓间充质干细胞密度为1×105个/mL细胞悬浮液加入到6孔板的每个孔中,并在湿润的5%CO2培养箱中于37℃保温。在接种后2小时和24小时分别检查细胞在预涂基板粘附和铺展,在玻片上的非贴壁细胞通过用PBS洗涤去除,贴壁细胞用甲醛固定和苏木精-伊红染色,得到染色的细胞显微照片。用INFINITY分析软件(lumenera公司,渥太华,加拿大)进行细胞形态学分析。使用单因素方差分析SNK-q检验进行统计分析。P<0.05的值被认为是显著。
(2)实验结果
实验结果见图2。由图2可见,骨髓间充质干细胞附着在TMV-IKVAV表面比TMV,laminin和APTES基质多4倍,附着增加有统计学意义(p<0.05);与在TMV,Laminin和APTES上的培养相比,TMV-IKVAV上培养的骨髓间充质干细胞增殖加快,24小时达到高汇合,TMV-IKVAV细胞密度增加达到近四倍,表明在TMV的表面引入高密度IKVAV抗原决定簇有利于更好的细胞粘附和增殖。
实施例3骨髓间充质干细胞向神经诱导分化实验
(1)实验方法
神经预诱导培养基为含10%胎牛血清,1%青霉素/链霉素和15μM维甲酸的DMEM培养基。神经诱导培养基为含10%胎牛血清,1%青霉素/链霉素和30μM维甲酸的DMEM培养基。两种方案被用来评估神经源性分化。在方案一中,骨髓间充质干细胞以1×105细胞/毫升在原代培养基中接种在APTES-TMV,APTES-TMV-IKVAV,APTES-laminin和APTES基板上24小时,接着用预诱导培养基培养24小时后,然后将培养基替换为含30μM视黄酸的神经诱导培养基。在方案二中,骨髓间充质干细胞在神经诱导培养基中在ATI基板上以1×105cells/mL接种和培养两周,每3天换一次培养基。非诱导对照MSCs在同一时间内只喂含10%小牛血清和1%青霉素/链霉素的基础培养基。
(2)实验结果
骨髓间充质干细胞向神经诱导分化(方案一)实验结果见图3。由图3可见,TMV-IKVAV基质快速促进大量的神经突起生长,远远超过了在TMV、层粘连蛋白、APTES基板上所观察到的。实时荧光定量RT-PCR结果表明,在TMV-IKVAV基质上向神经分化的MSCs中Otx1呈27.4倍上调,比在烟草花叶病毒、层粘连蛋白和APTES基质上的表达高3倍多,此外,TMV-IKVAV基质上的细胞nestin的表达也比在TMV,laminin和APTES基质上的细胞上调2-3倍。也就是说,正如所期望的,TMV-IKVAV显著上调Otx1和nestin表达。
实施例4脊髓横断大鼠移植向神经诱导分化骨髓间充质干细胞动物实验
(1)实验方法
所有的动物实验由南方医科大学动物护理和伦理委员会批准(ACEC,SMU)。共有30只成年SD大鼠(150-200g)为研究对象。将大鼠麻醉,在显微镜下进行完全的T10横断损伤产生的一个2.0mm的完全横断腔,病变腔仔细地检查,确保腹侧和横向完全横断病变。动物随机分为五组,每组6只,如下:组1,在脊髓损伤部位注射0.1ml生理盐水,作为非治疗对照组,称为损伤对照(LC)组;组2,在脊髓横断部位植入0.1毫升1×108细胞/毫升在TMV板上培养的似神经骨髓间充质干细胞,命名为TMV组;组3,在脊髓横断部位植入0.1毫升1×108细胞/毫升在TMV-IKVAV板上培养的神经源性骨髓间充质干细胞,命名为TMV-IKVAV组;组4,在脊髓横断部位植入0.1毫升1×108细胞/毫升在Laminin板上培养的神经源性骨髓间充质干细胞,命名为Laminin组;组5,在脊髓横断部位植入0.1毫升1×108细胞/毫升在APTES板上培养的神经源性骨髓间充质干细胞,命名为APTES组。逐层缝合关闭切口。手术后,大鼠分笼饲养,在12h光/暗周期获得食物和水自由采食。抗生素给药,每天两次,持续两周和膀胱轻轻按摩手动清空直至反射性膀胱排空约10天后恢复。
行为评价是以开放场BBB评分系统进行(Basso等人,1995,1996)。在一个开放场(75×120厘米),动物的行为,由两名研究人员观察。从0(完全瘫痪)到21(正常步态)的分数,在手术后的第一个星期每天记录,此后每周三次,直到手术后13周。
(2)实验结果
实验结果如图4所示。由图4可见,LC组导致永久性瘫痪和损伤部位以下的感觉丧失。APTES组和Laminin组后肢运动恢复慢,在3个月的过程中,左、右后肢BBB评分没有比4更高的;且Laminin组大鼠术后背部皮肤反复脱毛,进食少,蜷缩畏寒;而APTES组大鼠出现明显驼背。相比之下,TMV-IKVAV组大鼠术后第二周后肢肚关节即可向前弯曲,BBB评分最高,并在康复期继续稳步上升至3个月时BBB评分达12.2-16.4,且后肢肌无萎缩,无鼓槌指,很活跃,能跨上笼壁伸展身体。TMV组能慢步跛行,缓慢扶笼壁站立,康复情况优于Laminin组和APTES组,但不及TMV-IKVAV组。
在本研究中,TMV纳米管被设计用来向骨髓间充质干细胞多价呈递促进神经突起抗原决定簇IKVAV。我们的研究表明TMV-IKVAV可以用于增强骨髓间充质干细胞的粘附和增殖,并能促进其向神经诱导分化;在TMV-IKVAV上骨髓间充质干细胞衍生的神经性细胞移植入严重脊髓损伤部位可以整合入宿主神经网络并促进恢复神经连接。我们的研究结果勾勒出一种形式的中枢神经系统再生,对治疗外伤性脊髓损伤等中枢神经系统疾病具有重要意义。
Claims (8)
1.一种促进骨髓间充质干细胞分化的支架粒子,所述支架粒子为烟草花叶病毒与异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽的缀合物。
2.权利要求1所述的支架粒子的制备方法,其包括如下步骤:
1)将烟草花叶病毒将加入到缓冲液中,使烟草花叶病毒的重量浓度为15-30mg/ml,得到含烟草花叶病毒的储存液;
2)取3-乙炔苯胺1原位生成的重氮盐;
3)烟草花叶病毒的储存液与3-乙炔苯胺1原位生成的重氮盐在处理形成炔烃接枝烟草花叶病毒粒子;
4)将溴-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽用NaN3衍生化为叠氮-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽;5)将步骤3)所得炔烃接枝烟草花叶病毒粒子和步骤4)所得叠氮-端基-甘氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸六肽加入含CuSO4和抗坏血酸钠的Tris缓冲溶液中,在2-10℃下孵育后纯化,获得异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸五肽接枝烟草花叶病毒支架粒子。
3.一种促进骨髓间充质干细胞分化的培养基,其包含烟草花叶病毒或权利要求1所述的一种促进骨髓间充质干细胞分化的支架粒子。
4.权利要求3所述的培养基,其是将包含烟草花叶病毒或权利要求1所述的一种促进骨髓间充质干细胞分化的支架粒子涂覆在氨基丙基三乙氧基甲硅烷涂层基板。
5.权利要求3或4所述的培养基,其还含维甲酸,优选维甲酸的含量为10-50μM,更优选为15-30μM。
6.权利要求5所述的培养基,其还含有胎牛血清、青霉素和/或链霉素。
7.权利要求1所述的支架粒子在促进骨髓间充质干细胞向神经诱导分化的应用。
8.权利要求1所述的支架粒子在制备促进骨髓间充质干细胞向神经诱导分化的培养基的应用。
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