CN112409456B - 干细胞细胞因子在制备化妆品或药物中的用途 - Google Patents
干细胞细胞因子在制备化妆品或药物中的用途 Download PDFInfo
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Abstract
本发明涉及干细胞细胞因子在制备化妆品或药物中的用途。通过前期筛选，制备并获得了能够促进脂肪干细胞分泌细胞因子的多肽，所述多肽经过使用优化的方法能够高水平的促进脂肪干细胞分泌细胞因子，特别是能够促进伤口愈合的IL‑10因子以及其他多种类的活性因子。将制备的含有细胞因子的上清液作为药物对糖尿病皮肤创伤修复就有很好的愈合作用，可快速的发挥疗效，与偶极好的应用价值。
Description
技术领域
本发明涉及生物领域，具体的，涉及一种干细胞细胞因子在制备化妆品或药物中的用途。
背景技术
皮肤组织的损伤常由机械性、物理性、化学性和生物性等因素引起，机体对形成的损伤进行修补的过程称为组织修复。组织修复还是一系列不同类型的修复细胞、结构蛋白和生长因子等形成网络式交互作用的结果。
研究表明干细胞能够具有皮肤组织修复功能，例如皮肤干细胞。在毛囊隆突区移植皮肤干细胞时，第一次发现皮肤干细胞可分化为皮肤滤泡间上皮、皮脂腺和毛囊细胞。皮肤干细胞包括表皮干细胞、毛囊干细胞、真皮干细胞、皮脂腺干细胞和汗腺干细胞等，主要存在于毛囊隆突区，这些干细胞主要维护皮肤的自我更新和创伤后的再生。表皮的自我更新能力主要取决于表皮基底层的干细胞，表皮干细胞(ESC)的增殖主要取决于皮肤成纤维细胞分泌的生长因子，包括FGF-4、KGF和IL-6等。多项研究显示，ESC可治疗不同类型的皮肤缺损，如皮肤的烧伤和下肢慢性溃疡等，并将成为新的治疗方法。毛囊干细胞能分化形成毛囊细胞和表皮细胞，当表皮损伤时毛囊干细胞又可分化形成ESC。研究证明，人的毛囊干细胞可促进外周或脊髓神经组织损伤的修复和再生。ESC不仅能维持正常细胞的自我更新，而且使损伤的皮肤得以再生，普遍认为这些干细胞较易进入受损的皮肤组织促进其修复和再生，对以后的治疗具有重要意义。
脂肪干细胞也是常用的皮肤修复用细胞，其是另一种被广泛应用的多能干细胞，与BM-MSC有相似特性，可分化为不同谱系的细胞。另外，脂肪干细胞能分泌多种生长因子并作用于创缘的各种细胞。脂肪干细胞培养上清液可激活皮肤成纤维细胞和角质细胞，并通过旁分泌作用促进皮肤损伤的修复和再生。目前，应用人类吸脂术后的脂肪组织培养脂肪干细胞，并成功诱导其分化为软骨细胞。最近研究表明，脂肪干细胞可通过细胞与细胞间的相互作用和旁分泌作用促进人类皮肤成纤维细胞增殖，进而加快损伤皮肤的再生；该研究还发现脂肪干细胞不仅可分泌多种胶原蛋白、纤连蛋白和生长因子，还可促进细胞外基质蛋白mRNA的表达和成纤维细胞的迁移。Di Rocco等发现，局部应用脂肪来源的干细胞治疗能诱导细胞的分化，增加修复细胞的归巢和生长因子的旁分泌，从而促进小鼠糖尿病溃疡愈合。
研究表明，干细胞之所以能够治疗皮肤疾病，这与间充质干细胞可分泌多种细胞因子，如VEGF(血管内皮生长因子)、bFGF(碱性成纤维细胞生长因子)、
NGF(神经生长因子)、HGF(肝细胞生长因子)、IGF-1(胰岛素样生长因子1)、BDNF(脑源性神经营养因子)、GDNF(胶质源性神经营养因子)、IL-6(白细胞介素6)、IL-11(白细胞介素11)等有密切关系。间充质干细胞分泌活性因子具有改善炎性环境、调节机体免疫状态、抑制细胞凋亡、促进细胞增殖、促进血管再生、促进神经干细胞迁移和分化、促进轴突生长及突触连接形成、促进髓鞘形成的功能，通过脑白质损伤的动物模型证实可以抑制宿主细胞及移植细胞凋亡、改善模型鼠的神经功能，通过心梗动物模型证实可以促进心功能恢复，通过皮肤损伤实验证实可以促进伤口愈合。
目前提高干细胞细胞因子产量的方法虽然已有研究，但是用于大规模使用的方法还不多，应用场景也不充分，需要进一步的提高。
发明内容
本发明一方面，根据之前的研究基础，提供了一个能够促进干细胞细胞因子分泌的多肽，所述多肽能够特异性的促进细胞因子的分泌。
具体的，所述的多肽为CJYZ-7多肽，其序列如SEQ ID NO：1所示。
本发明另外一方面，提供一种提高干细胞细胞因子产量的方法，所述方法包取脂肪干细胞，将细胞接种于T25培养瓶中，完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养，待细胞浓度融合至80％后，吸弃原来的培养基，用PBS洗涤2次，再每瓶加入配制含多肽CJYZ-7的完全培养基(含DMEM—HG-F12培养基和10％胎牛血清)37℃、5％CO2、5％O2，继续培养36h后，半量相同的新鲜培养基换液，收集换下的细胞代谢的培养基备用；继续培养36h后收集培养基并合并之前的半量换下的细胞代谢的培养基，其中即含有制备的高含量细胞因子。
此外，本发明还提供了细胞因子的制备方法，具体的，包括将干细胞培养的上清液进行离心，随后将上清液通过中空纤维滤膜进行循环过滤，后泵至切向流超滤膜包，循环过滤浓缩，将浓缩液经0.22μm滤膜过滤除菌，分装到西林瓶，置于-80℃冰箱速冻；随后将西林瓶置于压盖式真空冷冻干燥机，浮盖盖好，启动冷冻干燥机，设置真空度1.05mbar，干燥时间24h；干燥结束，真空压盖，西林瓶取出-20℃保存。
更进一步的，本发明提供所述方法制备的细胞因子在制备皮肤修复的药物中的应用。
所述药物还可以包含其他的能够促进皮肤损伤修复的第二物质。
进一步地，所述的药物为液体制剂或者固体制剂。
本发明另一目的在于提供上述的含有干细胞因子的药物在促进糖尿病皮肤创伤修复中的用途。
更具体的，所述含有干细胞因子的药物是采用如下方法制备获得：取脂肪干细胞，将细胞接种于T25培养瓶中，完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养，待细胞浓度融合至80％后，吸弃原来的培养基，用PBS洗涤2次，再每瓶加入配制含多肽的完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)37℃、5％CO2、5％O2，继续培养36h后，半量相同的新鲜培养基换液，收集换下的细胞代谢的培养基备用；继续培养36h后收集培养基并合并之前的半量换下的细胞代谢的培养基，其中即含有制备的高含量细胞因子；将培养基进行离心，随后将上清液通过中空纤维滤膜进行循环过滤，后泵至切向流超滤膜包，循环过滤浓缩，将浓缩液经0.22μm滤膜过滤除菌，分装到西林瓶，置于-80℃冰箱速冻；随后将西林瓶置于压盖式真空冷冻干燥机，浮盖盖好，启动冷冻干燥机，设置真空度1.05mbar，干燥时间24h；干燥结束，真空压盖，西林瓶取出-20℃保存。
更进一步的，所述药物中还含有赋形剂、防腐剂以及其他可用的活性物质。
本发明具有以下优点：
1.本发明通过前期筛选，制备并获得了能够促进脂肪干细胞分泌细胞因子的多肽，所述多肽经过使用优化的方法能够高水平的促进脂肪干细胞分泌细胞因子，特别是能够促进伤口愈合的IL-10因子以及其他多种类的活性因子。
2.本发明将制备的含有细胞因子的上清液药物主要活性成分为人干细胞因子，对糖尿病皮肤创伤修复就有很好的愈合作用，可快速的发挥疗效。
附图说明
图1多肽对干细胞生长的促进效果图
图2细胞因子基因表达量检测结果图
具体实施方式
为了更进一步的说明本发明的目的、技术方案和优点，我们结合以下具体实施例来阐述本发明，这些实施例仅为了更好的说明本发明专利，而不用于限制本发明范围。基于本发明中的实施例，本领域的技术人员在没有做出创造性的情况下所得到的其他所有实施方式均属于本发明所保护的范围。
实施例1脂肪间充质干细胞的制备
无菌条件下将健康成年人皮下脂肪10g，经组织冲洗液(Hank’s液加150μg/ml庆大霉素和1μg/ml两性霉素)冲洗，培养皿中剔除结缔组织和小血管，使用剪刀剪成细小组织碎片，PBS磷酸盐缓冲液反复冲洗5次除去红细胞。.0.15％I型胶原酶与胰蛋白酶按l:1配制成组织消化液，按组织4倍体积加入进行消化，37℃恒温摇床中振荡150r/min消化25min。组织悬液经200目筛网进行机械分离，得细胞悬液经1000r/min离心5min，弃上清。所得细胞沉淀经PBS反复冲洗，离心3次。最终所得细胞沉淀以含20％FCS的DMEM-F12培养基进行培养，待观察到贴壁细胞后首次换液。细胞传至3代进行流式细胞分析检测免疫表型。用体积分数0.25％的胰蛋白酶消化收集P3代CD31+细胞，取2×106个细胞于流式管中，PBS洗涤2次，将荧光标记单克隆抗体CD29，CD73，CD90和CD34(美国Bioscience公司)加入到流式管中，鼠同型对照抗体作为参照。轻轻混匀，4℃避光孵育30min。流式细胞仪(英国BD公司)行表面抗原标记鉴定。结果如下表1所示。
表1分离的脂肪干细胞表面分子鉴定结果
| 表面分子 | 结果 |
| CD29 | (99.33％±0.11％) |
| CD73 | (99.74％±0.20％) |
| CD90 | (99.96％±0.15％) |
| CD34 | (2.01％±0.04％) |
从表1的脂肪干细胞鉴定情况可以看出，P3代脂肪干细胞其表达阳性细胞比例分别为CD29(99.33％)、CD73(99.74％)和CD90(99.96％)，而几乎不表达造血干细胞表面标志CD34(2.01％)。这说明成功的分离并制备得到了脂肪干细胞。
实施例2多肽CJYZ-7对脂肪间充质干细胞细胞因子分泌的影响
收集实施例1制备的P3代脂肪干细胞，将细胞接种于T25培养瓶中，完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养，待细胞浓度融合至80％后，吸弃原来的培养基，用PBS洗涤2次，在每瓶加入配制含多肽CJYZ-7(序列如SEQ ID NO：1所示)终浓度为50ng/mL的完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)37℃、5％CO2、5％O2，继续培养36h后，半量相同的新鲜培养基换液，收集换下的细胞代谢的培养基备用；继续同条件培养36h后收集培养基并合并之前的半量换下的细胞代谢的培养基，离心上清液中即含有制备的高含量细胞因子。以不含多肽的培养基同条件作为对照。使用ELISA试剂盒(美国R&D公司)测定收集的培养基上清液中血管内皮细胞生长因子、脑源性神经营养因子和肝细胞生长因子水平。
收集实施例1制备的P10代脂肪干细胞，将细胞接种于T25培养瓶中，完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养，待细胞浓度融合至80％后，吸弃原来的培养基，用PBS洗涤2次，在每瓶加入配制含多肽CJYZ-7终浓度为50ng/mL的完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)的完全培养基(含DMEM7HG/F12培养基和10％胎牛血清)37℃、5％CO2、5％O2，继续培养36h后，半量相同的新鲜培养基换液，收集换下的细胞代谢的培养基备用；继续同条件培养36h后收集培养基并合并之前的半量换下的细胞代谢的培养基，离心上清液中即含有制备的高含量细胞因子。以不含多肽的培养基同条件作为对照。使用ELISA试剂盒(美国R&D公司)测定收集的培养基上清液中血管内皮细胞生长因子、脑源性神经营养因子和肝细胞生长因子水平。
(1)脑源性神经营养因子的测定：50μL标准品及样品加入已包被好的反应杯中，室温下孵育2h，不洗板直接加入脑源性神经营养因子结合液，室温孵育1h，洗板后加入底物室温30min，然后再加终止液，30min内，在450nm处测吸光度；(2)血管内皮细胞生长因子的测定：200μL标准品及样品加入已包被好的反应杯中，室温下孵育2h，洗板后加入血管内皮细胞生长因子结合液，室温孵育2h，洗板后加入底物室温20min，然后再加终止液，30min内在450nm处测吸光度；(3)肝细胞生长因子的测定：50μL标准品及样品加入已包被好的反应杯中，室温下孵育2h，洗板后加入肝细胞生长因子结合液，室温孵育1.75h，洗板后加入底物室温30min，然后再加终止液，30min内在450nm处测吸光度。根据标准曲线计算相应血管内皮细胞生长因子、脑源性神经营养因子和肝细胞生长因子水平。结果如表2所示。
表2多肽对细胞因子产量的影响
| 类型 | VEGF(μg/L) | BDNF(μg/L) | HGF(μg/L) |
| P3细胞不加多肽 | 988.2±36.9 | 123.4±13.5 | 1588.6±98.6 |
| P3细胞加多肽 | 1645.1±55.7 | 197.4±28.7 | 2711.9±130.2 |
| P10细胞不加多肽 | 1023.5±45.8 | 92.5±18.6 | 1236.5±84.2 |
| P10细胞加多肽 | 1711.9±52.6 | 175.3±19.2 | 2281.2±90.1 |
从表1的结果可以看出，P3代和P10代的干细胞在添加了多肽的培养基中各种细胞因子的分泌量随着多肽浓度的升高均得到了显著的提高。而没有添加所述多肽的干细胞相应的细胞因子产量没有显著提高。
实施例3多肽对干细胞生长以及相应细胞因子产量的影响
绘制脂肪干细胞生长曲线：将上述P3代脂肪干细胞分别用CJYZ-7多肽终浓度为50ng/mL的完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)和不含多肽的完全培养基分别重悬为6×106L-1单细胞悬液，接种于5块96孔培养板中，每孔200μL(2.4×104/孔)每板设5个复孔，完全培养基做空白对照，测定吸光度值后取其均值。在培养0，24，48，72，96h，分别更换相应的培养基后加入CCK-8试剂继续培养2h，用酶标仪测定吸光度值，将吸光度值代入标准曲线获得细胞数后，绘制细胞生长增殖曲线。结果如图1所示。
从图1的结果可以看出，在培养96h后，OD值达到了3.2，比对照的2.9有显著的提高，这说明多肽不仅能够增加细胞因子的胞外分泌，还能够提高细胞的增殖效果。
实施例4细胞因子基因表达量检测
将实施例3采用CJYZ-7多肽培养以及没有采用多肽培养的干细胞，用PBS洗细胞3次，采用Trizol法提取总RNA，反转录为cDNA。PCR反应体系：SYBR Premix Ex TaqTM II 25μL，Forward Primer2μL，Reverse Primer 2μL，ROX Reference Dye 1μL，cDNA 4txL，dH2O16μL。反应条件：95℃15s，95℃10S，61℃30S，循环40次。反应完成后，计算机自动分析荧光信号，并将其转换为ct值(循环阈值)，根据公式计算肝细胞生长因子(HGF)、尿激酶型纤溶酶原激活物(uPA)、基质金属蛋白酶-1(MMP-1)、血管内皮生长因子(VEGF)的相对表达量。引物序列见表2。
表2引物序列表
结果如图2所示，经过多肽处理后的干细胞其中IL-10、HGF、uPA、MMP-1、VEGF均得到了显著的提高，其中特别是IL-10的提高量最高达到了2.4倍，但是其他因子表达量相较IL-10的提高效果相对减弱，但是也比未经多肽处理的干细胞的细胞因子的表达量有显著的的提高。
实施例5、本发明修复液促进糖尿病皮肤创伤修复试验
1、多肽处理的上清液的制备
将实施例1制备的P3代脂肪干细胞，以5×103/mL密度接种于12孔板，每孔1mL，各组重复3次，用CJYZ-7多肽终浓度为50ng/mL的完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养3d后，收集细胞上清液500g离心10min，随后进行浓缩；所述浓缩步骤为将上清液通过中空纤维滤膜进行循环过滤3遍，后泵至切向流超滤膜包，循环过滤浓缩，干细胞因子浓缩液，将浓缩液经0.22μm滤膜过滤除菌备用；部分长期保存的可以分装到西林瓶，置于-80℃冰箱速冻8h；随后将西林瓶置于压盖式真空冷冻干燥机，浮盖盖好，启动冷冻干燥机，设置真空度1.05mbar，干燥时间24h；干燥结束，真空压盖，西林瓶取出-20℃保存。1、多肽处理的上清液的制备
2、未用多肽处理的上清液的制备
将实施例1制备的P3代脂肪干细胞，以5×103/mL密度接种于12孔板，每孔1mL，各组重复3次，用完全培养基(含DMEM-HG/F12培养基和10％胎牛血清)培养3d后，收集细胞上清液500g离心10min，随后进行浓缩；所述浓缩步骤为将上清液通过中空纤维滤膜进行循环过滤3遍，后泵至切向流超滤膜包，循环过滤浓缩，干细胞因子浓缩液，将浓缩液经0.22μm滤膜过滤除菌备用。
3、模型制备：db/db小鼠(二型糖尿病小鼠)(货号J220，南京君科生物工程有限公司)先用脱毛膏将小鼠背部双侧毛发脱除，再用皮肤打孔器在双侧做8mm的圆形全层皮肤切除伤口。将上述皮肤损伤模型的小鼠，随机分为3组：空白对照组(10只，不加任何处理)、多肽处理上清实验组(10只，伤口处涂抹多肽培养的上清浓缩液0.2mL)、未用多肽处理上清对照组(10只，伤口处涂抹未用多肽处理的上清组0.2mL)。给药后每天观察创口愈合情况，并统计愈合率，结果如表3所示。
表3试验结果
从表3可以明显看出，涂抹本发明实施例多肽处理上清浓缩液后第7天愈合率较对照组明显显著(**P＜0.01)，尤其是涂抹后第14天，愈合率达到了94.17±2.01％以上，而对照组仅仅为79.88±2.63％左右，空白组仅仅为49.52％左右，并且所有试验小鼠均存活至试验完成。这充分说明本发明多肽促进生产的细胞因子能够加速皮肤损伤的修复，具有较好的应用价值。
序列表
<110> 北京欣颂生物科技有限公司
<120> 干细胞细胞因子在制备化妆品或药物中的用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Ser Ile His Leu Phe Lys Gly Gln Lys Ile Trp Trp His Val Ser
1 5 10 15
Trp His Thr
Claims (4)
1.一种促进脂肪干细胞分泌细胞因子的多肽，所述多肽的氨基酸序列如SEQ ID NO：1所示。
2.一种促进脂肪干细胞分泌细胞因子的方法，其特征在于：取脂肪干细胞，将细胞接种于T25培养瓶中，用含DMEM-HG/F12培养基和10％胎牛血清的完全培养基进行过培养，待细胞浓度融合至80%后，吸弃原来的培养基，用PBS洗涤2次，再每瓶加入配制含终浓度为50ng/mL多肽的DMEM-HG/F12培养基和10％胎牛血清完全培养基 37℃、5％CO2、5％O2，继续培养36h后，半量相同的新鲜培养基换液，收集换下的细胞代谢的培养基备用；继续培养36h后收集培养基并合并之前的半量换下的细胞代谢的培养基，其中即含有制备的高含量细胞因子；其中多肽为权利要求1所述的多肽。
3.如权利要求2所述的方法，其特征在于：还包括如下步骤：将收获的培养基进行离心，随后将上清液通过中空纤维滤膜进行循环过滤，后泵至切向流超滤膜包，循环过滤浓缩，将浓缩液经0.22μm滤膜过滤除菌，分装到西林瓶，置于-80℃冰箱速冻；随后将西林瓶置于压盖式真空冷冻干燥机，浮盖盖好，启动冷冻干燥机，设置真空度1.05mbar，干燥时间24h；干燥结束，真空压盖，西林瓶取出-20℃保存。
4.氨基酸序列如SEQ ID NO：1所示的多肽在体外促进脂肪干细胞分泌细胞因子中的用途。
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