CN114591419B - 一种热水解蛋白及其制备方法和应用 - Google Patents
一种热水解蛋白及其制备方法和应用 Download PDFInfo
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- CN114591419B CN114591419B CN202210225116.XA CN202210225116A CN114591419B CN 114591419 B CN114591419 B CN 114591419B CN 202210225116 A CN202210225116 A CN 202210225116A CN 114591419 B CN114591419 B CN 114591419B
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Abstract
本发明提供一种热水解蛋白及其制备方法和应用,属于医学技术领域。本发明运用热改性技术得到一种可溶于水的降解蛋白,使得液态水解蛋白产品的生产更加经济,同时pH值更接近人体创面环境,利于创面愈合。同时,本发明可实现废弃皮肤的回收利用,提高了烧伤皮肤的回收利用率。此外,该种改性蛋白可以在4℃时凝固,便于塑型,在接近人体温度时成为液态,利于与创面更好贴合。此外,利用该技术可制备生物活性支架,对较大创面的愈合提供新的技术。而且,得到的水解改性蛋白保留了传统生产技术下其相对分子量较小吸收利用率更高的优势,具有较好的生物相容性,能够较好的促进创面愈合,因此具有极为广泛的应用空间。
Description
技术领域
本发明属于医学技术领域,具体涉及一种热水解蛋白及其制备方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
在临床外科,各种原因导致的创面生成是十分常见的。创面愈合过程是一个动态交互的过程,主要包括止血、炎症、细胞增殖和重塑4步。对患者而言,创面愈合的过程会导致患者生活质量下降和医疗费用增加,从而对社会和经济产生不良影响。因此,创面愈合是成为外科治疗皮肤创伤和组织损伤的重中之重,研究者也正在不断探索新方法治愈未闭合伤口或加速伤口愈合。
随着科学技术的进步,组织工程皮肤作为创面愈合的一种全新的治疗方法,其发展备受关注。其基本原理和方法是将体外扩增的自体或异体皮肤细胞,同体外构建的细胞外基质模拟支架相结合,形成支架与细胞的复合物,然后移植到创面。通过植入皮肤细胞的黏附、增殖与分化以及细胞外基质模拟支架的逐渐吸收与降解,最终形成结构、功能与正常皮肤相一致的新皮肤,从而达到创面修复和皮肤组织功能重建的目的。
根据临床创面愈合需求,发现用于组织工程支架制作的理想生物材料应具有良好的生物相容性和生物安全性、一定的空间结构、适宜的力学强度、可控的降解速率、良好的塑性及加工性能、来源广泛价格便宜、方便灭菌和储存等特点。但目前临床应用的传统生物材料(如新鲜异种皮、戊二醛处理的异种皮、辐照处理的异种皮、冻干异种皮等)不同程度的存在抗原性强、存活时间短、细菌污染率大、弹性差、透水性高等缺点。种子细胞作为组织工程皮肤研究的前提,其增殖和细胞外基质的分泌决定了创面愈合的速度和效果。而在实践中,种子细胞的存活是组织工程皮肤生产和应用过程中一个较大的阻碍。
目前,市面上有许多商品化的真皮替代物可应用于基础研究或临床治疗。由Integra生命科学公司开发生产的采用牛胶原为原料制成的胶原海绵,具有弹性、韧性好,色素沉着清等优点;由日本郡是株式会社公司生产 是抗原性极低的无端胶原蛋白海绵与一层硅胶膜组成的双层结构移植物;与相似的是由中国兰度生物材料公司生产的仿生材料,研究证明在移植后2周可实现胶原海绵的血管化。这些产品的共性在于大量胶原蛋白的使用,胶原蛋白的提取技术主要有酸、碱提取法,酶提取法。虽然酸法和碱法水解都迅速彻底,但是这两种方法都具有很大的局限性,酸水解法会使色氨酸全部被破坏,丝氨酸和酪氨酸部分被破坏,且产品得率低,设备腐蚀严重,并产生二次污染,而碱水解法则使含羟基和巯基的氨基酸,全部被破坏且产生消旋。酶提取法具有反应温度低,反应时间短,无环境污染等优点,但随着需求量的不断增大,该方法的经济效益大打折扣,所以不得不探索一种经济简便的技术来实现水解胶原蛋白的批量生产。
此外,从种子细胞增殖和迁移效率角度来看,由高级组织科学公司生产的D是一种由儿童包皮提取的成纤维细胞、细胞外基质和可降解生物材料构成的人工真皮,其优点在于能有效减少创面收缩,具有良好的生物活性和组织相容性,但缺点在于成纤维细胞的需求量大。由此可见,借助少量活性细胞使产品具有较高的生物学活性也是该组织工程技术的另一大需求。
综上所述,当前在高分子科学与生命科学和生物医学工程等交叉融合的领域内进行组织工程皮肤的研究,而现有技术存在的问题有:
(1)在实际临床操作中,因烧伤变性皮肤会有大量炎症因子浸润,会存在一定的生物学危害,不利于创面愈合,如果回收利用作为支架种植细胞有一定的可行性但仍存在一定的难度,故在大面积烧伤患者切痂手术中的变性皮肤多会直接作废,取而代之的是采取用其他部位植皮或者天然生物材料。异位植皮会损害患者正常皮肤,带来一定的痛苦,大众接受度较低;天然生物材料往往会因来源限制存在一定的免疫原性和微生物易感性,因而临床使用存在一定的限制。
(2)现有生物材料制作工艺复杂,价格昂贵,给使用者带来一定的经济负担。
(3)种子细胞作为组织工程皮肤研究的前提,其增殖和细胞外基质的分泌决定了创面愈合的速度和效果,而在组织工程皮肤制备与临床应用中的需求量较大。
发明人发现,解决上述问题的难度在于:
(1)现有ADM制备工艺化学物质残留导致的生物学毒性作用;
(2)蛋白热改性技术对温度和时间的选择;
(3)异体来源的生物材料存在一定的免疫原性和微生物易感性。
发明内容
针对现有技术的上述问题,本发明提供一种热水解蛋白及其制备方法和应用。本发明运用热改性技术得到一种可溶于水的降解蛋白,产物无毒性,无免疫原性。根据温度与作用时间的不同可以控制降解蛋白的结构,本发明所用材料来源广泛,具有良好的实际应用之价值。
具体的,本发明涉及如下技术方案:
本发明的第一个方面,提供一种热水解蛋白的制备方法,所述制备方法包括:将皮肤材料进行脱细胞处理后进行热改性。
其中,所述皮肤材料可以为自体皮肤和/或者异种皮;
所述热改性具体方法包括:将上述经脱细胞处理后的皮肤材料置于水中,在50-90℃加热条件下进行处理,得到热水解蛋白溶液。
本发明的第二个方面,提供上述制备方法获得的热水解蛋白。
本发明的第三个方面,提供上述热水解蛋白在如下任意一种或多种中的应用:
a)组织工程皮肤和/或制备组织工程皮肤;
b)提高废弃皮肤的回收利用。
其中,应用b)中,废弃皮肤包括变性皮肤,进一步包括烧伤变性皮肤。
上述一个或多个技术方案的有益效果:
上述技术方案运用热改性技术得到一种可溶于水的降解蛋白,上述技术方案突破传统的酸、碱、酶制备液态水解蛋白(胶原、明胶等)的限制条件,使得液态水解蛋白产品的生产更加经济,同时pH值更接近人体创面环境,利于创面愈合。同时,上述改性蛋白的技术方案可实现废弃皮肤的回收利用,提高了烧伤皮肤的回收利用率。此外,该种改性蛋白可以在4℃时凝固,便于塑型,在接近人体温度时成为液态,利于与创面更好贴合。此外,利用该技术可制备生物活性支架,对较大创面的愈合提供新的技术。
而且,上述技术方案得到的水解改性蛋白保留了传统生产技术下其相对分子量较小吸收利用率更高的优势,具有较好的生物相容性,能够较好的促进创面愈合,因此具有极为广泛的应用空间。
附图说明
图1为本发明实施例中热改性处理前后实物图,其中(a)为热改性处理前,(b)为热改性处理后;
图2为本发明实施例中分子量测定(电泳条带);其中,1-9对应泳道对应加热温度及加热时间为:1.50℃,2小时;2.60℃,1小时;3.70℃,1小时;4.70℃,2小时;5.70℃,3小时;6.80℃,1小时;7.80℃,2小时;8.80℃,3小时;9.90℃,2小时。
图3为本发明技术流程图。
具体实施方式
需要指出的是,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明。
本发明的一个典型具体实施方式中,提供一种热水解蛋白的制备方法,所述制备方法包括:将皮肤材料进行脱细胞处理后进行热改性。
其中,所述皮肤材料可以为自体皮肤和/或者异种皮;
本发明的又一具体实施方式中,所述自体皮肤可以是切痂手术废弃的自体皮肤,所述异种皮可以为鱼皮、猪皮等;更具体的,在进行脱细胞处理之前,所述皮肤材料置于缓冲液(如PBS缓冲液)中进行保存备用。
本发明的又一具体实施方式中,所述脱细胞处理的具体方法包括:将皮肤材料置于消毒剂中进行震荡洗涤消毒,然后缓冲液震荡清洗后,使用中性蛋白酶消化去除表皮,随后用EDTA和胰蛋白酶的混合溶液持续震荡处理,然后使用缓冲液震荡清洗后即得。
本发明的又一具体实施方式中,所述消毒剂可以为苯扎溴铵,更具体的,为0.05-0.2%(优选0.1%)苯扎溴铵溶液,震荡消毒10-60min,优选为30min;
本发明的又一具体实施方式中,所述脱细胞处理中,缓冲液可以为PBS缓冲液;
本发明的又一具体实施方式中,所述中性蛋白酶消化去除表皮具体方法包括:采用0.1-0.5%(优选为0.25%)中性蛋白酶在低温(如4℃)进行消化处理1-10h(优选为5h),去除表皮;
本发明的又一具体实施方式中,所述EDTA和胰蛋白酶的混合溶液中,EDTA浓度为0.01-0.05%,优选为0.02%;所述胰蛋白酶浓度为0.01-0.05%,优选为0.025%;具体的,持续震荡处理10-30h,优选为24h;
所述热改性具体方法包括:将上述经脱细胞处理后的皮肤材料置于水中,在50-90℃加热条件下进行处理,处理时间控制为0.5-5小时,进一步为1-3小时,得到热水解蛋白溶液。
本发明的又一具体实施方式中,提供上述制备方法获得的热水解蛋白。经研究发现,采用上述制备方法获得的热水解蛋白,其为多种分子量大小的混合物,分子量大致在25-120kDa;且随着热处理温度升高,高分子量的产物增多;作用时间的延长,分子量未见明显差异,但蛋白浓度明显升高;此外,如果通过控制调整加热时间及温度,该种蛋白热改性技术可得到分子量不同的水解产物以适应不同的需求。同时,该种改性蛋白可以在4℃时凝固,便于塑型,在接近人体温度时成为液态,利于与创面更好贴合。
本发明的又一具体实施方式中,提供上述热水解蛋白在如下任意一种或多种中的应用:
a)与脱细胞真皮基质(ADM)联合使用;
b)伤口敷料、组织工程支架和/或制备伤口辅料、组织工程支架;
c)递送系统和/或制备递送系统;
d)活性支架和和/或制备活性支架;
e)食品、药品、化妆品领域;
f)与细胞联合制备细胞悬液。
其中,所述a)中,ADM具有良好的柔韧性,易于修剪,可以切割、重叠、搓成卷状,亦可制成微粉状进行皮内或皮下注射,可以为成纤维细胞的移入及胶原的沉积提供一定的空间结构。ADM对代谢的要求很低,ADM血管化后会出现不同程度的吸收,其作为一种真皮替代物可为创面提供足够量的真皮组织,从而减轻瘢痕的形成和挛缩。ADM中细胞外基质结构完整,可为组织细胞的再生提供一个良好的支架,细胞外基质蛋白可促进表皮细胞的附着和增殖。因此将上述热水解蛋白与ADM联合应用,可以在为创面细胞移入和增殖提供空间结构的同时,提高细胞的成活率,促进细胞增殖,加快创面愈合。
所述b)中,伤口敷料以及组织工程支架可以通过上述热水解蛋白联合壳聚糖制备得到。由于壳聚糖是一种来源于甲壳素的天然线性阳离子多糖,具有优异的生物相容性、生物降解性、止血性能、抗菌活性和伤口愈合性能。此外,壳聚糖的结构与天然细胞外基质相似,为细胞黏附和增殖提供了有利的微环境。与联合ADM使用促进创面愈合的机制相似,该技术得到的水解蛋白与壳聚糖联合使用,制备人工真皮,可以使人工真皮来源更加广泛,经济成本更低。比如利用这两种材料制作的电纺纤维结构显示出高孔隙率、高比表面积、优异的机械性能和易于表面功能化、细菌屏障等特性,使其能够广泛应用于生物医学领域,如伤口敷料和组织工程支架。
所述c)中,由于胶原蛋白已广泛应用于组织工程领域,并在一定程度上应用于递送系统,它可塑性强,可以制成任何需要的物理结构形式(如多孔支架,膜,凝胶等),而生长因子和其他活性剂甚至药物可以与胶原制备的递送系统结合使用,以延长因子或药物的释放速率并提高其治疗效果。
所述d)中,热水解蛋白-壳聚糖复合支架还可以负载特定的细胞(如成纤维细胞、干细胞等)成为活性支架,为大面积难愈创面的愈合提供新思路。
所述e)中,具体的,由于水解蛋白作为一种已经被许可的食品添加剂和药品添加成分(例如它可以作为药品的塑形成份),目前还没有发现任何副作用。美国食品药品管监督管理理局(FDA)在开展了对胶原产品(包括明胶和水解胶原蛋白)的安全性研究之后,将其归类为最高安全等级的目录(GRAS:Generally RecognizedAs Safe)内。在FDA于2001年12月21日发布的公告函中是如此阐述的:“根据第170.35项条款的规定,委员会建议将胶原产品确认为作为直接使用的食品添加成分并赋予其最高安全等级GRAS”。这样,水解胶原蛋白产品就被赋予了与基本氨基酸同等水平的安全等级。因此,本发明制备的水解胶原蛋白可用于制作口服制剂(如胶原多肽螯合钙),促进软骨组织修复,改善骨关节活动,无有害添加,安全可靠。当然,本发明的水解胶原蛋白还可以制成凝胶剂用于促进创面愈合。
此外,许多高档化妆品中添加有胶原蛋白,化妆品中的水解胶原蛋白分子量要求一般在1000-5000Da,最佳吸收浓度范围为3-5%。水解胶原蛋白可应用于各类化妆品中(如面膜、面霜、眼霜、发膜等),可以发挥保湿、去色斑、修复毛发等作用。
本发明实施例中所用的试验材料均为本领域常规的试验材料,均可通过商业渠道购买得到。
实施例
一种热水解蛋白的制备方法,所述制备方法包括:
(1)废弃皮肤取下后浸泡在PBS中存放;
(2)对皮肤进行脱细胞处理(ADM制备):取出PBS中的皮肤,于0.1%苯扎溴铵溶液中震荡洗涤消毒30min,后PBS震荡清洗3遍,0.25%中性蛋白酶4℃消化5h,去除表皮,随后用0.02%EDTA+0.025%胰蛋白酶溶液持续震荡处理24h,后PBS震荡清洗6h,重复清洗4次后得到ADM,4℃保存备用;
(3)热改性处理:取ADM于无菌水中,分别在50-90℃加热条件下处理,得到热水解蛋白溶液,4℃保存备用;
(4)水解蛋白分子量测定
取100μL水解蛋白溶液与等量样品缓冲液均匀混合,在100℃水浴中加热3-5min,做成SDS-PAGE垂直板电泳,刚开始电压为80V,待胶跑成一条蓝色均匀的直线时,将电压调整为200V,电泳时间1.5h,用0.2%考马斯亮蓝染色,染色后以甲醇、冰醋酸及水混合过夜脱色,每4小时更换脱色液,同时以市售蛋白、明胶作为标准对照。
图1是利用本技术热处理脱细胞真皮前(a)后(4℃)(b)对比图,其中a为脱细胞真皮,b为热处理得到的水解蛋白4℃储存;制备的脱细胞真皮呈乳白色,质韧,柔韧性与延展性良好。热处理后的水解产物呈透明状,室温下为液体,4℃时可凝固。
图2是凝胶电泳显示该技术制作热改性蛋白的分子量大小,该产物为多种分子量大小的混合物,分子量大致在25-120kDa;且随着处理温度升高,高分子量的产物增多;作用时间的延长,分子量未见明显差异,蛋白浓度明显升高(Control组:为浓度0.5mg/ml,分子量55kDa的标准蛋白);此外,如果通过控制调整加热时间及温度,该种蛋白热改性技术可得到分子量不同的水解产物以适应不同的需求。
综上,本发明扩大了组织工程生物材料的来源,极大降低了异体乃至异种材料的免疫原性,简化了生物材料的生产工艺,降低生产成本,并可以在一定程度上实现废弃皮肤的回收利用。
以上所述仅为本发明的优选实施例,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (16)
1.一种热水解蛋白的制备方法,所述制备方法包括:
(1)废弃皮肤取下后浸泡在PBS中存放;
(2)对皮肤进行脱细胞处理:将皮肤材料置于消毒剂中进行震荡洗涤消毒,然后缓冲液震荡清洗后,使用中性蛋白酶消化去除表皮,随后用EDTA和胰蛋白酶的混合溶液持续震荡处理,然后使用缓冲液震荡清洗后即得脱细胞真皮基质,4℃保存备用;
(3)热改性处理:取脱细胞真皮基质于无菌水中,分别在50-90℃加热条件下处理,处理时间为1-3小时,得到热水解蛋白溶液,4℃保存备用;
热水解蛋白在4℃时凝固,便于塑型,在接近人体温度时成为液态,利于与创面更好贴合。
2.如权利要求1所述的制备方法,其特征在于,所述皮肤材料为自体皮肤和/或者异种皮。
3.如权利要求2所述制备方法,其特征在于,所述自体皮肤是切痂手术废弃的自体皮肤,所述异种皮包括鱼皮、猪皮。
4.如权利要求1所述的制备方法,其特征在于,所述消毒剂为0.05-0.2%苯扎溴铵,震荡消毒10-60min。
5.如权利要求4所述的制备方法,其特征在于,苯扎溴铵溶液的浓度为0.1%。
6.如权利要求4所述的制备方法,其特征在于,震荡消毒时间为30min。
7.如权利要求1所述的制备方法,其特征在于,所述脱细胞处理中,缓冲液为PBS缓冲液。
8.如权利要求1所述的制备方法,其特征在于,所述中性蛋白酶消化去除表皮具体方法包括:采用0.1-0.5%中性蛋白酶在4℃低温进行消化处理1-10h,去除表皮。
9.如权利要求8所述的制备方法,其特征在于,中性蛋白酶浓度为0.25%。
10.如权利要求8所述的制备方法,其特征在于,消化处理时间为5h。
11.如权利要求1所述的制备方法,其特征在于,所述EDTA和胰蛋白酶的混合溶液中,EDTA浓度为0.01-0.05%;胰蛋白酶浓度为0.01-0.05%;持续震荡处理10-30h。
12.如权利要求11所述的制备方法,其特征在于,EDTA浓度为0.02%。
13.如权利要求11所述的制备方法,其特征在于,胰蛋白酶浓度为0.025%。
14.如权利要求11所述的制备方法,其特征在于,震荡处理时间为24h。
15.权利要求1-14任一项所述制备方法获得的热水解蛋白。
16.权利要求15所述热水解蛋白在如下任意一种或多种中的应用:
a)制备伤口敷料、组织工程支架;
b)制备递送系统;
c)制备活性支架;
d)与细胞联合制备细胞悬液。
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