CN114533935A - 一种用于解决青春痘的细胞因子敷料及其制备方法 - Google Patents
一种用于解决青春痘的细胞因子敷料及其制备方法 Download PDFInfo
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- CN114533935A CN114533935A CN202210191032.9A CN202210191032A CN114533935A CN 114533935 A CN114533935 A CN 114533935A CN 202210191032 A CN202210191032 A CN 202210191032A CN 114533935 A CN114533935 A CN 114533935A
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Abstract
本发明涉及医用敷料技术领域，具体涉及一种用于解决青春痘的细胞因子敷料及其制备方法，细胞因子冻干粉1‑2份、聚谷氨酸5‑15份、甘油2‑6份、柠檬酸0‑2份、胶原蛋白10‑20份、海藻酸钠0‑15份、角蛋白1‑2份、壳聚糖5‑10份、碳酸氢钠0‑2份，通过免疫细胞与干细胞进行共培养，能够诱导干细胞与免疫细胞分泌的物质进行反应，从而生成具有一定抗菌作用的外泌体；碳酸氢钠能够与柠檬酸反应使敷料中存在大量细小孔洞，吸收皮肤脓液和组织液的效果，加快敷料中细胞因子的渗出，起到更好的消毒抗炎的作用，且能够加快角质促进伤口愈合，海藻酸钠、甘油、聚谷氨酸、角蛋白、胶原蛋白和壳聚糖能够增加皮肤保湿能力，缓解皮肤红肿的现象，从而促使青春痘快速好转。
Description
技术领域
本发明涉及医用敷料技术领域，具体涉及一种用于解决青春痘的细胞因子敷料及其制备方法。
背景技术
在日常生活和医疗工作中经常会碰到青少年、青壮年因头额、面颊及胸背等处青春痘、毛囊炎频频发作而烦恼不已，他们千方百计求医问药、美容整形，不惜耗资但收效甚微，青少年青壮年是一个庞大的群体，这个群体随着年龄的增长，他们的皮肤病可能会因为生活节奏的加快，生活中营养摄入的丰富多样，疾病和病毒的流行等等因素而复发或者变化，病程还常常缠绵不愈。
青春痘的发生主要与皮脂分泌过多、毛囊皮脂腺导管堵塞、细菌感染和炎症反应等因素密切相关，进入青春期后人体内雄激素特别是睾酮的水平迅速升高，促进皮脂腺发育并产生大量皮脂，同时毛囊皮脂腺导管的角化异常造成导管堵塞，皮脂排出障碍，形成角质栓，毛囊中多种微生物尤其是痤疮丙酸杆菌大量繁殖，痤疮丙酸杆菌产生的脂酶分解皮脂生成游离脂肪酸，同时趋化炎症细胞和介质，最终诱导并加重炎症反应。
发明内容
本发明要解决的问题是提供了一种促进伤口愈合、抑菌抗炎的用于解决青春痘的细胞因子敷料及其制备方法。
为解决上述技术问题，本发明采用的技术方案是一种用于解决青春痘的细胞因子敷料，按照重量份数包括以下组分：
细胞因子冻干粉1-2份、聚谷氨酸5-15份、甘油2-6份、柠檬酸0-2份、胶原蛋白10-20份、海藻酸钠0-15份、角蛋白1-2份、壳聚糖5-10份、碳酸氢钠0-2份。
通过免疫细胞与干细胞进行共培养，能够诱导干细胞与免疫细胞分泌的物质进行反应，从而生成具有一定抗菌作用的外泌体；碳酸氢钠能够与柠檬酸反应从而生成柠檬酸钠，水和二氧化碳，从而能够使敷料中存在大量细小孔洞，能够起到一定吸收皮肤脓液和组织液的效果，且能够加快敷料中细胞因子的渗出，起到更好的消毒抗炎的作用，而生成的柠檬酸钠对于皮肤也具有较好的效果，且减少了柠檬酸对于皮肤的刺激，使敷料更为温和，且能够起到加快角质更新的作用，从而能够起到促进伤口愈合的作用，且柠檬酸钠能够促进皮肤黑色素的剥落，也具有一定的杀菌效果，从而能够增强敷料消毒抗炎的效果；
海藻酸钠、甘油和聚谷氨酸能够增加皮肤保湿能力，且能够起到一定的抗菌作用，促进皮肤健康生长，
通过角蛋白、胶原蛋白和壳聚糖，能够起到促进细胞生长，促进伤口愈合的作用，缓解皮肤红肿的现象，从而促使青春痘快速好转
按照重量份数包括以下组分：
细胞因子冻干粉1.2-1.8份、聚谷氨酸7-13份、甘油3-5份、柠檬酸1份、胶原蛋白12-18份、海藻酸钠3-12份、角蛋白1-2份、壳聚糖6-9份、碳酸氢钠1份。
所述细胞因子冻干粉内含有细胞因子，所述细胞因子为干细胞与免疫细胞共培养的外泌体，所述细胞因子的活性单位为5000U/g。
所述细胞因子冻干粉的制备方法为：将干细胞与免疫细胞共培养后，再单独收集获取干细胞的培养上清，进行冻干，得到细胞因子冻干粉。
干细胞与免疫细胞共培养，具体为：
S1：将干细胞进行传代培养；
S2：取骨髓细胞，加入红细胞裂解液进行裂解，离心，获得免疫细胞；
S3：采用培养基洗吹三次，离心，采用培养基进行吹打获得重悬细胞，转移至培养瓶中培养24-48h；
S4：将所述干细胞与所述免疫细胞分别转移至共培养装置中，进行共培养。
进一步的，所述干细胞为间充质干细胞。
进一步的，制备细胞因子冻干粉包括如下步骤：
将空白培养基加入S4所述的共培养装置中，24h-48h后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
将所述培养上清液与外泌体提取液以体积比2:1进行混合，在4℃条件下孵育过夜，得到孵育液，将孵育液在4℃，10000g的条件下离心60min，吸取底部沉淀，获得含外泌体的浓缩液；
将浓缩液用PBS缓冲液进行溶解，得到外泌体盐溶液，将所述外泌体盐溶液进行冻干，得到细胞因子冻干粉。
进一步的，所述冻干温度为-80℃，冻干12h。
制备用于解决青春痘的细胞因子敷料的方法，其特征在于：包括如下步骤：
步骤一：将角蛋白加入50-60份去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸、胶原蛋白和碳酸氢钠，搅拌均匀获得溶液A；
步骤二：将壳聚糖加入50-60份去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油、柠檬酸、细胞因子冻干粉和海藻酸钠，搅拌均匀获得溶液B；
步骤三：将所述溶液A和溶液B进行混合搅拌1-2min，至于培养皿中，进行冻干即得成品；
进一步的，步骤三中，冻干温度为-80℃，冻干6-8h。
进一步的，所述免疫细胞含有粒细胞和巨噬细胞等，所述干细胞、免疫细胞的培养基均为RPMI1640培养基，共培养的培养基也为RPMI1640培养基。
与现有技术相比，本发明具有的优点和积极效果是：
本发明通过通过免疫细胞与干细胞进行共培养，能够诱导干细胞与免疫细胞分泌的物质进行反应，从而生成具有一定抗菌作用的外泌体；碳酸氢钠能够与柠檬酸反应从而生成柠檬酸钠，水和二氧化碳，从而能够使敷料中存在大量细小孔洞，能够起到一定吸收皮肤脓液和组织液的效果，且能够加快敷料中细胞因子的渗出，起到更好的消毒抗炎的作用，而生成的柠檬酸钠对于皮肤也具有较好的效果，且减少了柠檬酸对于皮肤的刺激，使敷料更为温和，且能够起到加快角质更新的作用，从而能够起到促进伤口愈合的作用，且柠檬酸钠能够促进皮肤黑色素的剥落，也具有一定的杀菌效果，从而能够增强敷料消毒抗炎的效果；
海藻酸钠、甘油和聚谷氨酸能够增加皮肤保湿能力，且能够起到一定的抗菌作用，促进皮肤健康生长，
通过角蛋白、胶原蛋白和壳聚糖，能够起到促进细胞生长，促进伤口愈合的作用，缓解皮肤红肿的现象，从而促使青春痘快速好转。
本发明具有修复皮肤屏障的作用，在表皮肤形成膜防护，导致角质层细胞活性加强和表皮脂质合成速度加快，从内部改善皮肤屏障的完善，修复肌肤组织损伤，激活细胞再生，能够针对痤疮、粉刺、青春痘、皮炎，湿疹等皮肤问题，缓解灼痛，消除红斑，缩短修复时间，高效创面愈合，抑菌抗炎，修复肌肤组织损伤。
具体实施方式
实施例1：
步骤一：S1：将间充质干细胞进行传代培养，用0.25％的胰蛋白酶对间充质干细胞进行消化，加入RPMI1640培养基终止消化，将其接种至T25培养瓶中，培养24h后更换培养基，培养7d；
用RPMI1640培养基将股骨中的骨髓细胞冲洗出来，使用巴氏吸管将骨髓吹打成单细胞悬液，1000转/min离心5min收集细胞，加入红细胞裂解液进行裂解，离心，获得免疫细胞，采用培养基洗吹三次，离心，采用培养基进行吹打获得重悬细胞，转移至培养瓶中培养24-48h，将免疫细胞采用加入血浆和白介素-2和沙培林的RPMI1640培养基中进行培养，血浆的含量为体积的10％；
将间充质干细胞与免疫细胞分别转移至具有共培养膜的可调节压力和流量的共培养装置的两侧，采用含有10％血清的培养基进行共培养；
S2:将空白培养基加入共培养装置中，24hh后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
S3:将培养上清液与外泌体提取液以体积比2:1进行混合，在4℃条件下孵育过夜，得到孵育液，将孵育液在4℃，10000g的条件下离心60min，吸取底部沉淀，获得含外泌体的浓缩液；
将浓缩液用PBS缓冲液进行溶解，得到外泌体盐溶液，将外泌体盐溶液进行冻干，冻干温度为-80℃，冻干12h，得到细胞因子冻干粉。
步骤二：将角蛋白1g加入50g去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸5g、胶原蛋白10g和碳酸氢钠1g，搅拌均匀获得溶液A；
步骤三：将壳聚糖5g加入50g去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油2g、柠檬酸1g、细胞因子冻干粉1g，搅拌均匀获得溶液B；
步骤四：将溶液A和溶液B进行混合搅拌1min，至于培养皿中，冻干温度为-80℃，冻干8h进行冻干，即得成品。
实施例2：
步骤一：S1：与实施例1相同；
S2:将空白培养基加入共培养装置中，36h后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
S3:与实施例1相同。
步骤二：将角蛋白1.5g加入55g去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸8g、胶原蛋白15g，搅拌均匀获得溶液A；
步骤三：将壳聚糖7g加入55g去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油4g、细胞因子冻干粉1.5g和海藻酸钠8g，搅拌均匀获得溶液B；
步骤四：将溶液A和溶液B进行混合搅拌1.5min，至于培养皿中，冻干温度为-80℃，冻干7h进行冻干，即得成品。
实施例3：
步骤一：S1：与实施例1相同；
S2:将空白培养基加入共培养装置中，48h后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
S3:与实施例1相同。
步骤二：将角蛋白2g加入60g去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸15g、胶原蛋白20g和碳酸氢钠2g，搅拌均匀获得溶液A；
步骤三：将壳聚糖10g加入60g去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油6g、柠檬酸2g、细胞因子冻干粉2g和海藻酸钠15g，搅拌均匀获得溶液B；
步骤四：将溶液A和溶液B进行混合搅拌2min，至于培养皿中，冻干温度为-80℃，冻干6h进行冻干，即得成品。
实施例4：
步骤一：S1：与实施例1相同；
S2:将空白培养基加入共培养装置中，48h后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
S3:与实施例1相同。
步骤二：将角蛋白2g加入60g去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸8g、胶原蛋白12g和碳酸氢钠1g，搅拌均匀获得溶液A；
步骤三：将壳聚糖8g加入60g去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油4g、柠檬酸1g、细胞因子冻干粉1g和海藻酸钠8g，搅拌均匀获得溶液B；
步骤四：将溶液A和溶液B进行混合搅拌2min，至于培养皿中，冻干温度为-80℃，冻干8h进行冻干，即得成品。
实施例1-4中组分如表1所示：
表1.实施例1-4中各组分质量(g)
实验例1：
该实验例考察了各成分浓度对成品消炎效果的影响。
对照组：采用的是壳聚糖8g与胶原蛋白12g加入100g去离子水中，搅拌2min，于-80℃下，冻干8h得到的；
实验组A、实验组B、实验组C、实验组D、实验组E:按照实施例4的方法制备用于解决青春痘的细胞因子敷料，所不同的是细胞因子冻干粉的克数分别为0g、0.5g、1g、1.5g、2g；
实验组F、实验组G、实验组H、实验组I按照实施例4的方法制备用于解决青春痘的细胞因子敷料，所不同的是碳酸氢钠的克数分别为0g、0.5g、1.5g、2g；
实验组J、实验组K、实验组L、实验组M：按照实施例4的方法制备用于解决青春痘的细胞因子敷料，所不同的是柠檬酸的克数分别为0g、0.5g、1.5g、2g；
实验组N、实验组O、实验组P、实验组Q：按照实施例4的方法制备用于解决青春痘的细胞因子敷料，所不同的是聚谷氨酸的克数分别为0g、5g、15g、20g。
将180个年龄18-25岁的重度痤疮患者，随机组合分组，,每组各10人，实验者进行脸部清洗，将实验组A-Q以及空白对照组中提供的敷料敷在患者的右脸30min，将敷料移除并进行脸部清洗，通过VISIA皮肤检测仪对患者的面部进行检测，通过VISIA皮肤检测红区指标分析，检测使用痤疮治疗前后皮肤中面部炎症改善情况，结果如表2所示。
表2.面部炎症改善情况
通过炎症改善情况的测定，能够看出实验组A-E中能够起到消炎的效果主要的成分为细胞因子冻干粉，消炎效果也随着细胞因子冻干粉的含量增加而增加；
实验组F-I通过改变碳酸氢钠的含量，来测定碳酸氢钠对敷料效果的影响，碳酸氢钠能够与柠檬酸反应从而生成柠檬酸钠，水和二氧化碳，从而能够使敷料中存在大量细小孔洞，能够起到一定吸收皮肤脓液和组织液的效果，且能够加快敷料中细胞因子的渗出，起到更好的消毒抗炎的作用，因此当碳酸氢钠的含量较少时，使敷料内的空隙较少，结构更为致密，使其渗出作用较差，而通过添加碳酸氢钠能够增加敷料内的空隙，而碳酸氢钠添加至1.5g时，此时柠檬酸已经反应完全，难以释放出过多碳酸氢钠中的碳酸根，因此当碳酸氢钠含量大于柠檬酸的含量时则难以对敷料效果进行增强；
实验组J-M：碳酸氢钠能够与柠檬酸反应从而生成柠檬酸钠对于皮肤也具有较好的效果，柠檬酸钠相较于柠檬酸对皮肤的刺激较小，使敷料更为温和，且能够起到加快角质更新的作用，从而能够起到促进伤口愈合的作用，能够促进皮肤黑色素的剥落，也具有一定的杀菌效果，从而能够增强敷料消毒抗炎的效果，但柠檬酸钠具有一定的刺激性，当柠檬酸钠含量过高时则能够引起皮肤的过敏现象，且由于柠檬酸钠具有一定的抗凝血作用，当柠檬酸钠含量过高时会影响皮肤愈合；
实验组N-Q：中能够看出聚谷氨酸能够对于消炎具有一定的辅助作用，且能够通过增加聚谷氨酸的含量能够加强其消炎作用，聚谷氨酸能够增加皮肤保湿能力，且能够起到一定的抗菌作用，促进皮肤健康生长。
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明范围所作的均等变化与改进等，均应仍归属于本专利涵盖范围之内。
Claims (10)
1.一种用于解决青春痘的细胞因子敷料，其特征在于：按照重量份数包括以下组分：
细胞因子冻干粉1-2份、聚谷氨酸5-15份、甘油2-6份、柠檬酸0-2份、胶原蛋白10-20份、海藻酸钠0-15份、角蛋白1-2份、壳聚糖5-10份、碳酸氢钠0-2份。
2.根据权利要求1所述的一种用于解决青春痘的细胞因子敷料，其特征在于：按照重量份数包括以下组分：
细胞因子冻干粉1.2-1.8份、聚谷氨酸7-13份、甘油3-5份、柠檬酸1份、胶原蛋白12-18份、海藻酸钠3-12份、角蛋白1-2份、壳聚糖6-9份、碳酸氢钠1份。
3.根据权利要求1所述的一种用于解决青春痘的细胞因子敷料，其特征在于：所述细胞因子冻干粉内含有细胞因子，所述细胞因子为干细胞与免疫细胞共培养的外泌体，所述细胞因子的活性单位为5000U/g。
4.根据权利要求3所述的一种用于解决青春痘的细胞因子敷料，其特征在于：所述细胞因子冻干粉的制备方法为：将干细胞与免疫细胞共培养后，再单独收集获取干细胞的培养上清，进行冻干，得到细胞因子冻干粉。
5.根据权利要求4所述的一种用于解决青春痘的细胞因子敷料，其特征在于：干细胞与免疫细胞共培养，具体为：
S1：将干细胞进行传代培养；
S2：取骨髓细胞，加入红细胞裂解液进行裂解，离心，获得免疫细胞；
S3：采用培养基洗吹三次，离心，采用培养基进行吹打获得重悬细胞，转移至培养瓶中培养24-48h；
S4：将所述干细胞与所述免疫细胞分别转移至共培养装置中，进行共培养。
6.根据权利要求5所述的制备用于解决青春痘的细胞因子敷料的方法，其特征在于：所述干细胞为间充质干细胞。
7.根据权利要求5所述的一种用于解决青春痘的细胞因子敷料，其特征在于：制备细胞因子冻干粉包括如下步骤：
将空白培养基加入S4所述的共培养装置中，24h-48h后收取干细胞培养上清液；在4℃，2000g的条件下离心30min，弃去沉淀，得到培养上清液；
将所述培养上清液与外泌体提取液以体积比2:1进行混合，在4℃条件下孵育过夜，得到孵育液，将孵育液在4℃，10000g的条件下离心60min，吸取底部沉淀，获得含外泌体的浓缩液；
将浓缩液用PBS缓冲液进行溶解，得到外泌体盐溶液，将所述外泌体盐溶液进行冻干，得到细胞因子冻干粉。
8.根据权利要求7所述的一种用于解决青春痘的细胞因子敷料，其特征在于：所述冻干温度为-80℃，冻干12h。
9.制备权利要求1-8任一所述的用于解决青春痘的细胞因子敷料的方法，其特征在于：包括如下步骤：
步骤一：将角蛋白加入50-60份去离子水中，进行搅拌，搅拌均匀后加入称量好的聚谷氨酸、胶原蛋白和碳酸氢钠，搅拌均匀获得溶液A；
步骤二：将壳聚糖加入50-60份去离子水中，进行搅拌，搅拌均匀后加入称量好的甘油、柠檬酸、细胞因子冻干粉和海藻酸钠，搅拌均匀获得溶液B；
步骤三：将所述溶液A和溶液B进行混合搅拌1-2min，至于培养皿中，进行冻干即得成品。
10.根据权利要求9所述的制备用于解决青春痘的细胞因子敷料的方法，其特征在于：步骤三中，冻干温度为-80℃，冻干6-8h。
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