CN113563452A - 一种生物活性肽及其与脂肪干细胞外泌体在皮肤增殖修复中的应用 - Google Patents
一种生物活性肽及其与脂肪干细胞外泌体在皮肤增殖修复中的应用 Download PDFInfo
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Abstract
本发明属于生物制药技术领域,具体涉及一种生物活性肽及其与脂肪干细胞外泌体在皮肤增殖修复中的应用,该生物活性肽可同时高亲和力结合VEGFR和bFGFR,促进上皮细胞增殖;该活性肽可与脂肪干细胞外泌体联用,治疗皮肤损伤,促进细胞因子分泌,还可以辅助脂肪细胞移植,抑制免疫排斥现象,降低炎症因子表达,提高透明质酸分泌水平,改善皮肤生理活性,维持皮肤组织正常生理状态,为皮肤的修复和养护提供了新思路。
Description
技术领域
本发明属于生物技术领域,具体涉及一种生物活性肽及其与脂肪干细胞外泌体在皮肤增殖修复中的应用。
背景技术
皮肤修复与护理是美容保健和皮肤疾病治疗领域中的重要分支,尽管已经出现了化学小分子、天然提取物、生物大分子等多种类型的皮肤护理产品,但是仍难以满足市场和用户的多样性需求,尤其是市售的化妆品或皮肤护理产品中,含有大量化学物质,虽然具有一定的治疗皮肤损伤功能,但一方面其治疗或养护效果有效,另一方面长期大量使用会对机体造成二次损伤,诱发皮肤干裂、过敏、炎症等不良反应,甚至诱发皮肤癌变。
为解决上述困境,研究人员将研发重点逐步调整为皮肤细胞治疗或养护产品的开发,较多获得关注的是胚胎干细胞、间充质干细胞、皮肤祖细胞、IPS细胞等具有多向分化能力的细胞,这些细胞的增殖能力强,且具有多向分化潜能,通过体内或体外的刺激,其本身可分化为多种不同类型的细胞,诸如真皮细胞、表皮细胞、间质细胞、血管上皮细胞等,从而有效促进皮肤损伤后修复,改善皮肤生理机能。但是上述细胞也面临制备条件苛刻,存储和运输也面临困难,使得成本居高不下,相关产品的推广和市场化开发受到限制。20世纪末,研究分离获得了干细胞外泌体,最初认为外泌体是细胞代谢废物,但后来发现外泌体中富含蛋白、核酸、糖类、脂类等多种营养物质,具有相当广泛的生理活性,可以应用于皮肤衰老、皮肤损伤、皮肤养护中,然而由于外泌体成分复杂,单纯使用干细胞外泌体仍难以取得令人满意的治疗效果。
脂肪细胞最早被用于临床治疗始于1893年,此后被广泛用于整形修复、美容手术和美容产品中(自体脂肪移植中脂肪处理与临床应用的研究进展,丁飞雪、朱朱等,组织工程与重建外科,2020,16(5):418-420),自体脂肪细胞来源于受试者自身组织,如背部、腹部、臀部、四肢等脂肪丰厚部位,资源丰富,无伦理风险,且脂肪细胞来自受试者自身,免疫排斥反应较低,安全性较高,制备成本低廉且易于培养,故近年来受到业内青睐。但是自体脂肪细胞往往需要通过原代培养方式制备,细胞的存活率和增殖能力较低(miR-210/YWHAG分子轴与自体脂肪移植后促进脂肪细胞增殖及血管形成,姚炎燚、陈晓玲等,中国组织工程研究,2021,26(1):59-63),使用过程中主要采用过量注射和重复注射方式维持治疗效果,增加了经济成本,降低了受试者的生活治疗。脂肪细胞移植可治疗多种疾病,也可用于面部凹陷、小乳畸形、乳房再造、器官再造、瘢痕修复等美容用途,但是单独移植脂肪细胞仍可能诱发免疫排斥、皮肤过敏、皮肤皲裂等并发症,限制了其使用。
本发明基于上述现有技术中存在的问题,提供了一种新型生物活性肽,该肽为VEGF和bFGF的生物类似肽,能够与VEGFR和bFGFR高水平结合,兼具两种细胞因子的生理活性,与脂肪干细胞外泌体联用可以提高上皮细胞增殖能力,促进皮肤组织损伤后修复,还可以抑制脂肪细胞移植过程中免疫排斥,促进胶原物质分泌。
发明内容
本发明的主要目的是提供一种新型皮肤养护产品,具体提供了一种新型生物活性肽,能够促进脂肪细胞增殖,改善皮肤生理功能,促进皮肤损伤后修复,详细技术方案如下:
提供了一种生物活性肽,其氨基酸序列如SEQ ID NO:1所示。
该活性肽兼具血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维细胞生长因子(basic fibroblastic growth factor,bFGF)的生理功能,可与相关受体高水平结合,其氨基酸结构得到简化,适用于固相合成法大规模制备,降低了生产成本;且该活性肽能够促进脂肪细胞增殖,防止其凋亡,能够与脂肪干细胞外泌体发挥协同作用。
提供了一种能够促进皮肤增殖修复的组合物,其特征在于包括本发明中所述的活性肽和脂肪干细胞外泌体。
进一步的,所述自体脂肪细胞为人自体脂肪细胞。
进一步的,所述自体脂肪细胞采用如下步骤制备:
(1)采用抽吸法取脂肪组织;
(2)将脂肪组织通过物理法粉碎,然后加入胶原酶溶液,充分搅拌并振荡混匀,37℃消化;
(3)将酶解后脂肪组织5000-10000rpm条件下离心5-10min,离心后分为三层,分别为顶层油脂、中间层混合物和底层液,去除脂肪上层油脂和底层液,获取中层纯脂肪;
(4)将获取脂肪干细胞,使用无菌生理盐水冲洗,于3000-5000rpm条件下离心10min,收取沉淀细胞,重复三次;
(5)将所述细胞接种于细胞培养瓶中,置于37℃5%CO2培养箱中培养;
(6)待细胞融合度达到80%以上时,进行传代培养,共培养3-5代,获得足够量的脂肪干细胞;
(7)将脂肪干细胞上清液于4℃10000rpm超速离心60-90min,收获脂肪干细胞外泌体。
进一步的,所述物理法粉碎包括超声粉碎、机械粉碎和热裂解。
进一步的,所述胶原酶为I型胶原酶、II型胶原酶、IV型胶原酶、V型胶原酶或其混合物。
进一步的,所述(5)步骤中使用DMEM培养液,培养液中还包括KGF、bFGF、HGF,每2-3天更换一次培养基。
进一步的,所述(5)步骤中KGF浓度为10-100mg/mL,bFGF浓度为50-150mg/mL,HGF浓度为10-80mg/mL。
提供了一种本发明中所述活性肽、组合物在制备美容产品中的应用。
提供了一种本发明中所述活性肽、组合物在制备化妆品中的应用。
本发明中所提供的活性肽可与脂肪干细胞外泌体产生协同作用,促进皮肤损伤后修复,提供相关细胞因子表达水平;促进脂肪细胞移植后吸收,提高透明质酸表达水平,移植免疫排斥反应。
本发明的有益效果包括:
(1)本发明提供了一种新型活性肽,能够与VEGFR和bFGFR高效结合,兼具VEGF和bFGFR的生理功能,不仅能够促进皮肤后的损伤修复过程,还能够改善脂肪细胞移植效果;
(2)本发明提供的活性肽与脂肪干细胞外泌体,可有效促进皮肤组织损伤后修复,提高KGF等细胞因子表达水平;
(3)本发明提供的活性肽与脂肪干细胞外泌体可辅助脂肪细胞移植,减低炎症因子表达水平,抑制免疫排斥反应,提高透明质酸分泌能力,改善皮肤生理状态。
附图说明
图1 HaCaT细胞增殖能力图;
图2 大鼠皮肤损伤病理切片,其中A为PBS组,B为外泌体组,C为MD组,D为VEGF组,E为bFGF组,F为联合组;
图3 大鼠皮肤损伤模型中KGF表达水平图;
图4 脂肪细胞移植大鼠模型中IL-2表达水平图;
图5 脂肪细胞移植大鼠模型中IL-6表达水平图;
图6 脂肪细胞移植大鼠模型中HA表达水平图;
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂生物材料、检测试剂盒,如无特殊说明,均可从商业途径获得。
实施例1生物活性肽的制备
所述生物活性肽的其氨基酸序列如SEQ ID NO.1所示,命名为MD肽,该肽氨基酸结构简单,适用于化学合成方式进行大规模制备,本实施例中采用固相合成方式,为了有效缩短反应时间提高合成效率,采用“二段法”制备目标活性肽,具体步骤如下:
(1)制备第一肽段“Asn-Phe-Gly-Tyr-Lys-Leu-Ser-Gly-Thr-COOH”:以王树脂和Fmoc保护的Fmoc-Asn-Wang树脂为原料,使用DMF浸泡溶胀树脂,室温反应3-5小时,按照固相合成方法依次加入Fmoc-Phe–COOH、Fmoc-Gly–COOH、Fmoc-Tyr–COOH、Fmoc-Lys–COOH、Fmoc-Leu–COOH、Fmoc-Ser–COOH、Fmoc-Gly–COOH、Fmoc-Thr–COOH,使其逐个偶联到树脂上,采用茚三酮法检测是否反应完全。当反应完全后,加入全保护切割试剂25%三氟乙醇/DCM,室温反应2-3小时,收集切割液,旋蒸去除液体,得到全保护的第一肽段;
(2)制备第二肽段“Thr-Ser-Tyr-Glu-Leu-Leu-Val-Pro-Thr-Tyr-COOH”,方法同步骤(1),依次连接缩合氨基酸树脂,经切割反应后得到全保护的第二肽段;
(3)完整活性肽的制备:将固相合成的第一肽段溶于DCM中,加入缩合试剂进行C端活化,反应1-2小时;将第二肽段加入到上述经活化的溶液中,反应时间4-8小时;茚三酮法检测完成后,旋干反应液后,加入切割试剂,室温反应2-4小时;切割完成后,加入-4℃乙醚沉淀,获得多肽粗品。
(4)活性肽粗品的纯化:采用高效液相色谱进行纯化,以0.1%TFA/水溶液-0.1%TFA/乙腈溶液为流动相,采用梯度系统洗脱循环进样纯化,得到MD肽纯化产品,经HPLC-质谱鉴定多肽纯度均达99.53%以上。
实施例2活性肽与目标受体亲和力测定
本发明中采用表面等离子共振成像(SPR)方法检测多肽VEGFR和bFGFR的亲和力。
2.1多肽与VEGFR亲和力测定
以固定重组人VEGFR的通道作为检测通道,空白通道作为对照通道进行亲和力分析实验,具体步骤包括:(1)通道表面平衡,使用HBS-EP缓冲液,以10μL/min的流速平衡芯片表面5-10min;(2)表面活化,加入NHS+EDC 1∶1混合液,以10μL/min的流速活化芯片表面5-10min;(3)偶联受体蛋白,加入重组人VEGFR(稀释在10mM醋酸钠pH5.0缓冲液中),以10μL/min的流速耦联约10min,对照通道不做处理;(4)表面封闭,加入乙醇胺,以10μL/min的流速封闭表面5-10min;(5)样品结合,将多肽样品注入SPR系统中,通道内与重组人VEGFR孵育,结合到VEGFR蛋白上的多肽被回收,以含有5%DMSO的PBS缓冲溶液为流动相,样品流速为5μL/min;(6)样品检测与数据处理,使用Agilent 1290液相色谱系统样品进行分析,基于Agilent MassHunter B.02.00软件对数据进行处理与分析。
2.2多肽与bFGFR亲和力测定
以固定重组人bFGFR的通道作为检测通道,空白通道作为对照通道进行亲和力分析实验,具体检测步骤参照2.1节。
结果如表1所示,MD多肽与VEGFR、bFGFR均展示出了较高亲和力,其亲和力水平与重组VEGF和重组bFGF水平相近,并且与重组bFGF相比,与bFGFR亲和力似乎更高。
表1多肽亲和力检测
实施例3脂肪干细胞外泌体制备
本发明中以人脂肪组织为材料,制备脂肪干细胞外泌体,具体步骤包括:
(1)采用抽吸法取脂肪组织,向实验者大腿内侧注射2%盐酸利多卡因40mL,进行局部麻醉,麻醉成功后,在抽吸部位做3~5mm皮肤切口,用吸脂针沿切口插入皮下脂肪组织,吸脂针呈扇形均匀抽取深层脂肪,至足够量。
(2)将脂肪组织通过超声法粉碎,过滤除去大颗粒物质;然后加入1%浓度的I型胶原酶溶液,充分搅拌并振荡混匀,37℃消化1-2h;
(3)向酶解后脂肪组织于5000rpm条件下离心5-10min,离心后分为三层,分别为顶层油脂、中间层混合物和底层液,则弃去底层肿胀液,过滤去除顶层油脂,获取中层脂肪干细胞;
(4)将获取脂肪干细胞,使用无菌生理盐水冲洗,于3000rpm条件下离心10min,收取沉淀细胞,重复三次;
(5)将所述细胞用含10%FBS的DMEM培养基重悬,接种于细胞培养瓶中,加入适量含10%FBS的DMEM培养基,所述培养液中还包括KGF浓度为50mg/mL,bFGF浓度为100mg/mL,HGF浓度为20mg/mL,置于37℃5%CO2培养箱中培养,每隔12h观测一次细胞培养状况,每2-3天更换一次培养基。
(6)至细胞融合度达到80%,进行传代培养,共培养3-5代,获得足够量的脂肪干细胞;
(7)将脂肪干细胞接种于洁净培养皿中,待细胞融合度达80%以上,更换为无血清培养基,培养24h,收集细胞上清,4℃2000r/min,离心20min,取上清去掉细胞碎片,重悬后经0.22μm滤器过滤,转移至超速离心管中,4℃10000r/min,离心80min,取沉淀;PBS冲洗后,4℃10000r/min,离心20min,重复3次,收集沉淀即为脂肪干细胞外泌体,于-80℃冰箱中保存。
实施例4上皮细胞体外增殖实验
为了考察本发明中所提供的活性肽和外泌体对于皮肤的修复和养护功能,选用人上皮细胞HaCaT细胞作为实验对象,在体外环境下研究其对于细胞的促进作用。
将本实验室保存的HaCaT细胞接种于含10%FBS的DMEM培养基,37℃,5%CO2培养箱中进行培养。取状态良好处于指数生长期的HaCaT细胞,消化,计数,将细胞接种于96孔板,每孔含有200μL新鲜培养基,接种浓度为0.5×105个/孔,置于37℃,5%CO2培养箱中继续培养12小时。将上述细胞分为组,各组分别加入:
PBS组:50μL PBS;
外泌体组:50μL含浓度为10mg/mL的脂肪干细胞外泌体的PBS;
MD组:50μL含浓度为10mg/mL的脂肪干细胞外泌体和10mg/mL的MD多肽的PBS;
VEGF组:50μL含浓度为10mg/mL的脂肪干细胞外泌体和10mg/mL的重组VEGF的PBS;
bFGF组:50μL含浓度为10mg/mL的脂肪干细胞外泌体和10mg/mL的重组bFGF的PBS;
联合组:50μL含浓度为10mg/mL的脂肪干细胞外泌体、10mg/mL的重组VEGF和10mg/mL的重组bFGF的PBS。
上述细胞置于37℃,5%CO2培养箱中继续培养24小时。
采用CCK-8法测定细胞存活率,按CCK-8检测试剂盒说明书指示,每孔加入CCK-8溶液10μL,在5%CO2,37℃条件下孵育2h,用酶标仪在450nm波长下测定吸光值,进而计算细胞增殖能力。如图1所示,MD多肽可显著促进上皮细胞增殖,相比于阴性对照组(PBS组)细胞增殖能力提高近1倍,bFGF也能够显著促进上皮细胞增殖,但提高幅度低于MD多肽,而VEGF似乎对于上皮细胞增殖无明显促进作用,联合施用VEGF和bFGF也未展现出协同作用,其改善幅度与单独使用bFGF水平相当。
实施例5大鼠皮肤损伤的修复作用
5.1大鼠皮肤损伤模型制备及治疗
选取健康SD大鼠,SPF级,雌雄各半,体重200±20g,随机分组,每组10只,共6组,分别为生理盐水组、脂肪细胞组、MD组、VEGF组、bFGF组和联合组,各组大鼠背部毛发剃净,选择面积约为2cm×2cm的目标区域,注射盐水利多卡因局部麻醉,使用无菌手术剪剪去皮肤约2cm×2cm面积的创口。造模成功后,于患处周边多点注射相关药物,每周一次,共给药三次。各组给药方案如下:
生理盐水组:0.2mL生理盐水;
外泌体组:0.2mL含终浓度为100mg/mL脂肪干细胞外泌体的生理盐水;
MD组:0.2mL含终浓度为100mg/mL脂肪干细胞外泌体和100mg/mL MD肽的生理盐水;
VEGF组:0.2mL含终浓度为100mg/mL脂肪干细胞外泌体和100mg/mL重组VEGF的生理盐水;
bFGF组:0.2mL含终浓度为100mg/mL脂肪干细胞外泌体和100mg/mL重组bFGF的生理盐水;
联合组:0.2mL含终浓度为100mg/mL脂肪干细胞外泌体、100mg/mL重组VEGF和100mg/mL重组bFGF的生理盐水。
5.2大鼠皮肤病理检查
最后一次给药1周后,脱颈处死大鼠,取大鼠背部患处位皮肤组织约1cm2,置于4%多聚甲酵溶液中固定48小时后石蜡包埋,制备病理切片,进行HE染色。如图2所示,生理盐水组中,新生表皮发育迟缓,炎性细胞呈现聚集性分布,成纤维细胞扩散分布,毛细血管、胶原纤维较少;而各个治疗组中,皮肤正常生理结构逐渐恢复,炎症细胞减少,其中MD组和联合组中恢复最快,炎性细胞明显减少,胶原纤维明显增多,毛细血管较为丰富,说明MD多肽,以及bFGF、VEGF与脂肪干细胞外泌体联用可显著促进皮肤组织的损伤后修复。
5.3大鼠皮肤组织中细胞因子的表达
最后一次给药1周后,脱颈处死大鼠,取大鼠背部患处位皮肤组织,经无菌PBS清洗干净,取约0.5g组织,使用无菌手术剪将其剪碎,而后加入约5mL无菌PBS溶液,置冰浴中经匀浆器进行充分研磨,获得组织匀浆液,4℃,4000r/min离心10分钟,收集上清进行后续检测。
皮肤组织的损伤后修复与多种细胞因子有关,其中角质细胞生长因子(keratinocyte growth factor,KGF)是其中重要的一员。KGF是人体皮下的组织细胞分泌的一种碱性蛋白生长因子,能特异刺激上皮细胞的新陈代谢等生理过程,包括细胞的再生、分化和迁移等,能够特异性的结合到上皮细胞表面的特异受体,经过复杂的信号传递过程,启动上皮细胞内参与分裂生长的基因表达,从而刺激上皮组织的新陈代谢,目前KGF已经广泛用于皮肤损伤修复和化妆品产品中。
本发明中采用ELISA法检测大鼠皮肤组织匀浆中的KGF表达水平,如图3所示,各个治疗组中KGF表达量均有所提高,相比于单独使用外泌体,联合施用VEGF和bFGF可明显提高,而与MD多肽联用KGF表达水平达到最高值,这可能是由于不仅MD多肽能够与VEGFR和bFGFR结合,而且其分子量较低,易于在细胞间传递,并配合脂肪干细胞外泌体的滋养作用,刺激上皮组织的细胞分泌KGF,加速损伤后修复过程。
实施例6促进脂肪细胞移植
为研究本发明中所提供的脂肪干细胞外泌体和活性肽对脂肪细胞移植的相关作用,本节中以大鼠为研究对象,考察本发明所提供的外泌体和活性肽在细胞移植中的功效。
6.1大鼠皮肤组织中脂肪细胞移植
参考姜筱唐(自体脂肪细胞移植技术在面部年轻化的应用效果分析,智慧健康,2018,4(24):85-86)、王大勇(自体脂肪细胞移植隆乳术的临床效果观察,世界最新医学信息文摘,2019,9(36):64-65)等提供的方法,制备人脂肪细胞。
选取健康SD大鼠,SPF级,雌雄各半,体重200±20g,随机分组,每组10只,共6组,分别为生理盐水组、脂肪细胞组、MD组、VEGF组、bFGF组和联合组,各组大鼠背部毛发剃净,选择面积约为5cm×5cm的目标区域,注射人脂肪细胞和医用水凝胶混合物,脂肪细胞剂量为106个/只。注射完成6h后,于目标区域周围多点注射相关药剂,各组给药方案同5.1节。
6.2大鼠血浆中验证因子检测
各组大鼠注射相关药剂24h和48h后,尾静脉取血,离心收集血清,采用ELISA法检测血清中的IL-2和IL-6含量,具体操作步骤按照试剂盒说明书进行。
注射人脂肪细胞后,大鼠血浆中IL-2和IL-6含量迅速升高,经过治疗后上述验证因子的表达含量有不同程度的降低,如图4所示,IL-2迅速升高,经过脂肪干细胞外泌体治疗后,IL-2表达水平开始下降,采用bFGF细胞因子与外泌体联用可产生明显协同效应,IL-2水平进一步下降,但是VEGF却未见明显促进作用,在与外泌体联用,以及配合bFGF、脂肪细胞施用中,VEGF均未见明显的IL-2抑制作用,这可能是因为VEGF主要功能在于促进血管形成和发育,对于炎症因子的表达无明显抑制作用,而bFGF可广泛作用于中胚层和神经外胚层细胞,可通过多种信号途径影响炎症因子的分泌;外泌体与MD肽联用产生了更加明显的协同作用,IL-2水平降低明显,而且作用迅速,在给药24h后即可见明显下降。如图5所示,在IL-6的表达中,虽然使用脂肪细胞和相关细胞因子、多肽能够显著抑制IL-6的表达,但是各个因子或多肽之间的作用却未见统计学差异,这说明对于IL-6的抑制作用可能主要来自于脂肪干细胞外泌体自身的影响。
6.3大鼠皮肤组织中透明质酸表达
透明质酸(hyaluronic acid,HA),又称玻尿酸,可调节血管通透性,促进蛋白质和电解质转运,增强皮肤光泽,防止皮肤干涩、皲裂,维持皮肤组织正常生理机能。本节中取给药1周后,给药处大鼠皮肤组织,提取组织匀浆,方法同第5.3节。按照试剂盒说明书中所述方法检测上清液中的透明质酸水平。如图6所示,脂肪干细胞外泌体可显著促进皮肤组织中的透明质酸分泌,在MD多肽与外泌体联用中作用更强,明确高于外泌体与其他因子联用的情况,说明本发明中所提供的脂肪干细胞外泌体与活性肽在促进透明质酸分泌方面具有优势。
序列表
<110> 北京戴域生物技术有限公司
<120> 一种生物活性肽及其与脂肪干细胞外泌体在皮肤增值修复中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asn Phe Gly Tyr Lys Leu Ser Gly Thr Thr Ser Tyr Glu Leu Leu Val
1 5 10 15
Pro Thr Tyr
Claims (10)
1.一种生物活性肽,其特征在于氨基酸序列如SEQ ID NO:1所示。
2.一种能够促进皮肤增殖修复的组合物,其特征在于包括权利要求1中所述的活性肽和脂肪干细胞外泌体。
3.根据权利要求2所述的组合物,其特征在于,所述脂肪干细胞为人脂肪肝细胞。
4.根据权利要求2所述的组合物,其特征在于所述人脂肪肝细胞外泌体采用如下步骤制备:
(1)采用抽吸法取脂肪组织;
(2)将脂肪组织通过物理法粉碎,然后加入胶原酶溶液,充分搅拌并振荡混匀,37℃消化;
(3)将酶解后脂肪组织5000-10000rpm条件下离心5-10min,离心后分为三层,分别为顶层油脂、中间层混合物和底层液,去除脂肪上层油脂和底层液,获取中层纯脂肪;
(4)将获取脂肪干细胞,使用无菌生理盐水冲洗,于3000-5000rpm条件下离心10min,收取沉淀细胞,重复三次;
(5)将所述细胞接种于细胞培养瓶中,置于37℃5%CO2培养箱中培养;
(6)待细胞融合度达到80%以上时,进行传代培养,共培养3-5代,获得足够量的脂肪干细胞;
(7)将脂肪干细胞上清液于4℃10000rpm超速离心60-90min,收获脂肪干细胞外泌体。
5.根据权利要求4所述的自体脂肪细胞,其特征在于,所述物理法粉碎包括超声粉碎、机械粉碎和热裂解。
6.根据权利要求4所述的自体脂肪细胞,其特征在于,所述胶原酶为I型胶原酶、II型胶原酶、IV型胶原酶、V型胶原酶或其混合物。
7.根据权利要求4所述的自体脂肪细胞,其特征在于,所述(5)步骤中使用DMEM培养液,培养液中还包括KGF、bFGF、HGF,每2-3天更换一次培养基。
8.根据权利要求4所述的自体脂肪细胞,其特征在于,所述(5)KGF浓度为10-100mg/mL,bFGF浓度为50-150mg/mL,HGF浓度为10-80mg/mL。
9.权利要求1中所述活性肽、权利要求2-8中所述组合物在制备美容产品中的应用。
10.权利要求1中所述活性肽、权利要求2-8中所述组合物在制备化妆品中的应用。
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