CN111718894B - 一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法 - Google Patents
一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法 Download PDFInfo
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Abstract
本发明公开了一种联合利用hFDSPCs‑CM和HA促进糖尿病创面愈合的方法,联合利用***来源的真皮干细胞条件培养基和透明质酸促进糖尿病创面愈合,首次将***来源的真皮干细胞/祖细胞的条件培养基联合透明质酸应用于糖尿病鼠模型,与目前广泛应用的脂肪干细胞条件培养基相对比,具有更好的促进创面愈合的效果,尤其是在促进胶原合成方面有着明显优势,对临床治疗糖尿病难愈创面有着重要意义,同时操作简单有望在未来实现临床转化。本发明提供了***来源的真皮干细胞/祖细胞的条件培养基促进糖尿病创面愈合的相关数据,并且在体内体外实验中探究了相关机制。
Description
技术领域
本发明涉及一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法。
背景技术
糖尿病创面是临床常见的棘手问题,其特点是趋化因子和血管化不足,成纤维细胞迁移和增殖减少,以及异常的炎症反应。目前的针对该问题的常规治疗方法包括了定制辅料、外科清创、负压治疗以及抗生素和高压氧治疗等。但是,仍有近50%患者无法通过传统治疗方法治愈。
近年来大量研究证明了多种组织来源的间充质干细胞可以促进糖尿病创面愈合,例如:骨髓、脂肪、脐带血和皮肤组织。虽然不同组织来源的间充质干细胞有一定的相似性,但是在不同微环境中有不同的特征。并且已有研究证实了在体外实验中真皮干细胞比脂肪干细胞在促进创面愈合过程中更有优势。值得关注的是,***作为一种临床废弃组织,具有丰富的来源、容易获取以及无伦理争议的特点,具有作为临床应用间充质干细胞来源的的巨大潜能。
目前认为间充质干细胞促进创面愈合主要是靠旁分泌途径分泌丰富的“蛋白质组”包括:生长因子、miRNA、蛋白酶体和细胞外囊泡等。这些成分比细胞疗法更适合临床应用,因为它们规避了与细胞疗法相关的安全和伦理问题。
细胞外囊泡在创面治疗取得了较好的效果,但是制备过程繁琐限制了其进一步的应用。相比之下,条件培养基(conditionedmedium,CM)优势更加突出,如成本更加经济,制备过程更加简易,可以获得等效甚至更强效的产品。除CM外,创面敷料对创面愈合也有重要的作用。其中,透明质酸(hyaluronic acid,HA)作为一种皮肤中的天然成分,无毒副作用同时可以在局部有效传递CM中的活性成分,同时已经成为一种成熟的产品便于使用,因此,非常适合作为皮肤创面敷料。
目前多种组织来源的间充质干细胞条件培养基已经展现了对糖尿病创面愈合的促进作用。而***组织作为间充质干细胞的可靠来源同时具有来源广泛无伦理问题,在未来临床应用中有巨大潜力。同时相比于细胞外囊泡的复杂制备过程,条件培养基操作简单,同时更加经济,获得的蛋白质组成分更加丰富。
到目前为止,尚无研究报道人***来源的真皮干细胞/祖细胞条件培养基(humanforeskin-derived dermal stem/progenitor cells conditioned medium,hFDSPCs-CM)联合HA对糖尿病创面的作用效果。同时,由于人类脂肪来源干细胞条件培养基(Humanfat-derived stem cells conditions medium,hADSCs-CM)广泛应用于糖尿病创面治疗,选择hADSCs-CM作为阳性对照。
发明内容
本发明的目的是克服现有技术的缺陷,提供一种联合利用hFDSPCs-CM(***来源的真皮干细胞条件培养基)和HA(透明质酸)促进糖尿病创面愈合的方法,评估该方法对于促进糖尿病创面愈合的作用;分析了***来源的真皮干细胞条件培养基促进促进糖尿病创面愈合的具体机制。
实现上述目的的技术方案是:一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,包括以下步骤:
S1,细胞获取与培养,具体包括以下工序:
(1)hADSCs的提取:用胶原酶消化法消化抽脂术后废弃的颗粒脂肪,提取脂肪干细胞,长满后传代,第0~3代细胞用于实验;
(2)hFDSPCs的提取:用中性蛋白酶和胶原酶消化儿童***组织,用细胞克隆法培养细胞,第0~3代细胞用于实验;
(3)成纤维细胞的提取:用中性蛋白酶和胶原酶消化腹壁整形术后的皮肤组织,提取皮肤成纤维细胞,长满后传代,第3代细胞用于实验;
(4)人永生化角质形成细胞和人脐静脉内皮细胞采用市售产品;
S2,条件培养基制备:将hFDSPCs和hADSCs分别进行离心,然后分别用0.22μm滤网过滤,再分别用10kDa超滤管离心浓缩,并将hFDSPCs和hADSCs的浓缩液分别在负80度中保存;
S3,克隆形成实验:以50个细胞/cm2密度将细胞接种于6孔板,连续培养7天,固定并且统计克隆形成数;
S4,干细胞鉴定:利用细胞流式分析细胞标志物表达,并且通过三向分化诱导分析细胞多向分化能力;
S5,糖尿病小鼠创面模型的构建和干预:将雄性C57BLKS/J db/db糖尿病小鼠随机分为4组,每组5只:
(1)对照组:100μL PBS;
(2)HA组:10%HA;
(3)hADSCs-CM+HA组:100μg/mLhADSCs-CM混合在10%HA中;
(4)hFDSPCs-CM+HA组:100μg/mLhFDSPCs-CM混合在10%HA中;
每只小鼠吸入异氟醚麻醉,消毒备皮,在背部皮肤建立一个直径6毫米的圆形全层创面,所有小鼠在模型建立后1天给予1次治疗;
S6,糖尿病小鼠创面愈合评估,包括以下工序:
(1)创面愈合大体评估:在模型建立第0,3,7,10,14天采集所有小鼠照片,统计创面愈合率和自然愈合时间;
(2)组织学染色:模型建立第14天取材,HE、马松、天狼猩红染色,并且计算胶原/组织比例和I/III型胶原比例,CD31染色评估微血管形成,CD68染色评估炎症反应;
S7,体外实验评估:
(1)对细胞增殖能力影响分析:用CCK-8实验和EdU荧光染色分析hADSCs-CM和hFDSPCs-CM对HaCaT和成纤维细胞的增殖能力的影响;
(2)对细胞迁移能力影响分析:通过划痕实验分析hADSCs-CM和hFDSPCs-CM对HaCaT和成纤维细胞的迁移能力的影响;
(3)对成纤维细胞的ECM形成能力影响分析:
hADSCs-CM和hFDSPCs-CM分别作用于成纤维细胞后,分析细胞COL 1,COL 3,FN,α-SMA的表达情况,用RT-qPCR检测基因表达,免疫荧光染色和Western分析蛋白表达;
hADSCs-CM和hFDSPCs-CM分别作用于成纤维细胞后,RT-qPCR分析细胞MMP-1和MMP-3的基因表达情况;
相关通路分析:RT-qPCR检测hFDSPCs-CM作用后成纤维细胞TGF-β1的基因表达;ELISA检测hFDSPCs-CM作用后成纤维细胞分泌TGF-β1水平;Western分析hFDSPCs-CM作用后成纤维细胞TGF-β1/Smad通路蛋白表达;
(4)成管实验:分析hADSCs-CM和hFDSPCs-CM对HUVECs成管能力影响,并且统计成管数量和长度。
上述的一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,步骤S2中,将hFDSPCs和hADSCs分别进行离心时,离心加速度为300g,时间为10min;10kDa超滤管离心浓缩时的离心加速度为3000g,时间为1h。
上述的一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,步骤S5中,小鼠为8周龄,且体重为36.5~40.2克。
本发明的联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,首次将***来源的真皮干细胞/祖细胞的条件培养基联合透明质酸应用于糖尿病鼠模型,与目前广泛应用的脂肪干细胞条件培养基相对比,具有更好的促进创面愈合的效果,尤其是在促进胶原合成方面有着明显优势,对临床治疗糖尿病难愈创面有着重要意义,同时操作简单有望在未来实现临床转化。本发明提供了***来源的真皮干细胞/祖细胞的条件培养基促进糖尿病创面愈合的相关数据,并且在体内体外实验中探究了相关机制。
附图说明
图1为本发明的联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法的流程图;
图1A为hFDSPCs连续3代(P1-P3)成集落大体外观图;
图1B为hFDSPCs连续3代(P1-P3)成集落数统计图;
图1C为hFDSPCs连续3代集落形态镜下观察图;
图1D为细胞流式分析hFDSPCs免疫表型图;
图2A为hFDSPCs成骨诱导实验茜素红染色图;
图2B为hFDSPCs成骨相关基因表达分析图;
图2C为hFDSPCs成脂诱导实验油红O染色图;
图2D为hFDSPCs成脂相关基因表达分析图;
图2E为hFDSPCs成软骨诱导实验阿尔新兰染色图;
图2F为hFDSPCs成软骨相关基因表达分析图;
图3为hADSCs鉴定图;
图4A为糖尿病小鼠创面愈合情况图;
图4B为糖尿病小鼠第14天的创面比统计分析图;
图4C为糖尿病小鼠创面愈合时间统计图;
图5A为第14天HE染色组织愈合情况观察图;
图5B为第14天HE染色表皮再生情况观察图;
图5C为组织CD31染色微血管再生情况观察图;
图5D为组织CD31染色微血管再生情况统计图;
图5E为CD68阳性染色细胞观察图;
图5F为CD68阳性染色细胞数统计图;
图6为hFDSPCs-CM促进db/db小鼠创面胶原再生、成熟和重建实验图;
图7为hFDSPCs-CM体外促进HaCaT和成纤维细胞增殖实验图;
图8为hFDSPCs-CM体外促进HaCaT和成纤维细胞迁移实验图;
图9为hFDSPCs-CM促进HUVECs体外成管实验图;
图10为hFDSPCs-CM体外通过TGF-β/Smad通路促进细胞外基质形成实验图。
具体实施方式
为了使本技术领域的技术人员能更好地理解本发明的技术方案,下面结合附图对其具体实施方式进行详细地说明:
请参阅图1,本发明的最佳实施例,一种联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,包括以下步骤:
S1,细胞获取与培养,具体包括以下工序:
(1)hADSCs的提取:用胶原酶消化法消化抽脂术后废弃的颗粒脂肪,提取脂肪干细胞,长满后传代,第0~3代(P1~P3)细胞用于实验;
(2)hFDSPCs的提取:用中性蛋白酶和胶原酶消化儿童***组织,用细胞克隆法培养细胞,第0~3代(P1~P3)细胞用于实验;
(3)成纤维细胞的提取:用中性蛋白酶和胶原酶消化腹壁整形术后的皮肤组织,提取皮肤成纤维细胞,长满后传代,第3代细胞用于实验;
(4)人永生化角质形成细胞和人脐静脉内皮细胞采用市售产品;
S2,条件培养基制备:将hFDSPCs和hADSCs分别进行离心,离心加速度为300g,时间为10min;然后分别用0.22μm滤网过滤,再分别用10kDa超滤管离心浓缩,离心加速度为3000g,时间为1h;并将hFDSPCs和hADSCs的浓缩液分别在负80度中保存;
S3,克隆形成实验:以50个细胞/cm2密度将细胞接种于6孔板,连续培养7天,固定并且统计克隆形成数;
S4,干细胞鉴定:利用细胞流式分析细胞标志物表达,并且通过三向分化诱导分析细胞多向分化能力;
S5,糖尿病小鼠创面模型的构建和干预:将雄性C57BLKS/J db/db糖尿病小鼠随机分为4组,每组5只,小鼠为8周龄,且体重为36.5~40.2克:
(1)对照组(Control):100μL PBS;
(2)HA组:10%HA;
(3)hADSCs-CM+HA组:100μg/mLhADSCs-CM混合在10%HA中;
(4)hFDSPCs-CM+HA组:100μg/mLhFDSPCs-CM混合在10%HA中;
每只小鼠吸入异氟醚麻醉,消毒备皮,在背部皮肤建立一个直径6毫米的圆形全层创面,所有小鼠在模型建立后1天给予1次治疗;
S6,糖尿病小鼠创面愈合评估,包括以下工序:
(1)创面愈合大体评估:在模型建立第0,3,7,10,14天采集所有小鼠照片,统计创面愈合率和自然愈合时间;
(2)组织学染色:模型建立第14天取材,HE、马松、天狼猩红染色,并且计算胶原/组织比例和I/III型胶原比例,CD31染色评估微血管形成,CD68染色评估炎症反应;
S7,体外实验评估:
(1)对细胞增殖能力影响分析:用CCK-8实验和EdU荧光染色分析hADSCs-CM和hFDSPCs-CM对HaCaT和成纤维细胞的增殖能力的影响;
(2)对细胞迁移能力影响分析:通过划痕实验分析hADSCs-CM和hFDSPCs-CM对HaCaT和成纤维细胞的迁移能力的影响;
(3)对成纤维细胞的ECM形成能力影响分析:
hADSCs-CM和hFDSPCs-CM分别作用于成纤维细胞后,分析细胞COL 1,COL 3,FN,α-SMA的表达情况,用RT-qPCR检测基因表达,免疫荧光染色和Western分析蛋白表达;
hADSCs-CM和hFDSPCs-CM分别作用于成纤维细胞后,RT-qPCR分析细胞MMP-1和MMP-3的基因表达情况;
相关通路分析:RT-qPCR检测hFDSPCs-CM作用后成纤维细胞TGF-β1的基因表达;ELISA检测hFDSPCs-CM作用后成纤维细胞分泌TGF-β1水平;Western分析hFDSPCs-CM作用后成纤维细胞TGF-β1/Smad通路蛋白表达;
(4)成管实验:分析hADSCs-CM和hFDSPCs-CM对HUVECs成管能力影响,并且统计成管数量和长度。
一、请参阅图1A至图3,hFDSPCs具有多能干细胞特性以及hADSCs鉴定:
(1)hFDSPCs成克隆能力和表面标志物:
请参阅图1A至图1D,图1A显示了hFDSPCs连续3代(P1-P3)成集落大体观;图1B中通过成集落数统计显示连续3代成集落数在30以上;图1C显示了集落形态镜下观察图;图1D:细胞流式分析hFDSPCs免疫表型:hFDSPCs表达CD90(97.53%±0.48%),CD44(97.28%±0.52%),CD105(22.57%±2.09%),CD34(1.30%±0.75%),CD45(0.73%±0.39%),CD29(98.62%±1.18%),CD13(98.06%±0.26%),CD59(98.80%±1.02%),CD31(2.07%±0.31%)and CD133(0.88%±0.16%)。
(2)hFDSPCs具有多向分化潜能:
请参阅图2A至图2F,图2A显示了成骨诱导实验茜素红染色;图2B显示了骨相关基因表达分析;图2C显示了成脂诱导实验油红O染色;图2D显示了成脂相关基因表达分析;图2E显示了成软骨诱导实验阿尔新兰染色;图2F显示了成软骨相关基因表达分析,根据成脂相关基因表达分析和成软骨相关基因表达分析结果显示,hFDSPCs具有多向分化潜能。
(3)hADSCs鉴定:
图3中,3A为成骨诱导实验茜素红染色;3B为成脂诱导实验油红O染色;3C为成软骨诱导实验阿尔新兰染色;3D为细胞流式分析hADSCs免疫表型:CD19(1.17%±0.69%),CD34(0.83%±0.35%),CD11b(1.50%±0.86%),CD45(0.99%±0.54%),HLA-DR(1.20%±0.45%),CD73(97.97%±1.19%),CD90(97.03%±1.45%)and CD105(97.04%±1.12%)。
二、hFDSPCs促进db/db小鼠创面愈合:
请参阅图4A,糖尿病小鼠模型建立后分别给予100μLPBS,100μL 10%HA,100μg/mLhADSCs-CM混合在10%HA(hADSCs-CM+HA),100μg/mLhFDSPCs-CM混合在10%HA(hFDSPCs-CM+HA)在第0、3、7、10、14天观察创面愈合情况,结果显示4组愈合面积依次增加,hFDSPCs-CM+HA处理后第14天创面完全愈合;图4B显示了糖尿病小鼠第14天的创面比统计分析;图4C显示了糖尿病小鼠创面愈合时间统计。
三、hFDSPCs-CM促进db/db小鼠创面表皮再生、血管化和抑制炎症
请参阅图5A至图5F:图5A显示了对照组(Control)、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组,在第14天HE染色观察组织大体愈合情况;图5B显示了HE染色观察表皮再生情况:对照组几乎无表皮再生,HA组在创面边缘有较少的表皮再生,hADSCs-CM+HA组表皮再生明显但是仍不连续,hFDSPCs-CM+HA组表皮已经完全再生结构连续完整;请参阅图5C和5D:组织CD31染色,对照组微血管再生较少,HA组有所增加,hADSCs-CM+HA组合hFDSPCs-CM+HA组的微血管再生明显增多,但是两组之间没有明显统计学差异;请参阅图5E和5F对照组(Control)、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组CD68染色阳性依次减少,说明巨噬细胞减少,炎症反应得到抑制。
四、hFDSPCs-CM促进db/db小鼠创面胶原再生、成熟和重建
请参阅图6,图6中,6A为对照组、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组在第14天组织马松染色观察组织胶原再生情况:对照组中仅可见少量的稀疏新生胶原组织,HA组胶原含量有所增加但是结构仍不完整;hADSCs-CM+HA组局部可见胶原组织不连续且结构仍不规则;hFDSPCs-CM+HA组胶原合成明显增加同时结构连续规则;6B为天狼猩红染色分析:对照组、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组依次III型胶原(绿色荧光)减少,而I型胶原(橘色/红色)增加;6C为胶原成分与组织的比例:对照组、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组的胶原含量依次增加;6D显示对照组、HA组、hADSCs-CM+HA组和hFDSPCs-CM+HA组中I/III型胶原比例依次递增。
五、hFDSPCs-CM体外促进HaCaT和成纤维细胞增殖
请参阅图7,不同浓度梯度(0,5,10,20,50,100μg/mL)的hADSCs-CM和hFDSPCs-CM作用于HaCaT(A)和成纤维细胞(B)72小时后,CCK-8实验加测细胞活力,细胞活性呈现剂量依赖性增加,7A:HaCaT在hFDSPCs-CM20μg/mL和100μg/mL浓度作用下与hADSCs-CM相比有更强的细活性;7B:成纤维细胞在hFDSPCs-CM各浓度作用下都比hADSCs-CM有很强的细胞活性;选择20μg/mL作为工作浓度,hADSCs-CM和hFDSPCs-CM分别作用于HaCaT(C)和成纤维细胞(D)72小时,EdU荧光染色检测细胞增殖活力,7C:HaCaT在hFDSPCs-CM作用下比hADSCs-CM组EdU荧光阳性染色增加;7D:HaCaTEdU荧光阳性染色统计分析;7E:成纤维细胞在hFDSPCs-CM作用下比hADSCs-CM组EdU荧光阳性染色增加;7F:成纤维细胞荧光阳性染色统计分析。
六、hFDSPCs-CM体外促进HaCaT和成纤维细胞迁移
请参阅图8,选择20μg/mL作为工作浓度,hADSCs-CM和hFDSPCs-CM分别作用于HaCaT和成纤维细胞作用24小时,通过划痕实验分析细胞的迁移能力。8A:与对照相比HaCaT在hADSCs-CM和hFDSPCs-CM作用下细胞迁徙明显增加;8B:HaCaT 24小时候迁移面积;8C:与hADSCs-CM组相比,hFDSPCs-CM组成纤维细胞的迁移面积明显增加;8D:成纤维细胞24小时候迁移面积。
七、hFDSPCs-CM促进HUEVCs体外成管
请参阅图9,9A:选择20μg/mL作为工作浓度,hADSCs-CM和hFDSPCs-CM分别作用于HUVECs 6小时后观察细胞的成管能力,与对照组相比hADSCs-CM组和hFDSPCs-CM组细胞成管明显增加。9B:成管分支数量统计:与对照组相比较,hADSCs-CM和hFDSPCs-CM组成管分支数量明显增加,两组之间无明显统计学差异;9C:成管长度统计:与对照组相比较,hADSCs-CM和hFDSPCs-CM组成管长度明显增加,两组之间无明显统计学差异。
八、hFDSPCs-CM体外通过TGF-β/Smad通路促进细胞外基质(extracellularmatrix,ECM)形成
请参阅图10,hADSCs-CM和hFDSPCs-CM(20μg/mL)分别作用于通过成纤维细胞48小时后,通过RT-qPCR检测ECM成分COL 1(见图10中的10A),COL 3(见图10中的10B),FN(见图10中的10C)以及肌成纤维细胞标志物α-SMA(见图10中的10D)的基因表达,10I为免疫荧光检测以上指标的表达;10J为Westernblot检测以上指标的蛋白表达水平;结果证明以上成分在hFDSPCs-CM组的表达高于hADSCs-CM组。同时金属蛋白酶MMP-1(见图10中的10E)和MMP-3(见图10中的10F)的基因表达水平在hFDSPCs-CM组下降。随后检测TGF-β/Smad通路是否参与了hFDSPCs-CM的对成纤维细胞的调节过程,首先检测了TGF-β1的基因表达(见图10中的10G),ELISA检测TGF-β1细胞外分泌水平(见图10中的10H),结果表明了TGF-β1在hFDSPCs-CM组的表达高于hADSCs-CM组,随后分析相关通路;10K为将不同浓度的hFDSPCs-CM(0,10,20μg/mL)作用于成纤维细胞检测TGF-β/Smad通路中相关蛋白表达情况,结果表明随着hFDSPCs-CM浓度增加,通路中TGF-β1蛋白表达增加,以及Smad2和Smad3的磷酸化水平增加,证明了TGF-β/Smad通路参与了体外hFDSPCs-CM促进成纤维细胞合成胶原的过程。
综上所述,本发明联合利用hFDSPCs-CM和HA促进糖尿病创面愈合的方法,首次将***来源的真皮干细胞/祖细胞的条件培养基联合透明质酸应用于糖尿病鼠模型,与目前广泛应用的脂肪干细胞条件培养基相对比,具有更好的促进创面愈合的效果,尤其是在促进胶原合成方面有着明显优势,对临床治疗糖尿病难愈创面有着重要意义,同时操作简单有望在未来实现临床转化。本发明提供了***来源的真皮干细胞/祖细胞的条件培养基促进糖尿病创面愈合的相关数据,并且在体内体外实验中探究了相关机制。
本技术领域中的普通技术人员应当认识到,以上的实施例仅是用来说明本发明,而并非用作为对本发明的限定,只要在本发明的实质精神范围内,对以上所述实施例的变化、变型都将落在本发明的权利要求书范围内。
Claims (1)
1. 人***来源的真皮干细胞/祖细胞条件培养基和透明质酸在制备促进糖尿病创面愈合的药物中的用途,其特征在于,人***来源的真皮干细胞/祖细胞条件培养基的制备方法为:将人***来源的真皮干细胞/祖细胞的培养液上清进行离心,然后用0.22μm滤网过滤,再用10kDa超滤管离心浓缩,并将浓缩液在-80℃中保存;将人***来源的真皮干细胞/祖细胞的培养液上清进行离心时,离心加速度为300g,时间为10min;10kDa超滤管离心浓缩时的离心加速度为3000 g,时间为1h。
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