CN107446027B - 具有核定位能力的透皮短肽及其应用 - Google Patents
具有核定位能力的透皮短肽及其应用 Download PDFInfo
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- CN107446027B CN107446027B CN201710790765.3A CN201710790765A CN107446027B CN 107446027 B CN107446027 B CN 107446027B CN 201710790765 A CN201710790765 A CN 201710790765A CN 107446027 B CN107446027 B CN 107446027B
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Abstract
本发明涉及一种具有核定位能力的透皮短肽，其氨基酸序列如SEQ ID No.1所示；本发明还提供了一种具有核定位能力的融合蛋白，包括大分子蛋白，大分子蛋白的一端连接上述透皮短肽。本发明还公开了上述透皮短肽在制备治疗皮肤病的药物或透皮制剂中的应用。本发明进一步提供了一种治疗皮肤病的药物，包括上述透皮短肽以及药学上可接受的辅料。本发明的透皮短肽可自主进入细胞后定位于细胞核且具有透皮能力，能透过皮肤角质层，进入真皮层中的细胞中。该短肽人工合成方便、能够通过透皮给药，具有携带治疗皮肤疾病药物进行透皮给药治疗的潜力。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种具有核定位能力的透皮短肽及其应用。
背景技术
皮肤是人体最大的器官，它覆盖于全身的表面，约占体重的16％，总面积达1.2～2平方米。皮肤由表皮层、真皮层和皮下组织组成，含有丰富的血管、神经以及多种皮肤附属器，包括毛发、毛囊、汗腺、皮脂腺等。它是机体的第一道防线，防止组织和内脏器官受到外来的物理、化学、生物刺激因子对机体的侵袭。除此之外，皮肤还具有免疫、感受刺激、调节体温、吸收分泌和排泄等作用。角质层是表皮最外层的部分，主要由10-20层扁平、没有细胞核的死亡细胞组成。当这些细胞脱落时，其下方位于基底层的细胞会被推上来，形成新的角质层。角质层的功能是对皮肤进行物理性、机械性、化学性以及生物性的保护，能够阻挡绝大多数大分子物质的进入，使其免受有害物质的损伤，保持其功能的完整性。但是角质层这一屏障也阻止了表皮和真皮组织对通过表皮补给的外源营养物质和药物的吸收。
透皮促渗剂是一类是指可以加快外用药物穿透皮肤的化合物，如吡咯酮类、氮酮、萜类、氨基酸脂类等化合物。然而现有的透皮促渗化合物持续时间短，易被代谢掉，且透皮效果不理想，高浓度时具有皮肤毒性，尤其对于蛋白质等生物大分子的透皮几乎没有作用。
正是由于大分子蛋白质和多肽类药物均系亲水性大分子，几乎不能透过皮肤角质层的亲脂性屏障。即使在透皮促渗剂的帮助下，分子量大于500Da的蛋白质分子也难以透过角质层。因此，长期以来，具有生物活性的大分子蛋白质的透皮给药一直没有解决。如何使得具有重要价值的生物大分子能够通过表皮途径给药成为研究的难点与热点。采用物理方法介导蛋白质透皮研究广泛，包括超声、电击、微针等方法，但是由于上述方法对设备具有依赖性，难以得到大规模应用。
近年来采用短肽介导的蛋白质透皮引起了广泛的关注。寻找到新的能介导大分子透皮的短肽后，有望通过将功能性蛋白质、多肽及化合物与透皮短肽相连后，通过透皮给药的方式治疗相关疾病。因此，提供一种具有核定位能力的透皮短肽具有重要的现实意义。
细胞核(nucleus)是真核细胞内最大、最重要的细胞器，是细胞遗传与代谢的调控中心，是真核细胞区别于原核细胞最显著的标志之一。细胞膜及细胞核膜均是选择性通透的膜，这一方面对于细胞及细胞核是一种保护，但也组织了功能性细胞及细胞核保护分子的进入。因而筛选能介导关键分子进入细胞膜及核膜并定位于细胞核的短肽对于携带重要分子保护细胞核具有重要意义。但目前未出现关于同时具有核定位及透皮能力的短肽的报道。
发明内容
为解决上述技术问题，本发明的目的是提供一种具有核定位能力的透皮短肽及其应用，本发明的透皮短肽可自主进入细胞并定位于细胞核，且能透过皮肤角质层，进入真皮层中的细胞中；该短肽人工合成方便、能够通过透皮给药。
本发明提供了一种具有核定位能力的透皮短肽，其氨基酸序列如SEQ ID No.1所示，透皮短肽可自主进入真核细胞。
进一步地，透皮短肽还连接有荧光素。
进一步地，荧光素为异硫氰酸荧光素(FITC)、罗丹明或羧基荧光素(FAM)。
进一步地，真核细胞为HaCaT细胞、WS1细胞、TE-1细胞、Eca-109细胞、Hela细胞或原代皮肤细胞。
具有核定位能力的透皮短肽的等电点为10.92，分子量为2465Da。
透皮短肽以剂量依赖性及时间依赖性进入真核细胞，浓度为0.1-10μmol/L，在0.1-240min后可成功进入细胞。
本发明还提供了一种具有核定位能力的融合蛋白，包括大分子蛋白，大分子蛋白的一端连接上述具有核定位能力的透皮短肽(氨基酸序列如SEQ ID No.1所示)。
进一步地，透皮短肽还连接有荧光素。
进一步地，大分子蛋白的另一端连接有荧光蛋白。
进一步地，荧光蛋白为绿色荧光蛋白(EGFP)。
进一步地，大分子蛋白的分子量为10-50KD。
进一步地，大分子蛋白的氨基酸序列如SEQ ID No.2所示；或者大分子蛋白为人超氧化物歧化酶1(SOD1)。
本发明还公开了上述具有核定位能力的透皮短肽(氨基酸序列如SEQ ID No.1所示)在制备治疗皮肤病的药物或透皮制剂中的应用。
优选地，皮肤病为皮肤损伤，更优选地，皮肤病为放射性皮肤损伤。
进一步地，透皮短肽还连接有荧光素。
本发明还提供了一种治疗皮肤病的药物，包括上述具有核定位能力的透皮短肽以及药学上可接受的辅料。
进一步地，治疗皮肤病的药物的剂型为水浸剂、粉剂、洗剂、酊剂、油剂、乳剂、软膏、硬膏或气雾剂。
借由上述方案，本发明至少具有以下优点：
本发明公开的透皮短肽一方面具有自主进入细胞后定位于细胞核的能力；另一方面具有透皮能力，能透过皮肤角质层，进入真皮层中的细胞中。该短肽分子量小，具有人工合成方便、能够通过表皮给药等特点，具有携带治疗皮肤疾病治疗药物透皮的潜力。
本发明公开的透皮短肽可以与大分子蛋白融合后，所得到的融合蛋白能够进入细胞中并进行核定位，说明本发明的透皮短肽具有携带与其相连的大分子蛋白进入细胞并进入核定位的功能。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明的透皮短肽以及对照短肽经FITC标记后进HaCaT细胞及核定位能力检测结果；
图2是本发明的透皮短肽以及对照短肽经FITC标记后进WS1细胞及核定位能力检测结果；
图3是本发明的透皮短肽以及对照短肽经FITC标记后进TE-1细胞及核定位能力检测结果；
图4是本发明的透皮短肽经FITC标记后进HaCaT细胞百分比的剂量依赖性和时间依赖性检测结果；
图5是本发明的融合蛋白进HaCaT细胞并定位于细胞核的检测结果；
图6是本发明的透皮短肽的透皮作用结果示意图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
本发明提供的一种具有核定位能力的透皮短肽中所用原料及试剂均可由市场购得。
采用fmoc法合成N端为FITC标记的短肽，其氨基酸序列如SEQ ID NO.1所示。合成后的短肽溶于磷酸缓冲液(PBS，pH＝7.4)，中，配成浓度为500μmol/L的溶液。体外培养HaCaT细胞，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。为了作为对照，使用上述方法合成SEQ ID NO.3所示的对照多肽，其中对照多肽的N端同样用FITC标记。并使用对照多肽，按照上述方法培养细胞后测试荧光结果。与未处理的对照组细胞进行对比，结果如图1所示，图1表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入HaCaT细胞的功能。
实施例2
按照实施例1的方法合成本发明的透皮短肽以及对照多肽，且两种多肽的N端均采用FITC进行标记。按照实施例1的方法培养人成纤维细胞WS1，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液或对照多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。与未处理的对照组细胞进行对比，结果如图2所示，图2表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入WS1细胞的功能。
实施例3
按照实施例1的方法合成本发明的透皮短肽以及对照多肽，且两种多肽的N端均采用FITC进行标记。按照实施例1的方法培养TE-1细胞，待其生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液或对照多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。与未处理的对照组细胞进行对比，结果如图3所示，图3表明，加入本发明短肽的细胞(目的多肽处理组)内的细胞核中能观察到绿色荧光信号，而在加入了无关对照多肽的细胞(无关多肽处理组)中没有绿色荧光，这表明本发明的短肽具有进入TE-1细胞的功能。
实施例4
按照实施例1的方法合成本发明的透皮短肽，且多肽的N端采用FITC进行标记。按照实施例1的方法在多个培养皿中培养HaCaT细胞，待其生长到密度为50％时，在各培养基中分别加入终浓度为0μmol/L、3μmol/L、6μmol/L和10μmol/L的多肽溶液，4小时后加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。
或待HaCaT细胞生长到密度为50％时，在培养基中加入终浓度为10μmol/L的多肽溶液，于0、60、120和240分钟后，加入DAPI染核30min。弃掉培养液，用PBS漂洗三次，去除游离的荧光多肽后置于共聚焦荧光显微镜下拍照。结果如图4所示，加入本发明短肽的细胞内的细胞核中荧光信号的细胞百分比呈剂量依赖性(图4A所示)和加入时间依赖性(图4B所示)。随多肽剂量的增大以及时间的延长，进入细胞中的多肽越多。
实施例5
本实施例提供了一种融合蛋白，并对其进入细胞及核定位能力进行了检测，具体方法如下：
1、构建偶联蛋白与具有核定位和透皮能力短肽融合的基因
构建具有核定位和透皮能力短肽(氨基酸序列如SEQ ID NO.1所示)，并构建SEQID No.2所示的蛋白和和绿色荧光蛋白(EGFP)融合的蛋白质。其中上述蛋白的编码基因位于中间，EGFP位于其N端，具有核定位和透皮能力短肽基因位于其C端。依据多肽及蛋白质的序列设计并合成核酸，然后插入pET-28a载体(美国Novagen公司)中，构建成为pET-28a-EGFP-pep，插入序列经测序鉴定，序列正确。
2、融合基因在大肠杆菌中的表达
用构建的载体pET-28a-EGFP-pep转化E.coli BL21(DE3)。菌液生长至OD600＝0.6时，加入IPTG(终浓度为1mmol/L)，30℃下诱导10小时。
3、融合蛋白纯化
向上述诱导表达的菌液中加入溶菌酶处理一小时，经过超声破碎后，4℃12000rpm离心30分钟，表达产物溶于上清液中。取离心后上清至10mL Eppendorf管中，加入Ni-NTA(美国Novagen公司)，4℃，50rpm震荡2h。将上述混合物转移至层析柱中，待液体快流尽时加入4ml Wash Buffer(含有PBS及0.1M的咪唑)，洗涤层析柱。待液体快流尽时加入300μL洗脱液，(含有PBS及0.4M的咪唑)，在核酸蛋白质检测仪作用下收集流出的蛋白峰。纯化后液体经SDS-PAGE检验，分子量大小约为50KD，与计算值相符，上述方法可获得纯度大于90％的融合蛋白。
4、融合蛋白的进细胞及核定位能力检测
体外培养的HaCaT细胞，生长到密度为50％时，在培养基中加入终浓度为10μmol/L的上述纯化的融合蛋白，4小时后加入DAPI染核30min。用PBS漂洗三次，去除游离的融合蛋白后置于共聚焦荧光显微镜下拍照。结果显示：加入本发明短肽融合上述多肽及EGFP的融合蛋白后，在HaCaT细胞核中能观察到绿色荧光信号(图5)，这表明该短肽具有携带与之相连的大分子蛋白质分子进入细胞并进行核定位的功能。
实施例6
本实施例对短肽的透皮作用进行检测，具体方法如下：
采用8周龄SD大鼠(体重250g)进行实验。将SD大鼠背部毛剃掉，将实施例1制备的FITC标记的短肽和对照短肽分别溶于磷酸缓冲液(PBS，pH＝7.4)中，配成浓度为500μmol/L的溶液。将SD大鼠分为三组：1)对照组，背部不涂抹多肽溶液；2)大鼠背部表皮上涂抹SEQID NO.3所示的对照短肽；3)大鼠背部表皮上涂抹SEQ ID NO.1所示的短肽。组2)和组3)均涂抹50μL多肽溶液。三小时后洗去涂抹区域的多肽；处死上述SD大鼠，取涂抹多肽处的皮肤，冰冻切片后在分别置于荧光显微镜下观察。试验结果表明：对照组(未处理大鼠皮肤)和对照短肽组(涂抹无关对照组)大鼠皮肤的真皮组织中未见绿色荧光(图6中箭头指部位为具有自发荧光的角质层)；而涂抹本发明的短肽(涂抹目的多肽)的大鼠皮肤切片中，真皮区域的皮肤附属器中能明显观察到荧光信号(图6中椭圆圈中所示的荧光信号)，表明该短肽可透皮进入真皮组织中。
实施例7
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备水浸剂。
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)与人超氧化物歧化酶1(SOD1)融合表达后，涂抹在背部表皮丢失的小鼠表面，每日一次。对照组涂抹未与本发明多肽相连接的SOD1。结果表明，三天后，实验组小鼠创面较对照组减小38％，表明该发明短肽介导SOD1进入细胞可有效治疗皮肤创伤。
实施例8
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备粉剂。
实施例9
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备油剂。
实施例10
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备乳剂。
实施例11
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备软膏。
实施例12
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备硬膏。
实施例13
将本发明的透皮短肽(氨基酸序列如SEQ ID NO.1所示)用于制备治疗皮肤疾病的的药物，添加常规辅料后，制备气雾剂。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
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Claims (6)
1.一种具有核定位能力的透皮短肽，其特征在于：其氨基酸序列如SEQ ID No.1所示，所述透皮短肽可自主进入真核细胞。
2.根据权利要求1所述的透皮短肽，其特征在于：所述透皮短肽还连接有荧光素。
3.根据权利要求2所述的透皮短肽，其特征在于：所述荧光素为异硫氰酸荧光素、罗丹明或羧基荧光素。
4.根据权利要求1所述的透皮短肽，其特征在于：所述真核细胞为HaCaT细胞、WS1细胞、TE-1细胞、Eca-109细胞或Hela细胞。
5.根据权利要求1-4中任一项所述的透皮短肽在制备治疗皮肤创伤药物或透皮制剂中的应用。
6.一种治疗皮肤创伤的药物，其特征在于：包括权利要求1-4中任一项所述的透皮短肽以及药学上可接受的辅料。
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