CN116687837B - 一种促进创面愈合的干细胞制剂及其制备方法 - Google Patents
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Abstract
本发明公开了一种促进创面愈合的干细胞制剂及其制备方法,18.75mL卡波姆凝胶与6.25mL人脂肪间充质干细胞浓缩上清液混合,再加入1562.5μL甜菜碱工作溶液震荡至混匀后,即得一种促进创面愈合的干细胞制剂,该干细胞制剂于4℃条件下进行保存。经实验发现,本发明制得的干细胞制剂处理后的的伤口面积显著低于碘伏对照组,并且在第六天差异明显。第九天时,本发明处理后的伤口创面已经几乎完全填充,上皮愈合接近完全,伤口愈合可以认为基本完成。本发明制得的促进创面愈合的干细胞制剂在抑制炎症,减缓感染方面有一定的作用,有利于组织重塑,达到治疗皮肤创伤愈合的目的。
Description
技术领域
本发明属于生物制剂领域,公开了一种促进创面愈合的干细胞制剂的制备方法以及采用该制备方法制得的促进创面愈合的干细胞制剂。
背景技术
创伤愈合是指机体因多种原因产生创口后,由于一系列的调控作用下的组织重形成过程,包括可以自然愈合的急性伤口如轻度烧伤烫伤伤口、浅层皮外伤,也有必须依靠药物和手术治疗的如溃疡性伤口等。皮肤创伤修复过程主要分为凝血期、炎症反应期、细胞生长期、组织重形成几个阶段。创伤修复需要多种细胞的共同合作完成,在整个过程中任何一个环节的缺失或延后都导致皮肤伤口愈合过程不能正常的进行。目前,对于皮肤创伤治疗的基本方案是外科手术和药物治疗。外科手术一般对创面进行缝合,使皮肤缺损最终得到修复,是临床上常用的方法,见效速度快。但是当创伤面积较大、创口较深时,实施手术清创,容易加重病情,而对于药物治疗来讲,通常的治疗效果大多不尽人意。
干细胞本身具有分化和增殖等的特性,在创伤愈合过程中,可参与与伤口愈合相关的细胞增殖分化、转移以及细胞外基质(ECM)的重形成等过程,多方面发挥加速伤口愈合的作用,不仅加快了伤口愈合时间,还解决了皮肤附属器官如汗腺无法再生等问题,使皮肤恢复了原有的生理功能。脂肪间充质干细胞来源广泛易于提取,脂肪间充质干细胞具有自我更新能力和多向分化潜能,然而间充质干细胞移植治疗组织损伤时,单次移植细胞数量需要高达到1×109个才能有效发挥治疗作用,为了保证实验结果的稳定性和可比性,需要大量的均一来源的种子细胞,另外异种移植存在强烈的免疫反应,因此需要一种避免因细胞反复传代而导致的间充质干细胞老化、甚至恶变,并且易于获取,便于储存,移植后不诱发免疫排斥反应的新手段。间充质干细胞分泌多种生物活性因子,并且证实干细胞的旁分泌功能是其发挥作用的主要方式。
而甜菜碱,被称为三甲基甘氨酸,广泛分布于动物、植物和微生物中,由于无毒无害及其作为渗透保护剂和甲基供体的两种主要生理作用被广泛应用到疾病的治疗中,越来越多的证据表明甜菜碱在多种疾病中具有抗炎作用。从机制上看,甜菜碱可以改善硫氨基酸代谢对抗氧化应激,抑制核因子-κB活性和NLRP3炎症小体激活,对炎症性疾病起到治疗作用。甜菜碱与其它药物联合的可能性目前被研究,虽然其机制有待研究,但是这也是一个甜菜碱治疗疾病的新思路。因此,我们在传统的干细胞治疗皮肤创伤形式上进一步完善出一种快速、高效、安全的治疗皮肤伤口愈合的多组分成分的水凝胶,以发挥更好的疗效应用于皮肤伤口愈合。
发明内容
鉴于上述不足,本发明提出了一种促进创面愈合的干细胞制剂的制备方法,本发明是通过如下技术手段实现的:
一种促进创面愈合的干细胞制剂,包括:
卡波姆凝胶;
人脂肪间充质干细胞浓缩上清液;以及
甜菜碱工作溶液。
进一步地,所述卡波姆凝胶用量为18.75mL;人脂肪间充质干细胞浓缩上清液用量为6.25mL;甜菜碱工作溶液用量为1562.5μL。
本发明还公开了一种根据上述促进创面愈合的干细胞制剂的制备方法,包括:
取卡波姆凝胶与人脂肪间充质干细胞浓缩上清液混合相溶,得第一溶液;
向第一溶液中加入甜菜碱工作溶液,震荡至混匀后于4℃条件下进行保存,即得。
进一步地,所述卡波姆凝胶由如下方法制得:
称取0.05g卡波姆于称量纸中,紫外灭菌30min,置于50mL离心管中;
在无菌条件下依次加入13.75mL无菌去离子水和5mL浓度为0.1mol/L的NaOH溶液,静置12h,即得。
进一步地,所述人脂肪间充质干细胞浓缩上清液由如下方法制得:
从液氮中取出要复苏的细胞(购买自华拓生物,货号:HTX2309),在37℃、含有体积分数为5%的二氧化碳细胞培养箱中培养;
待细胞密度长到90%以上时更换为无酚红无血清培养液,加入NEAA(非必需氨基酸)和B27(细胞培养添加剂),随后置于培养箱中饥饿处理;
饥饿处理结束后回收上清液,经透析除盐后,再对其进行细胞破碎,过滤除菌;除菌的上清液经冻干后,用6.25mL无菌去离子水溶解,即得人脂肪间充质干细胞浓缩上清液。
进一步地,所述无酚红无血清培养液由包括:
无血清的含100U/mL青霉素和100U/mL链霉素的DMEM培养液。
进一步地,所述饥饿处理时间为9小时。
进一步地,所述细胞破碎次数为5次,每次破碎时间为10s,功率为40%。
进一步地,所述甜菜碱工作溶液由如下方法制得:
称取2.9g甜菜碱粉末,配置成1600g/L的甜菜碱工作溶液于4℃下保存。
本发明还公开了一种根据任一上述的制备方法制得的促进创面愈合的干细胞制剂。
本发明的有益效果在于:
1.经统计学分析发现,甜菜碱干细胞凝胶组的伤口面积显著低于碘伏对照组。并且在第六天差异明显。第九天时,各组伤口均有肉芽组织填充,其中,甜菜碱干细胞凝胶组伤口创面已经几乎完全填充,上皮愈合接近完全,伤口愈合可以认为基本完成。本研究在解决治疗皮肤损伤时相比于其它发明治疗疗程较短,易于治疗。
2.本研究使用人脂肪间充质干细胞卡波姆凝胶、甜菜碱工作溶液和人脂肪间充质干细胞浓缩上清液三者相溶治疗皮肤损伤,在抑制炎症,减缓感染方面有一定的作用,有利于组织重塑。人脂肪间充质干细胞浓缩上清液与甜菜碱混合制备皮肤修复剂能够双重发挥抗炎作用,达到治疗皮肤创伤愈合的目的。
附图说明
图1为各组小鼠治疗不同时间后皮肤创面愈合情况;
图2为各组小鼠治疗不同时间后皮肤创面愈合率比较;
图3为各组小鼠治疗后皮肤病理组织学切片染色结果。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
本发明实施例所使用的生物产品均能从市场购得,其中NEAA为非必需氨基酸,B-27为一种经过优化的无血清添加剂,是广泛应用的血清替代品,各大生物公司有售。
实施例1
一种促进创面愈合的干细胞制剂的制备方法
(1)制备卡波姆凝胶:称取0.05g卡波姆于称量纸中,紫外灭菌30min,置于50mL离心管中。在无菌条件下依次加入13.75mL无菌去离子水和5mL浓度为0.1mol/L的NaOH溶液,静置12h,即得。
(2)制备人脂肪间充质干细胞浓缩上清液:从液氮中取出要复苏的细胞,在37℃、含有体积分数为5%的二氧化碳细胞培养箱中进行培养,换液后待细胞密度长到90%以上时开始更换更换无酚红无血清培养液,加入NEAA和B27,加好以后放于培养箱中饥饿9小时,9小时后回收上清,透析除盐;同时回收人脂肪间充质干细胞,用透析除盐后的上清液破碎细胞,超声波细胞破碎仪进行细胞破碎,过滤除菌,冻干后用6.25mL无菌去离子水溶解,制成人脂肪间充质干细胞浓缩上清液。
(3)制备甜菜碱工作溶液:称取2.9g甜菜碱粉末于称量纸中,配置成1600g/L的甜菜碱工作溶液于4℃条件下进行保存,用于后续实验。
(4)制备促进创面愈合的干细胞制剂:18.75mL卡波姆凝胶与6.25mL人脂肪间充质干细胞浓缩上清液混合,再加入1562.5μL甜菜碱工作溶液震荡混匀后,即得一种促进创面愈合的干细胞制剂,该制剂于4℃条件下进行保存,用于后续实验。
应用例1
实验方法:
采用BABI/C小鼠6只,采用0.25%Avertin进行腹腔注射,用于麻醉小鼠,小鼠背部皮肤5cm×5cm区域进行脱毛处理,先用推子剃去长毛发,再涂抹脱毛膏去除短的毛发,擦去多余脱毛膏,采用酒精、碘伏对脱毛区进行消毒,选取距脊柱1cm的腰后部位置,以脊柱为中轴线,在两侧制作对称的等大的直径为5mm的圆形皮肤全层切除伤口。
将6只制作好伤口的小鼠随机分为两组,分别为碘伏组和甜菜碱干细胞凝胶组。甜菜碱干细胞凝胶组小鼠每天定时定量(即每天在同一时间涂抹50μL甜菜碱干细胞凝胶于伤口处)涂抹实施例1制得的促进创面愈合的干细胞制剂,碘伏组小鼠每天定时定量涂抹碘伏,将每只小鼠单笼饲养,自由进食,连续9天拍照观察,对创面面积进行统计学分析。
检测项目:
每日拍照观察愈合情况,计算小鼠各组皮肤创面愈合率。
小鼠皮肤常规病理组织学切片染色
实验结果:
每日拍照观察愈合情况,计算小鼠各组皮肤创面愈合率。
根据图1的结果可以观察到,甜菜碱干细胞凝胶组在愈合过程中伤口面积明显小于碘伏对照组。根据图2的统计学分析结果发现,甜菜碱干细胞凝胶组的伤口面积显著低于碘伏对照组(P<0.05)。
小鼠皮肤常规病理组织学切片染色
H&E染色结果如图3所示,第9d时,各组伤口均有肉芽组织填充,其中,甜菜碱干细胞凝胶组伤口创面已经几乎完全填充,上皮愈合接近完全,伤口愈合可以认为基本完成;碘伏对照组伤口创面处上皮组织较薄,再生不完全。
以上所述仅是本发明的优选实施方式,应当指出,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本技术领域的技术人员来应当理解,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围,不偏离本发明权利要求书所限定的范围。包括:
1.对于本发明技术方案中的甜菜碱这一抗炎药物,假设找到令外一种抗炎药物,按照甜菜碱与干细胞上清浓缩液的水凝胶制备方案,或许可达到治疗皮肤创伤愈合的目的;
2.对于本发明技术方案中的卡波姆凝胶作为水凝胶中的一部分被替代,形成液体制剂治疗皮肤创伤愈合。
Claims (7)
1.一种促进创面愈合的干细胞制剂的制备方法,包括:
(1)制备人脂肪间充质干细胞浓缩上清液:
人脂肪间充质干细胞,在37℃、含有体积分数为5%的二氧化碳细胞培养箱中培养;待细胞密度长到90%以上时更换为无酚红无血清培养液,加入NEAA和B27,随后置于培养箱中饥饿处理;饥饿处理结束后回收上清液,经透析除盐后,再对其进行细胞破碎,过滤除菌;除菌的上清液经冻干后,用6.25mL无菌去离子水溶解,即得人脂肪间充质干细胞浓缩上清液;
(2)取18.75mL卡波姆凝胶与6.25mL人脂肪间充质干细胞浓缩上清液混合相溶,再加入1562.5μL甜菜碱工作溶液,震荡至混匀后于4℃条件下进行保存,即得。
2.根据权利要求1所述的制备方法,其中:
步骤(1)所述无酚红无血清培养液为:
无血清的含100U/mL青霉素和100U/mL链霉素的DMEM培养液。
3.根据权利要求1所述的制备方法,其中:
步骤(1)所述饥饿处理时间为9小时。
4.根据权利要求1所述的制备方法,其中:
步骤(1)所述细胞破碎次数为5次,每次破碎时间为10s,功率为40%。
5.根据权利要求1所述的制备方法,其中:
步骤(2)所述甜菜碱工作溶液由如下方法制得:
称取2.9g甜菜碱粉末,配置成1600g/L的甜菜碱工作溶液于4℃下保存。
6.根据权利要求1所述的制备方法,其中:
步骤(2)所述卡波姆凝胶由如下方法制得:
称取0.05g卡波姆,紫外灭菌30min,置于离心管中;
在无菌条件下依次加入13.75mL无菌去离子水和5mL浓度为0.1mol/L的NaOH溶液,静置12h,即得。
7.一种根据权利要求1~6任一所述的制备方法制得的促进创面愈合的干细胞制剂。
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