CN111893085A - 一种通过干细胞体外分化获得人工皮肤的方法及其应用 - Google Patents

一种通过干细胞体外分化获得人工皮肤的方法及其应用 Download PDF

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CN111893085A
CN111893085A CN202010774689.9A CN202010774689A CN111893085A CN 111893085 A CN111893085 A CN 111893085A CN 202010774689 A CN202010774689 A CN 202010774689A CN 111893085 A CN111893085 A CN 111893085A
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mesenchymal stem
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曹毓琳
滕睿頔
张海林
白志惠
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Beijing Zhenxigu Medical Research Center LP
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Abstract

一种通过干细胞体外分化获得人工皮肤的方法及其应用。本发明提供了一种干细胞体外向皮肤细胞分化获得人工皮肤的方法及应用,该培养方法包括以下步骤:S1:将间充质干细胞传代培养至细胞60‑90%汇合时,收集P3代细胞,制成间充质干细胞悬液;S2:附着:将脱细胞真皮和间充质干细胞悬液放置于超低吸附反应器中使用完全培养基进行培养;S3:体外诱导脱细胞真皮负载的间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,所述诱导培养液以KFSM培养基为基础培养基,添加浓度为30‑50g/L的黄芪注射液和体积分数为1‑3%的胎牛血清,诱导培养时间为14天;TM4SF1和广谱细胞角蛋白表达均呈阳性,获得人工皮肤和组成皮肤组织的细胞更为相似,会起到更好的治疗效果。

Description

一种通过干细胞体外分化获得人工皮肤的方法及其应用
技术领域
本发明属于生物组织器官修复技术领域,特别涉及一种干细胞体外向皮肤细胞分化获得人工皮肤的方法及其应用。
背景技术
皮肤是人体最大的器官,外伤、烧伤、糖尿病足、肿瘤切除手术等均会造成其损伤。创伤修复一般分为3个相互重叠的阶段:炎性反应期-增生期-重塑期,其中任一阶段失控都会导致病理性愈合,如形成瘢痕疙瘩、伤口延迟愈合或不完全愈合;严重时可造成局部功能受限、失去皮肤屏障作用,引发脓毒症等感染性疾病,威胁患者生命。目前,临床上用于较深或大面积皮肤缺损的修复方法是自体皮片或皮瓣移植。但自体皮片或皮瓣移植面临供皮来源问题,且会造成供区损伤;同种异体皮片或皮瓣移植存在免疫排斥及使用安全性等问题;尤其是当患者全身情况差(如患糖尿病且控制欠佳、全身大面积重度烧伤等),局部微环境无法为皮片或皮瓣成活提供足够新生血管时,可能导致组织移植失败。在成年哺乳动物,真皮不能自发地再生,在缺少移植存活的真皮基质时,成纤维细胞便开始合成不成熟的基质,基质经重塑形成疤痕。部分疤痕收缩皮肤引起疼痛,部分还会影响相近关节活动,对患者的身心健康都有长期影响。
发明内容
为了解决以上技术问题,本发明提供了一种干细胞体外向皮肤细胞分化获得人工皮肤的方法,该培养方法制得的人工皮肤可进一步减少创面的深度,利用体外分化技术为脱细胞真皮基质补足了缺失的细胞组分,分化获得的皮肤细胞与脱细胞异体真皮构成组织工程学皮肤能够促进创面愈合,加快移植物炎性反应消退;该方法可用于外伤、烧伤、糖尿病足、肿瘤切除手术等均会造成的皮肤损伤。
本发明具体技术方案如下:
本发明提供了一种干细胞体外向皮肤细胞分化获得人工皮肤的方法,培养方法包括以下步骤:
S1:间充质干细胞悬液的制备:将间充质干细胞传代培养至细胞60-90%汇合时,收集P3代细胞,制成间充质干细胞悬液;
S2:附着:将脱细胞真皮和间充质干细胞悬液放置于超低吸附反应器中使用完全培养基进行培养,先在37±1℃条件下震荡培养20-40min,再静置继续培养2-3天,培养条件如下:温度为37.0±0.5℃、CO2浓度为5.0±0.2%,湿度为饱和湿度;
S3:体外诱导脱细胞真皮负载的间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,所述诱导培养液以KFSM培养基为基础培养基,添加浓度为30-50g/L的黄芪注射液和体积分数为1-3%的胎牛血清。
进一步地,该培养方法具体包括以下步骤:
S1:将间充质干细胞传代培养,用完全培养基培养至细胞90%汇合时,收集P3代细胞,制成间充质干细胞悬液;完全培养基为:以DMEM/F12为基础培养基,添加浓度为25μg/ml的人纤连蛋白、浓度为10ng/ml的碱性成纤维细胞生长因子、浓度为15ng/ml的人表皮细胞生长因子、浓度为1%的ITS、浓度为5%的人血白蛋白、浓度为1%的NEAA、浓度为0.1μmol/L的氢化考的松、浓度为0.1%的β-巯基乙醇;
S2:附着:将脱细胞真皮和间充质干细胞悬液放置于超低吸附反应器中使用完全培养基进行培养,先在37℃条件下震荡培养30min,摇床转速为50r/min,后停止震荡,静置继续培养2-3天,培养条件如下:温度为37.0±0.5℃、CO2浓度为5.0±0.2%,湿度为饱和湿度;
S3:体外诱导脱细胞真皮负载的间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,诱导培养液以KFSM培养基为基础培养基,添加浓度为40g/L的黄芪注射液和体积分数为2%的胎牛血清,诱导培养时间为14天。
其中,KFSM培养基购自Gibco,黄芪注射液购自正大青春宝药业有限公司产品,批号是0207114,每支装2ml(相当于原药材4g);实验发现,部分细胞经体外诱导培养14天后,由长梭形转变为扁圆形,呈表皮样细胞形态特征。
本发明研究发现,ADSCs可以在脱细胞异体真皮支架中得以良好存活并具有相应的生物学活性,将ADSCs与无免疫原性的脱细胞异体真皮支架相结合,所组成的人体组织工程学皮肤不仅能够满足以上要求,脱细胞异体真皮支架本身还能够提供可观的组织容量,可以进一步减少创面的深度,提供相对满意的愈合效果;本发明利用ADSCs为脱细胞真皮基质补足了缺失的细胞组分,间充质干细胞与脱细胞异体真皮支架构成组织工程学皮肤能够促进创面愈合,加快移植物炎性反应消退,经诱导分化后,TM4SF1和广谱细胞角蛋白表达均呈阳性,获得的人工皮肤和组成皮肤组织的细胞更为相似,会起到更好的治疗效果。
进一步地,间充质干细胞包括脂肪间充质干细胞、脐带间充质干细胞和宫内膜间充质干细胞。
进一步地,间充质干细胞为脂肪间充质干细胞,脂肪间充质干细胞悬液的制备方法如下:
(1)取脂肪细胞;
(2)将脂肪细胞放置于试管内,在试管中加入与脂肪组织量相同体积的消化液,消化液包括体积比为1:1的胰蛋白酶-EDTA和0.1%的I型胶原酶,将试管密封,放入37℃的恒温摇床中以190r/Min的速度震荡消化,分为3层;
(3)吸出离心管中的下层液体移入装有完全培养基的新离心管中终止消化,将离心管封闭,离心,分为2层;
(4)用吸管吸取新离心管中的上清后去掉,加入完全培养基,轻轻吹打制成细胞悬液;
(5)将细胞悬液按照0.5×106/ml的密度接种到培养瓶中,放入37℃、5%CO2培养箱培养;12-24小时后进行第一次换液,此后每隔3天换液,待细胞生长至融合后进行传代;
(6)吸去培养瓶内的旧培养基,加PBS冲洗2-3次,再加入消化液消化3-5min,消化液为胰蛋白酶-EDTA;轻轻吹打细胞,使细胞脱离瓶底得到单细胞悬液;将单细胞悬液离心吹散,按1:8传代。
进一步地,步骤(1)的脂肪细胞为通过抽脂直接获得的脂肪抽取液。
进一步地,步骤(1)的脂肪细胞的获取方法为:取脂肪组织挤压进入离心管中,吸取PBS缓冲液加入到装有组织的离心管中,每克脂肪组织使用14mlPBS缓冲液,用吸管吹打10-20次,然后静置0.5-1.5min,用吸管将组织下层的液体吸出,如此反复直到所吸出的液体呈透明状,不带有血细胞为止。
进一步地,脱细胞异体真皮支架放置于超低吸附反应器前需要进行预处理:将脱细胞真皮剪成边长为2cm的正方形小片,使用生理盐水冲洗三遍。
本发明还提供了一种人工皮肤,通过以上培养方法制备而成。
本发明还体供了人工皮肤在仿生材料医学领域中的应用。
本发明提供的人工皮肤的使用方法是将完成分化的人工皮肤取出后用生理盐水清洗干净,再手术移植到真皮缺损处。
本发明提供的培养方法具有以下技术效果:本发明提供的方法制备的人工皮肤可以进一步减少创面的深度,提供相对满意的愈合效果;本发明利用ADSCs为脱细胞真皮基质补足了缺失的细胞组分,间充质干细胞与脱细胞异体真皮支架构成组织工程学皮肤能够促进创面愈合,加快移植物炎性反应消退,经诱导分化后,TM4SF1和广谱细胞角蛋白表达均呈阳性,获得的人工皮肤和组成皮肤组织的细胞更为相似,会起到更好的治疗效果。
附图说明
图1.为实施例1步骤S3换液前的培养液的显微镜下图;
图2.为实施例1步骤S3诱导培养第14天的诱导培养液的显微镜下图;
图3.为对照例1步骤S3诱导培养第14天的诱导培养液的显微镜下图;
图4.为对照例2步骤S3诱导培养第14天的诱导培养液的显微镜下图;
图5.为对照例3步骤S3诱导培养第14天的诱导培养液的显微镜下图;
图6.为各组方法培养的细胞中TM4SF1表达直方图;
图7.为各组方法培养的细胞中角蛋白表达直方图;
具体实施方式
实施例1
本实施例提供了一脂肪干细胞体外向皮肤细胞分化获得人工皮肤的方法,该培养方法包括以下步骤:
S1:将间充质干细胞传代培养,用完全培养基培养至细胞90%汇合时,收集P3代细胞,制成间充质干细胞悬液,间充质干细胞悬液的制备方法是取2×106间充质干细胞经0.25%胰蛋白酶消化3分钟后制得;完全培养基为:以DMEM/F12为基础培养基,添加浓度为25μg/ml的人纤连蛋白、浓度为10ng/ml的碱性成纤维细胞生长因子、浓度为15ng/ml的人表皮细胞生长因子、浓度为1%的ITS、浓度为5%的人血白蛋白、浓度为1%的NEAA、浓度为0.1μmol/L的氢化考的松、浓度为0.1%的β-巯基乙醇;
S2:附着:将脱细胞真皮剪成边长为2cm的正方形小片,使用生理盐水冲洗三遍,与脂肪间充质干细胞悬液一起放置于超低吸附反应器中使用完全培养基进行培养,先在37℃条件下震荡培养30min,摇床转速为50r/min,后停止震荡,静置继续培养2天,培养条件如下:温度为37.0±0.5℃、CO2浓度为5.0±0.2%,湿度为饱和湿度;
S3:体外诱导脱细胞真皮负载的脂肪间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,诱导培养液以KFSM培养基为基础培养基,添加浓度为40g/L的黄芪注射液和体积分数为2%的胎牛血清,诱导培养时间为14天;
其中,ITS(insulin-transferrin-selenium),内含胰岛素转铁蛋白、硒溶液三种成分,购自Gibco,NEAA,中文名称非必需氨基酸,购自Gibco,KFSM培养基购自Gibco,黄芪注射液购自正大青春宝药业有限公司产品,批号是0207114,每支装10ml(相当于原药材20g);
其中,脂肪间充质干细胞悬液的制备方法如下:
(1)取脂肪细胞;
(2)将脂肪细胞分装于多个试管内,每个试管内分装5ml脂肪细胞,在试管中加入与脂肪组织量相同体积的消化液,消化液包括体积比为1:1的胰蛋白酶-EDTA和0.1%的I型胶原酶,将试管密封,放入37℃的恒温摇床中以190r/Min的速度震荡消化30min,此时液面分为3层,上层为黄色油状脂肪细胞层,中层为脂肪组织层,下层为含单个核细胞的液体;
本申请的胰蛋白酶-EDTA均购自Gibco;
(3)吸出离心管中的下层液体移入装有完全培养基的新离心管中终止消化,将离心管封闭,1500rpm离心10min,分为2层;
(4)用吸管吸取新离心管中的上清后去掉,加入1ml完全培养基,轻轻吹打20次制成细胞悬液;
(5)将各个试管内的细胞悬液按照0.5×106/ml的密度接种到ml的培养瓶中,该培养瓶的规格为150cm2,放入37℃、5%CO2培养箱培养;16小时后进行第一次换液,此后每隔3天换液,待细胞生长至融合后进行传代;
(6)吸去培养瓶内的旧培养基,加PBS冲洗2次,再加入3ml消化液消化4min,消化液为胰蛋白酶-EDTA;轻轻吹打细胞,使细胞脱离瓶底得到单细胞悬液;将单细胞悬液离心吹散,按1:8传代;
其中,步骤(1)的脂肪细胞的获取方法为:取500g脂肪组织挤压进入多个15ml的离心管中,每个离心管中的组织为5ml,吸取7ml的PBS缓冲液加入到装有组织的离心管中,用吸管吹打20次,然后静置1min,用吸管将组织下层的液体吸出,如此反复直到所吸出的液体呈透明状,不带有血细胞为止;
本发明使用的脱细胞真皮为市售的国产医疗器械商品,购自北京桀亚莱福。
对照例1
对照例1提供了一种脂肪干细胞体外向皮肤细胞分化获得人工皮肤的方法,与实施例1的区别之处在于,去掉诱导培养液中的黄芪注射液。
对照例2
对照例2提供了一种脂肪干细胞体外向皮肤细胞分化获得人工皮肤的方法,与实施例1的区别之处在于,去掉诱导培养液中的胎牛血清。
对照例3
对照例3提供了一种脂肪干细胞体外向皮肤细胞分化获得人工皮肤的方法,与实施例1的区别之处在于,诱导培养液为DMEM/F12。
试验例1
分别用显微镜观察实施例1步骤S3换液前的培养液、实施例1步骤S3诱导培养第14天的诱导培养液、对照例1-3步骤S3诱导培养第14天的诱导培养液,观察结果见图1-图5,放大倍数为100倍。
由图2可知,经过诱导后细胞由长梭形转变为扁圆形,呈表皮样细胞形态特征,图3即使不加入黄芪注射液,细胞仍会变形为扁圆、短伪足形,呈表皮样细胞形态特征,而图1、4、5细胞呈长梭形,伪足较长。
试验例2
分别检测实施例1和对照例1-3的人工皮肤中TM4SF1、广谱细胞角蛋白表达,检测方法如下:
1.将MSC细胞和各组人工皮肤消化为单细胞悬液,将各组细胞悬液分装到相应的对照管和测试管中,每组细胞悬液中含有0.5-1×106个细胞;
2.在每个测试管内加入100μl PBS重悬细胞;
3.向每个测试管中分别加入Mouse(G3A1)mAb IgG1 Isotype Control(PEConjugate)「鼠抗人(G3A1)单克隆抗体IgG1同型对照(PE结合物)」/Mouse(G3A1)mAb IgG1Isotype Control(FITC Conjugate)「鼠抗人(G3A1)单克隆抗体IgG1同型对照(FITC结合物)」和Keratin mAb(PE Conjugate)/TM4SF1 mAb(FITC Conjugate)「角蛋白单克隆抗体(PE结合物)/TM4SF1单克隆抗体(FITC结合物)」;
4.在室温孵育30分钟;
5.在每个测试管内添加5mlPBS并通过离心润洗;
6.在0.5mlPBS中重悬细胞,并用流式细胞术进行分析,试验结果见图6和图7。
由图6和图7可知,原始状态MSC细胞TM4SF1表达量为0.2%,对照例1、2、3的表达量分别为:52.5%、15.0%、0%。实施例1获得的细胞TM4SF1表达量为86.4%;另一个皮肤细胞表面标志光谱细胞物角蛋白(图中用角蛋白表示),MSC细胞表达量为0%,对照例1、2、3的表达量分别为:57.2%、13.4%、0%。实施例1获得的细胞骄傲蛋白表达量为95.7%。由上述数据可知,实施例1的细胞TM4SF1、广谱细胞角蛋白的表达均呈阳性,经过体外诱导,间充质细胞转分化为表皮样细胞,通过干细胞体外分化获得人工皮肤和组成皮肤组织的细胞更为相似,会起到更好的治疗效果,而对照例1-3修改诱导液后,对照例1中由于缺少黄芪成分,细胞分化不完全,仅有部分细胞表达TM4SF1、广谱细胞角蛋白;对照例2中由于缺少血清,细胞状态变差,出现死亡情况,且分化效果不佳,对照例3中细胞基本维持原状,仍为MSC细胞。
综上,仅为本发明之较佳实施例,不以此限定本发明的保护范围,凡依本发明专利范围及说明书内容所作的等效变化与修饰,皆为本发明专利涵盖的范围之内。

Claims (9)

1.一种干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,所述方法包括以下步骤:
S1:间充质干细胞悬液的制备:将间充质干细胞传代培养至细胞60-90%汇合时,收集P3代细胞,制成间充质干细胞悬液;
S2:附着:将脱细胞真皮和间充质干细胞悬液放置于超低吸附反应器中使用完全培养基进行培养,先在37±1℃条件下震荡培养20-40min,再静置继续培养2-3天,培养条件如下:温度为37.0±0.5℃、CO2浓度为5.0±0.2%,湿度为饱和湿度;
S3:体外诱导脱细胞真皮负载的间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,所述诱导培养液以KFSM培养基为基础培养基,添加浓度为30-50g/L的黄芪注射液和体积分数为1-3%的胎牛血清。
2.如权利要求1所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,所述培养方法具体包括以下步骤:
S1:间充质干细胞悬液的制备:将间充质干细胞传代培养,用完全培养基培养至细胞90%汇合时,收集P3代细胞,制成间充质干细胞悬液;所述完全培养基为:以DMEM/F12为基础培养基,添加浓度为25μg/ml的人纤连蛋白、浓度为10ng/ml的碱性成纤维细胞生长因子、浓度为15ng/ml的人表皮细胞生长因子、浓度为1%的ITS、浓度为5%的人血白蛋白、浓度为1%的NEAA、浓度为0.1μmol/L的氢化考的松、浓度为0.1%的β-巯基乙醇;
S2:附着:将脱细胞真皮和间充质干细胞悬液放置于超低吸附反应器中使用完全培养基进行培养,先在37℃条件下震荡培养30min,摇床转速为50r/min,后停止震荡,静置继续培养2-3天,培养条件如下:温度为37.0±0.5℃、CO2浓度为5.0±0.2%,湿度为饱和湿度;
S3:体外诱导脱细胞真皮负载的间充质干细胞向皮肤细胞分化:采用诱导培养液进行换液,所述诱导培养液以KFSM培养基为基础培养基,添加浓度为40g/L的黄芪注射液和体积分数为2%的胎牛血清,诱导培养时间为14天。
3.如权利要求1或2所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,所述间充质干细胞包括脂肪间充质干细胞、脐带间充质干细胞和宫内膜间充质干细胞。
4.如权利要求3所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,所述间充质干细胞为脂肪间充质干细胞,所述脂肪间充质干细胞悬液的制备方法如下:
(1)取脂肪细胞;
(2)将所述脂肪细胞放置于试管内,在试管中加入与脂肪组织量相同体积的消化液,所述消化液包括体积比为1:1的胰蛋白酶-EDTA和0.1%的I型胶原酶,将试管密封,放入37℃的恒温摇床中以190r/Min的速度震荡消化,分为3层;
(3)吸出离心管中的下层液体移入装有完全培养基的新离心管中终止消化,将离心管封闭,离心,分为2层;
(4)用吸管吸取新离心管中的上清后去掉,加入完全培养基,轻轻吹打制成细胞悬液;
(5)将细胞悬液按照0.5×106/ml的密度接种到培养瓶中,放入37℃、5%CO2培养箱培养;12-24小时后进行第一次换液,此后每隔3天换液,待细胞生长至融合后进行传代;
(6)吸去培养瓶内的旧培养基,加PBS冲洗2-3次,再加入消化液消化3-5min,所述消化液为胰蛋白酶-EDTA;轻轻吹打细胞,使细胞脱离瓶底得到单细胞悬液;将单细胞悬液离心吹散,按1:8传代。
5.如权利要求4所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,步骤(1)所述的脂肪细胞为通过抽脂直接获得的脂肪抽取液。
6.如权利要求3所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,步骤(1)所述的脂肪细胞的获取方法为:取脂肪组织挤压进入离心管中,吸取PBS缓冲液加入到装有组织的离心管中,每克脂肪组织使用14mlPBS缓冲液,用吸管吹打10-20次,然后静置0.5-1.5min,用吸管将组织下层的液体吸出,如此反复直到所吸出的液体呈透明状,不带有血细胞为止。
7.如权利要求1所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法,其特征在于,所述脱细胞异体真皮支架放置于超低吸附反应器前需要进行预处理:将脱细胞真皮剪成边长为2cm的正方形小片,使用生理盐水冲洗三遍。
8.一种人工皮肤,其特征在于,由权利要求1-6任一所述的干细胞体外向皮肤细胞分化获得人工皮肤的方法制备而成。
9.权利要求8所述的人工皮肤在仿生材料医学领域中的应用。
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