CN112980940A - 表皮生长因子Betacellulin在制备周围神经再生调控药物中的应用 - Google Patents
表皮生长因子Betacellulin在制备周围神经再生调控药物中的应用 Download PDFInfo
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- Tropical Medicine & Parasitology (AREA)
Abstract
本发明提供了表皮生长因子Betacellulin在制备周围神经再生调控药物中的应用。本发明首次揭示生长因子Betacellulin能够调控施万细胞迁移和轴突再生,可通过Betacellulin的敲降抑制施万细胞的迁移和神经元轴突生长或者通过外源性给予Betacellulin能够促进施万细胞的迁移和轴突再生。Betacellulin可以作为药物设计靶点,用于治疗周围神经损伤修复有关的疾病。
Description
技术领域
本发明涉及生物医药领域,特别涉及Betacellulin在制备周围神经再生调控药物中的应用。
背景技术
人体神经系统损伤修复属于世界性重大医学难题,尽管与中枢神经相比,周围神经系统表现出更强的再生能力,但周围神经损伤,尤其是伴有长距离神经缺损的周围神经损伤仍然是一个严重危害人类健康的问题。周围神经的再生依赖于神经元内源生长能力的激活和再生微环境的成功构建。在损伤局部给予具有神经营养和促神经再生特性的蛋白质和分子,在很大程度上有利于神经再生,被认为是一种很有前途的促再生策略。
当周围神经遭受创伤后,施万细胞去分化为未成熟细胞类型,大量分裂增殖以产生更大的群体,并消化轴突和髓鞘碎片以清扫随后轴突再生的通道,然后施万细胞开始沿着再生轴突迁移形成Büngner带,并引导轴突再生,并进行再分化和髓鞘形成,同时分泌神经营养因子、生长因子、细胞粘附分子、细胞外基质等,为神经再生提供营养物质,重建神经再生微环境。
生长因子是一类调节细胞行为的天然分子。许多生长因子,如神经生长因子、脑源性神经营养因子、胶质细胞系源性神经营养因子和成纤维细胞生长因子已被确定为促进神经修复的必需神经营养因子。Betacellulin是一种分泌性生长因子,属于表皮生长因子家族。Betacellulin已被鉴定为许多不同类型细胞的有效有丝分裂原,包括视网膜色素上皮细胞、血管平滑肌细胞、β细胞、视网膜祖细胞和神经干细胞。
发明内容
本发明联合使用高通量测序数据和生物信息学工具首次发现编码β-乙酰球蛋白(Betacellulin)的基因Betacellulin在损伤的大鼠坐骨神经显著上调,并进一步揭示了Betacellulin与施万细胞迁移和轴突再生之间的关系,证明Betacellulin能调节周围神经损伤后再生。
本发明具体技术方案如下:
表皮生长因子Betacellulin在制备周围神经损伤再生调控药物中的应用。
Betacellulin基因在人和大鼠间高度保守,本发明所述表皮生长因子Betacellulin的编码基因核苷酸序列如SEQ ID NO:1所示。
所述Betacellulin可以作为药物设计靶点设计抑制Betacellulin表达的药物,抑制施万细胞的迁移和神经元轴突生长;或者以Betacellulin作为药物设计靶点设计促进Betacellulin表达的药物,或者以Betacellulin作为外源性活性物质,促进施万细胞的迁移和神经再生。
人源重组蛋白是模拟生物体内源的蛋白,运用生物基因工程方法获取的蛋白质药物,能增强内源性蛋白的功能。而小干扰RNA(siRNA)是化学修饰的专门针对细胞中特异性的靶基因的抑制剂。
本发明的一个示例,所述抑制Betacellulin表达的药物是Betacellulin基因的小干扰RNA(Betacellulin的抑制剂)。本发明根据靶基因Betacellulin的mRNA目的片段位置的反义序列设计了双链小干扰RNA,其正义链序列如SEQ ID NO:2、4或6所示。
本发明所述周围神经再生调控药物包括促周围神经再生药物、治疗周围神经损伤和中枢神经损伤疾病药物、治疗施万细胞过度增殖疾病药物,所述施万细胞过度增殖疾病包括神经鞘瘤以及神经损伤后施万细胞大量增殖引发施万细胞的分化障碍。本发明所述的抑制Betacellulin表达的药物为治疗施万细胞过度增殖疾病药物。所述施万细胞过度增殖疾病包括神经鞘瘤以及神经损伤后施万细胞大量增殖引发施万细胞的分化障碍(即大量增殖的施万细胞无法正常分化形成髓鞘)。施万细胞的分化障碍会引发再生的轴突无髓鞘包绕,导致运动和感觉功能恢复受阻,抑制施万细胞增殖也是这类疾病治疗的关键。
所述促进Betacellulin表达的药物或者Betacellulin作为活性药物(如Betacellulin(人源)重组蛋白)为促进施万细胞迁移和治疗神经再生相关疾病药物。
施万细胞作为周围神经系统中特有的神经胶质细胞,为神经元提供必要的物理和营养支持,并围绕轴突形成髓鞘,对维持周围神经的正常生理功能十分重要。周围神损伤后,施万细胞除了开始大量增殖和去分化,还迁移形成条索引导轴突再生,同时施万细胞的表型可以被微环境中的各种细胞和因子所调节。当神经遭受损伤后,加速施万细胞迁移将有益于神经再生和功能恢复。
本发明为了评价Betacellulin(Btc)在周围神经再生中的生物学功能,培养并获得了高纯度的原代大鼠施万细胞,使用anti-Btc抗体对培养的施万细胞进行免疫染色显示,大多数Btc阳性信号与S100β阳性信号重叠,表明Btc蛋白在施万细胞中表达并存在(图1)。采用针对Btc的siRNA片段转染培养的施万细胞或在培养的施万细胞中加入Btc重组蛋白,利用Transwell迁移实验和划痕愈合实验发现,Btc促进施万细胞的迁移能力(图2A-C和图3A&B)。由于生长因子Btc是一种分泌性蛋白,将施万细胞与神经元进行间接共培养,以检测分泌的Btc蛋白对神经元行为的影响,与转染siRNA-Btc组的神经元相比,对照组的神经元具有更长的轴突,表明施万细胞分泌的Btc可以促进轴突的生长(图4A-C)。进一步评估Btc在体内的生物学效应,在大鼠坐骨神经夹伤模型和横断模型的损伤部位局部注射Btc重组蛋白,与生理盐水组相比,注射Btc重组蛋白组大鼠再生神经纤维的长度和施万细胞的迁移距离更长,提示Btc蛋白可在体内促进周围神经损伤后施万细胞迁移和神经再生(图5和图6A-D)。
本发明所述Betacellulin可以直接作为药物或者作为药物的靶点设计促进或抑制其表达的药物,通过与药物的相互作用,提高或抑制机体Betacellulin的表达,进而发挥调节周围神经再生的作用。
本发明的优点:本发明研究表明,可通过调节Betacellulin的表达影响施万细胞的行为,从而促进或抑制神经再生,可以广泛用于临床上有促进周围神经再生或抑制施万细胞不良增殖需求的疾病的治疗,特别是神经再生。考虑到组织工程神经移植已被广泛应用于周围神经损伤的治疗。如神经支架的成分/局部结构的调节和干细胞的加入,已经被用来优化组织工程神经移植物,生长因子具有强烈的损伤后效应,可触发周围神经再生。因此,促进神经再生的生长因子将有助于构建生长因子复合的组织工程神经移植物,有利于周围神经损伤的治疗。
附图说明
图1为体外培养的大鼠施万细胞Btc的免疫荧光。(红色表示S100β,绿色表示Btc,蓝色表示细胞核)。
图2为体外抑制Btc表达对施万细胞迁移的影响。(图2A为施万细胞中siRNA-Btc三个片段的敲降效率图;图2B为siRNA对照和siRNA-Btc转染施万细胞的典型Transwell迁移图像和归一化平均迁移率统计,紫罗兰色代表已迁移的施万细胞;图C为siRNA对照和siRNA-Btc转染施万细胞后代表性划痕愈合图像和空白区域面积的统计)。
图3为Btc重组蛋白对体外培养施万细胞迁移的影响。(图3A经Btc重组蛋白处理和未经Btc重组蛋白处理的施万细胞的典型Transwell迁移图像和归一化平均迁移率,紫罗兰色代表已迁移的施万细胞;图3B为经Btc重组蛋白处理和未经Btc重组蛋白处理的施万细胞代表性划痕愈合图像和空白区域面积的统计)。
图4为施万细胞分泌Btc对轴突生长的影响。(图4A为转染siRNA对照和siRNA-Btc的施万细胞分泌Btc的浓度统计;图4B为施万细胞和背根神经节神经元体外共培养示意图;图4C为与转染了siRNA对照和siRNA-Btc的施万细胞共培养24小时后神经元的代表性免疫荧光图像和标准化平均轴突长度统计,红色表示NF200)。
图5为大鼠坐骨神经挤压伤模型中Btc重组蛋白对神经再生的体内作用。(左侧为大鼠坐骨神经挤压伤后生理盐水处理组和Btc重组蛋白处理组损伤段神经的免疫荧光图像,白色代表SCG10,蓝色代表NF-H;右侧为SCG10在距离压伤部位1、2、3、4、5和6毫米处的标准化平均强度,蓝色线代表生理盐水组,红色线代表Btc重组蛋白组)。
图6为大鼠坐骨神经离断伤及硅胶管桥接后局部应用Btc重组蛋白的体内效应。(图6A&6B分别代表生理盐水和Btc重组蛋白处理后损伤段神经的代表性免疫荧光图像,绿色表示NF-H,红色表示S100β,蓝色表示细胞核;图a&b为高倍镜下图6A&6B种白色方框状a、b区域的放大图,箭头分别表示施万细胞在神经桥内形成条索;图C为再生神经纤维的平均长度统计;图D为施万细胞的平均迁移距离统计)。
具体实施方式
以下通过实施例说明本发明的具体步骤,但不受实施例限制。
在本发明中所使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解,该实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进一步说明。
实施例1
Betacellulin的抑制剂和抑制剂对照均由广州市锐博生物科技有限公司合成。序列如下:
siRNA-1:5’UUUGCGAUGUUUCCGAAGATT 3’(SEQ ID NO:2);
3’TTUCUUCGGAAACAUCGCAAA 5’(SEQ ID NO:3)。
siRNA-2:5’AUGGAUGCAGUAAUGCUUGTT 3’(SEQ ID NO:4);
3’TTCAAGCAUUACUGCAUCCAU 5’(SEQ ID NO:5)。
siRNA-3:5’AAAGAGAGCCAUUGGUUUCTT 3’(SEQ ID NO:6);
3’TTGAAACCAAUGGCUCUCUUU 5’(SEQ ID NO:7)。
抑制剂对照为无意义随机序列。
转染试剂lipofectamine RNAimax由Invitrogen公司生产。
实施例2细胞培养和转染
取新生1天的SD大鼠的坐骨神经,解剖镜下剥除血管膜后放入盛有1mL 3mg/mL胶原酶I的5mL EP管中,将神经组织剪碎,于37℃水浴中消化30min。去除胶原酶加入0.25%的胰酶1mL,37℃水浴消化5-10min后加入3mL完全培养基终止消化,用移液器轻柔吹打至肉眼基本看不到组织块。离心(800rpm×5min、常温),弃上清,用完全培养基清冼两次,以1×106个/mL的细胞密度种至预先用PLL包被中皿中,放入37℃、5%CO2培养箱中。培养24h后换成含有10mM阿糖胞苷的完全培养基。继续培养36h-48h后,PBS清洗2遍,换成含有2μMforskolin和10ng/mL HRG的完全培养基,每3-4d换一次液。待细胞长至融合时,弃去培养皿中的培养基,用PBS清冼两遍,加入0.25%的胰酶,轻轻摇晃,使消化液流遍所有细胞表面,镜下观察发现细胞胞体回缩,细胞间隙增大后,加入2mL完全培养基终止消化,吹散后用完全培养基清冼2遍,加入anti-thy1.1(1:1000)冰上孵育2h,离心弃上清,加入含有250μLrabbit complement和750μL DMEM培养液(1:3)的混合液,37℃孵育45min。完全培养基清冼2遍后吹散细胞以2-4×105个/mL的密度种至预先PLL包被好的培养皿中,并放入培养箱。培养24h后换成含有2μM forsokolin和10ng/mL HRG的完全培养基,每隔2-3d换一次液,待细胞长满培养皿即可使用。化学合成的Betacellulin抑制剂和抑制剂对照物如实施例1所示,转染使用lipofectamine RNAimax试剂(Invitrogen,Carlsbad,CA,USA),根据说明书操作。
实施例3real-time RT-PCR(qRT-PCR)
从实施例2转染siRNA-Btc的施万细胞中提取RNA,采用Oligo dT primer(Invitrogen)进行逆转录。Real-time RT-PCR采用SYBR Green Premix Ex Taq(TaKaRa)在Applied Biosystems Stepone real-time PCR System上进行操作。Betacellulin特异性引物序由一对qRT-PCR引物组成,序列如下:
Btc(forward):5’-TCTCCAGTGCGTGGTGG-3’(SEQ ID NO:8)。
Btc(reverse):5’-CGAGAGAAGTGGGTTTTCGATT-3’(SEQ ID NO:9)。
qRT-PCR反应程序:95℃预变性2min;40个PCR循环(95℃,5s;60℃,10s),在每个循环的延伸阶段收集荧光值;PCR扩增反应结束后,进行产物的溶解曲线分析,以确保PCR产物的质量。用ΔΔCT法分析结果,CT设为反应达到域值时的循环数,则每个基因相对于标准内参的表达量可以用方程2-ΔCT表示,所有反应设置三个复孔,GAPDH作为内参。
结果如图2A所示,结果显示转染Betacellulin抑制剂(siRNA-Btc)的施万细胞中Betacellulin的相对表达量明显低于抑制剂对照(siRNA-Con)组,结果表明本发明设计的Betacellulin抑制剂能够抑制Betacellulin的表达。选择转染siRNA-3的施万细胞用于后续实验研究。
实施例4Betacellulin(人源)重组蛋白体外暴露实验
Betacellulin(人源)重组蛋白由PeproTech公司生产,采用0.1%BSA配制10ng/mlBtc重组蛋白,将重组蛋白加入施万细胞培养基中预处理24小时,对照组使用0.1%BSA进行预处理。
实施例5Transwell迁移实验
Transwell小室的下室表面包被Fibronectin,分别收取实施例2转染siRNA-Btc的施万细胞和实施例4经Betacellulin重组蛋白处理的施万细胞,用DMEM培养基调整细胞密度为1×104个/mL,在transwell上室中加入100μL细胞悬液。下室加入500μL含10%胎牛血清的完全培养基。常规培养24h后结晶紫染色,染色后Leica DC300F正置显微镜进行观察和拍照,随机选取10个视野计数细胞个数。最后用33%冰醋酸将结晶紫完全洗脱下来,洗脱液可在酶标仪上570nm,测其OD值,间接反映迁移细胞数。
图2B&图3A为高倍显微镜下细胞迁移实验图(比例尺为50μm),图2B为转染Betacellulin抑制剂的施万细胞的迁移速率,结果显示,转染Betacellulin抑制剂的施万细胞的迁移速率低于抑制剂对照组,结果表明Betacellulin抑制剂能降低施万细胞的迁移速率。图3A为Betacellulin重组蛋白处理施万细胞的迁移速率,结果显示,Betacellulin重组蛋白处理组施万细胞的迁移速率高于对照组,结果表明Betacellulin增效剂能提高施万细胞的迁移速率。
实施例6细胞划痕愈合实验
分别取实施例2和实施例4获得的施万细胞,调整细胞密度为2×105个/mL,接种到置于6孔板中的1mm宽的模型室中。在移除模型室后的0h和9h分别拍摄相对空白区域,并采用Image Pro Plus(Media Cybernetics,Rockville,MD,USA)来统计相对空白区域的面积。
图2C&图3B为高倍显微镜下细胞划痕实验图(比例尺为100μm),图2C为转染Betacellulin抑制剂的施万细胞的迁移速率,结果显示,转染Betacellulin抑制剂的施万细胞的迁移速率低于抑制剂对照组,结果表明Betacellulin抑制剂能降低施万细胞的迁移速率。图3B为Betacellulin重组蛋白处理施万细胞的迁移速率,结果显示,Betacellulin重组蛋白处理组施万细胞的迁移速率高于对照组,结果表明Betacellulin增效剂能提高施万细胞的迁移速率。
实施例7酶联免疫吸附测定实验(ELISA)
分别取实施例2中转染siRNA-Btc和siRNA-Con的施万细胞,调整细胞密度为6×104个/mL在DMEM培养基中培养24h后,收集施万细胞培养上清液并通过0.22μm过滤器去除细胞碎片和杂质。根据说明书采用Btc ELISA试剂盒(Arigo biolaboratories),使用SynergyTM 2多模式微孔板读取器(BioTek)测定Btc蛋白的分泌量。
图4A为ELISA法测定施万细胞分泌Btc蛋白的含量。ELISA结果显示,转染siRNA-Btc的施万细胞Btc蛋白的分泌量明显少于转染siRNA-Con的施万细胞的分泌量,表明Betacellulin抑制剂能降低施万细胞分泌Betacellulin蛋白。
实施例8神经元和施万细胞体外共培养实验
取新生1天SD大鼠,用显微镊将椎孔中的背根节逐个取出并修剪背根节多余神经根,将剪碎后的神经节组织置入1mL 3mg/mL的I型胶原酶的EP管中,37℃消化30min,去除胶原酶加入0.25%的胰酶1mL,37℃消化5min后加入3mL完全培养基终止消化,采用15%BSA纯化神经元,采用含2%B27补充剂(Gibco)、2mM L-谷氨酰胺(ThermoFisher Scientific)和1%青霉素和链霉素(Invitrogen)的Neurobasal培养基重悬细胞,以1×106个/mL的密度种至预先用PLL包被的Transwell下室的载玻片上,同时将转染siRNA-Btc或siRNA-Con的施万细胞接种到上室,放入37℃,5%CO2培养箱中共培养24小时,用4%多聚甲醛固定神经元,对神经元进行免疫染色。使用Zeiss Axio Imager M2(Zeiss)拍摄神经元图像,Image J测量轴突长度。
图4C为神经元轴突长度测定。结果显示与转染siRNA-Btc的施万细胞共培养的神经元相比,与转染siRNA-Con的施万细胞共培养的神经元具有更长的轴突,表明施万细胞分泌的Btc可以促进神经元轴突生长。
实施例9大鼠坐骨神经夹伤实验
取健康成年雄性SD大鼠(180-220g),腹腔内注射复合麻醉剂,构建大鼠左后肢坐骨神经3mm夹伤模型,将溶解于0.1%BSA中的100ng Btc重组蛋白稀释于5μL生理盐水中,微量注射器将Btc重组蛋白或等量生理盐水(都需与Matrigel等体积混和均匀)注入压伤部位的神经外膜。在挤压伤后4天收集大鼠坐骨神经段,保留坐骨神经夹伤处及上下各0.5cm组织,冰冻切片,厚度12μm,切片用轴突标记物SCG10和NF-H进行免疫荧光染色。
图5为Btc重组蛋白对坐骨神经夹伤后轴突再生的影响。结果显示再生轴突穿过夹伤部位向其靶器官生长,Btc重组蛋白治疗组多个测量位点的SCG10强度比生理盐水治疗组更高,特别是距离夹伤位点1、2和4mm处。表明Btc蛋白促进坐骨神经损伤后再生。
实施例10大鼠坐骨神经横断后桥接硅胶管实验
取健康成年雄性SD大鼠(180-220g),腹腔内注射复合麻醉剂,构建大鼠坐骨神经横断伤形成6mm的神经间隙,植入相应长度的硅胶管,以桥接神经间隙。将溶解于0.1%BSA中的100ng Btc重组蛋白稀释于8μL生理盐水中,分别在管中填充Btc重组蛋白或等量生理盐水(都需与Matrigel等体积混和均匀)。在大鼠坐骨神经横断后10天收集桥接段神经并进行免疫荧光染色。采用Zeiss显微镜软件zen2.3计算轴突延伸长度和施万细胞迁移距离。
图6为Btc重组蛋白对坐骨神经横断伤后施万细胞迁移和轴突再生的影响。相对于坐骨神经夹伤,6mm的神经切断伤则是一种相对更严重的损伤。术后10天,Btc重组蛋白组和生理盐水组均观察到一定程度的施万细胞迁移和轴突延长(图6A和6B)。相对于生理盐水组,注射Btc重组蛋白组的大鼠的神经桥中有更多的施万细胞和相对完整的施万细胞索(图6B和6b),并且Btc重组蛋白处理组的大鼠再生神经纤维的长度和施万细胞的迁移距离更长(图6C和6D)。表明Btc蛋白可促进周围神经损伤后施万细胞迁移和神经再生。
序列表
<110> 南通大学
<120> 表皮生长因子Betacellulin在制备周围神经再生调控药物中的应用
<141> 2021-02-26
<160> 9
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accaatggct ctctttgtgg agctcctggg gaaaactgca caggtaccac ccctagacag 180
aaatcgaaaa cccacttctc tcggtgcccc aagcaataca agcattactg catccatggg 240
agatgccgct tcgtgatgga cgaacaaact ccctcctgca tctgtgagaa aggctacttt 300
ggggcccggt gtgagcaggt ggacctgttt tatctccagc aggacagggg gcagatcctg 360
gtggtctgct tgataggcgt catggtgctg ttcatcattt tagtcattgg cgtctgcacc 420
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Claims (5)
1.表皮生长因子Betacellulin在制备周围神经再生调控药物中的应用,所述表皮生长因子Betacellulin的编码基因核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的应用,其特征在于以Betacellulin作为药物设计靶点设计抑制Betacellulin表达的药物,抑制施万细胞的迁移和神经元轴突生长;或者以Betacellulin作为药物设计靶点设计促进Betacellulin表达的药物,或者以Betacellulin作为外源性活性物质的药物,促进施万细胞的迁移和神经再生。
3.如权利要求2所述的应用,其特征在于所述抑制Betacellulin表达的药物是Betacellulin基因的小干扰RNA。
4.根据权利要求3所述的应用,其特征在于所述Betacellulin基因的小干扰RNA为双链小干扰RNA,正义链序列如SEQ ID NO:2、4或6所示。
5.根据权利要求1所述的应用,其特征在于周围神经再生调控药物包括促周围神经再生药物、治疗周围神经损伤和中枢神经损伤疾病药物、治疗施万细胞过度增殖疾病药物,所述施万细胞过度增殖疾病包括神经鞘瘤以及神经损伤后施万细胞大量增殖引发施万细胞的分化障碍。
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