CN111388404A - 一种抗衰老的注射针剂及应用 - Google Patents
一种抗衰老的注射针剂及应用 Download PDFInfo
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Abstract
本发明的目的在于提供一种抗衰老的注射针剂,包括弹性蛋白肽,其特征在于,还包括人脂肪间充质干细胞培养基上清和海藻提取物。本发明利用脂肪干细胞与弹性蛋白肽联用辅以海藻萃取液,能够促进弹性蛋白的合成,抑制弹性蛋白酶的活性,配合维生素C促进表皮细胞的增殖和分化,清除氧自由基,促进受伤部位皮肤修复,维生素A促进皮肤细胞生长分化,增进表面皮肤造血功能,从本质上重建皮肤弹性组织的构造,延缓人皮肤衰老的发生。本发明采用脂肪干细胞与弹性蛋白联用并且辅以海藻萃取液进行面部提升,能够很好的从根源上解决皮肤衰老的导致的面部下垂,皮肤无弹性等问题,也有很好的去皱效果。
Description
技术领域
本发明涉及蛋白药物领域,特别涉及一种包括人脂肪间充质干细胞培养基上清的抗衰老的注射针剂。
背景技术
爱美是人的天性,因为地域、文化、历史、种族等差异,在不同国家、不同时期对美界定的标准有所差异,但是对保持年轻的期望始终不变。延缓衰老的研究开展至今己有悠久的历史,在古埃及、古罗马、古印度、古巴比伦时期,现如今返老还童、长生不老,仍是一个遥远的梦想,但是定格青春,逆转年龄却是可以企及的现实了。衰老是不可逃避的现实。其原因主要有光化学损伤及老龄性改变等两方面的原因。日常生活中慢性的阳光照射使皮肤弹力纤维异常排列方式堆积;胶原纤维数量下降,剩余的纤维逐无序顺序的排列;异常的真皮与表皮间形成境界带或称边缘带等改变。这些变化引发细小的皱纹、皮肤松弛、色素沉着、皮肤恶性肿瘤等问题。另一方面,随着年龄的增大,皮肤也发生着变化。真皮层变薄;成纤维细胞、肥大细胞、弹力纤维和血管等的减少。皮下的肌肉收缩产生的动力引起皮肤的皱纹,面部脂肪的减少松弛,重力引起较深的皱纹。
目前提升面部的方式主要有手术与非手术的治疗方式。手术提升下面部、线技术等能有效改善下面部松弛的问题,但该术式的适应人群仍是针对面部衰老下垂明显的人群,效果虽明显,但需要承担其创伤性及可能发生的严重的并发症的可能。美国整形外科医师协(American Society of Plastic Surgeons,ASPS)的统计结果也显示,美国的注射整形数量是700万例,是整形手术总数的4倍。比起手术“狠准”的治疗,微整形注射治在于“精细”确的治疗效果。
发明内容
本发明的目的在于提供一种应用于微整形的抗衰老的注射针剂,能够安全有效的进行微整形。
本发明采用的具体方案为:一种抗衰老的注射针剂,包括弹性蛋白肽,其技术方案在于,还包括人脂肪间充质干细胞培养基上清和海藻提取物。
所述的一种抗衰老的注射针剂,包括弹性蛋白肽的冻干粉与脂肪干细胞培养基上清混合,其中,脂肪干细胞培养基上清1000份,弹性蛋白肽0.04~0.06份、海藻提取物的浓度为19~21份、维生素A 49~51 份、维生素C 8~10 份。
所述的弹性蛋白肽的制备方法是:将弹性蛋白分散在去离子水中,调节pH值至pH8.0,然后进行酶解;将酶解所得混合物在 55 ℃下振荡温浴 10 h,酶灭活后进行离心,随后,将收集的上清液冻干并储存在-18℃直至使用。
所述的人脂肪间充质干细胞培养基上清的获取过程是:
1)将人体脂肪组织移入无菌大培养皿中,用无菌组织剪反复剪切至组织成糊状,移入离心管,加入等体积的0.075%的I型胶原酶,置于摇床上消化45min,得到消化后的脂肪颗粒;
2)消化后的脂肪颗粒加入等体积的含10%胎牛血清的间充质干细胞完全培养基终止消化,100目滤网过滤,滤液离心3min,弃上清后所得沉淀;
3)对步骤2)获得的沉淀用5mL间充质干细胞完全培养基重悬,细胞计数后稀释,以1×105/cm2的密度接种于培养皿中,置于37℃含5%CO2的恒温培养箱中培养,48 h后首次换液去除未贴壁细胞,此后每72h换液一次;
4)待细胞长至80%--90%融合时PBS漂洗两次,0.25%胰酶1mL,培养箱孵育2-3min,低倍镜下观察细胞缩小变圆,细胞脱壁后加入等体积完培终止消化,得到细胞悬液;
5)对步骤4)得到的细胞悬液离心3min后弃上清,以间充质干细胞完全培养基重悬后1:3传代,同法传代培养至P2代;采用无血清培养基第三代培养48小时后离心获取上清,保存于4度环境中待用。
所述的无血清培养基配方如下:按照重量份数,包含:a-MEM基础培养基1.5×104份、人血白蛋白4份、腺苷17份、胞苷15份、鸟苷15份、尿苷15份、2-脱氧腺苷16份、2′-脱氧胞苷盐酸16份、2-脱氧鸟苷16份、胸腺嘧啶15份、氢化可的松15份、维生素C 15份、碱性成纤维细胞生长因子0.03份、表皮生长因子0.02份、TGF-β0.03份、促红素0.02份和0.01份褪黑素。
有益效果:本发明利用脂肪干细胞与弹性蛋白肽联用辅以海藻萃取液,能够促进弹性蛋白的合成,抑制弹性蛋白酶的活性,配合维生素C促进表皮细胞的增殖和分化,清除氧自由基,促进受伤部位皮肤修复,维生素A促进皮肤细胞生长分化,增进表面皮肤造血功能,从本质上重建皮肤弹性组织的构造,延缓人皮肤衰老的发生。本发明采用脂肪干细胞与弹性蛋白联用并且辅以海藻萃取液进行面部提升,能够很好的从根源上解决皮肤衰老的导致的面部下垂,皮肤无弹性等问题,也有很好的去皱效果。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述。
2001年研究者发现在脂肪组织中存在一群具备多向分化能力的间充质干细胞,脂肪来源的间充质干细胞(Adipose derived mesenchymal stem cells,ADSCs) 具备间充质干细胞特性的hADSC相较于其他类别的间充质干细胞具有取材损伤小、来源丰富、易于提取、活性持久等优势,因此倍受广大学者的青睐。ADSCs的旁分泌作用机制得到了更多学者的支持。2007年学者发现体外培养脂肪来源间充质干细胞发现其能够分泌一系列的生长因子进入培养基中,形成脂肪来源间充质干细胞条件培养基(Adipose derived mesenchymalstem cells conditioned medium,ADSCs.CM),进而促进组织后真皮成纤维细胞的迁移,加速伤口的愈合,现已证实存在的细胞因子有碱性成纤维细胞生长因子(bFGF)、角质细胞生长因子(KGF)、转化生长因子D(TGF.p)、血管内皮生长因子(VEGF)等。2009年有学者进一步研究了ADSC条件培养基的抗氧化作用,该研究将培养了脂肪来源间充质干细胞72 h的培养基收集、离心、过滤后制备成ADSCs条件培养基,而后用其培养人真皮成纤维细胞(humandermal fibroblasts,HDF),结果发现ADSCs.CM能够减轻细胞凋亡、上调超氧化物歧化酶(SOD)、促使细胞从G0/G1期向S期转化,从而提高HDF应对氧应激的能力。
弹性蛋白,同胶原蛋白类似,也是皮肤结构蛋白的一种。弹性蛋白及其降解产物在皮肤衰老进程中发挥着重要的作用,如细胞相互作用和信号传导特性作用,促进细胞迁移,生长和增殖等。弹性蛋白是细胞外基质分子中最稳定的蛋白,与细胞外基质的其他分子成分如胶原蛋白、纤溶蛋白、纤维蛋白原、纤维连接蛋白和血小板反应蛋白等类似,弹性蛋白是一种存在于多个结缔组织中的重要结构,由成纤维细胞和角质形成细胞合成分泌,具有良好的弹性和独特的生理功能。虽然弹性蛋白仅占皮肤总蛋白含量的 2-4%,但它对皮肤结构的维持至关重要。弹性纤维由弹性微纤维(由微纤维相关糖蛋白、原纤维蛋白、纤蛋白和弹性蛋白受体等组成)和非晶弹性蛋白组成。胶原蛋白为皮肤提供抗拉强度,而弹性纤维为皮肤提供延展性和回弹性,使皮肤具有良好的柔韧性和弹性。皮肤中弹性蛋白酶和弹性蛋白的降解与皮肤衰老的关系已经被阐明,随着年龄的增大,弹性蛋白酶的活性及分泌量增加,进而导致弹性蛋白被降解。年轻人的皮肤在表皮层和真皮层之间的不规则边界上有垂直、网状的弹性纤维分布,但是在老化的皮肤中,乳头层消失,弹性纤维网络断裂成段状,并且分布密度也有很大程度的下降。其结果就是导致皮肤失去弹性,松弛,出现皱纹等现象的发生。
弹性蛋白在弹性蛋白酶的条件下进行酶促反应,降解成为弹性蛋白肽,这种弹性蛋白肽也被明确证实具有抑制弹性蛋白酶活性的作用,其本质原理为负反馈调节,其抑制作用与其分子量成正比。
海藻提取物(Sesmollient)是一种纯天然的海洋生物产品。SESMOLLIENT带有大量的阴离子,可以刺激纤维细胞生成胶原蛋白和弹性蛋白,促进皮肤的新陈代谢,抗皱,抗衰老。增进表面皮肤造血功能对体外皮肤抑菌效果,成为目前美容市场的新宠。主要来源为马尾藻科植物海蒿子Sargassum pallidum(Turn.) C. Ag.或羊栖菜S.fusiforme.(Harv.)Setch.的藻体。
因此,本发明利用脂肪干细胞与弹性蛋白肽联用辅以海藻萃取液,能够促进弹性蛋白的合成,抑制弹性蛋白酶的活性,配合维生素C促进表皮细胞的增殖和分化,清除氧自由基,从本质上重建皮肤弹性组织的构造,延缓人皮肤衰老的发生。
具体实施例I:制备过程:
1.弹性蛋白的纯化:取 10 g 人重组弹性蛋白粉末悬浮于 100 mL 0.1M NaOH溶液中,在95℃下搅拌1h,水洗至中性,弃上清液,冻干,储存在-18℃直至使用。
2.弹性蛋白水解物的制备:将弹性蛋白分散在去离子水中,弹性蛋白:水= 1:10,w/w,用1M NaOH将混合物调节至 pH8.0,然后加入 Alcalase®2.4L,2%,w/w,酶解。将所得混合物在 55 ℃下振荡温浴 10 h,沸水浴 15 min后在 8000g,4℃下离心 20 min。随后,将收集的上清液冻干并储存在-18℃直至使用。
3.人脂肪间充质干细胞(hADSCs)培养基上清的获取:
1)将术中获取的脂肪组织移入无菌大培养皿中,用无菌组织剪反复剪切至组织成糊状,移入50ml离心管,加入等体积的0.075%的I型胶原酶,置于摇床上消化45min,37℃,180r/min。
2)消化后的脂肪颗粒加入等体积的含10%胎牛血清的间充质干细胞完全培养基终止消化,100目滤网过滤,滤液离心3min,1000rpm,10min;
3)弃上清后所得沉淀用5mL完全培养基重悬,细胞计数后适当稀释,以1×105/cm2的密度接种于培养皿中,置于37℃含5%C02的恒温培养箱中培养,获取原代细胞。48 h后首次换液去除未贴壁细胞,此后每72h换液一次;
4)待细胞长至80%--90%融合时PBS漂洗两次,0.25%胰酶1mL,培养箱孵育2-3min,低倍镜下观察细胞缩小变圆,细胞脱壁后加入等体积完培终止消化;
5)细胞悬液离心3min,1000rpm/min,后弃上清,以间充质干细胞完全培养基重悬,充分轻柔吹打至均匀后1:3传代,同法传代培养至P2代。更换培养基采用无血清培养基第三代培养48小时后低速离心获取上清,保存于4度环境中待用。
其中,无血清培养基的配方如下:按照重量份数,包含:a-MEM基础培养基1.5×104份、人血白蛋白4份、腺苷17份、胞苷15份、鸟苷15份、尿苷15份、2-脱氧腺苷16份、2′-脱氧胞苷盐酸16份、2-脱氧鸟苷16份、胸腺嘧啶15份、氢化可的松15份、维生素C 15份、碱性成纤维细胞生长因子0.03份、表皮生长因子0.02份、TGF-β0.03份、促红素0.02份和0.01份褪黑素。
4.注射液的制备:将弹性蛋白肽的冻干粉与脂肪干细胞培养基上清混合,脂肪干细胞培养基上清1000份,弹性蛋白肽0.05份、海藻提取物的浓度为20份、维生素A 50 份、维生素C 9 份。
实验验证环节:
1.注射美容的实施:将受试者分为三组,实验组为采用本发明的注射液,对照组1采用上述注射液中用生理盐水代替干细胞上清,其余组分不变作为注射液,对照组2采用去除弹性蛋白肽的其余组分作为注射液,三组受试者注射量相同,注射部位相同,排除外部人为因素影响,每组20人。注射部位分别为降口角肌:鼻唇沟沿延长线、鼻翼点与口角连线的延长线与下颌缘围城的三角形区域内进行注射。下颌缘上方lcm处取2点行皮内注射,每点0.04mL,边距为0.5cm。颈阔肌下颌缘区:求美者做“呲牙”动作,观察颈阔肌的分布及特征,在下颌角及鼻翼点与口角连线的延长线与下颌缘的焦点区间进行皮内注射,每点0.04ml,两点间间隔0.5cm左右,注射2-3排。
2.效果评估:
皮肤弹性测试:采用仪器为德国CK公司皮肤弹性测试仪MPA580,采用2mmφ探头和450 mbar负压,测试部位为鼻唇沟,下颌缘等部位。R0=Uf即第一个最大振幅,第一条曲线的最高点,表明了皮肤的紧实度,这个参数表示皮肤对立的被动行为。R1=Uf-Ua=第一个最小振幅,第一条曲线的最低点即皮肤回到原始状态的能力。R2=Ua/Uf即回弹部分的弹塑性总量、拉伸部位的弹塑性总量。值越接近1(100%)曲线弹性越大。R3=最后一个最大振幅,最后一条曲线的最高点,和第一条曲线的最大振幅比较,显示皮肤的疲劳效应。R4=最后一个最小振幅,最后一次测量点,与第一条曲线的最小振幅比较,显示皮肤的疲劳效应。R5=Ur/Ue即回弹部分的弹性量/拉伸部分的弹性量,也称作净弹性,值越接近1(100%)曲线弹性越大。在某些情况下(如极具弹性,水分很多或非常薄的皮肤)值可能大于1,弹性松弛部分大于弹性吸入部分。R6=Uv/Ue=拉伸部分的塑性量/拉伸部分的弹性量,值越小弹性越大。R7=Ur/Uf即回弹部分的弹性量/拉伸部分的弹性总量,值越接近1(100%)曲线弹性越大。R8=第一条曲线的Ua,值越接近R0皮肤弹性越好。R9=R3-R0,表示皮肤经过反复吸入后的疲劳效应,值越小疲劳效应越小,这些参数的优点是不受皮肤厚度的影响。
在注射进行后的2,4,6,8,10个月内进行回访,记录测试结果
3.有效性评价:
下颌缘区有无提升,双侧嘴角有无提升评分为1-9分,9分为效果最好,1分为无效果或效果不可察。评分越高,面部提升效果越好。
皱纹情况评分为1-9分,1分为皱纹等级越低或无皱纹,9分为皱纹等级最高,评分越低效果越好。
4.效果验证:在三组实验中,均未出现皮肤红肿,瘙痒等过敏反应,一段时间后注射部位出现紧绷感,在皮肤弹性评价部分我们可以看出实验组的皮肤紧实度最好,疲劳效应较低,弹性及弹性总量远高于其余两组的数据,具体结果如下表I所示:
表I:
对于面部提升效果而言,实验组的效果也远高于两组对照组,同时针对除皱效果,实验组的数据也优于两个对照组,具体结果如下表II所示:
表II:
由表I和表II的实验数据可知,采用脂肪干细胞与弹性蛋白肽联用并且辅以海藻提取物进行面部提升,能够很好的解决皮肤衰老的导致的面部下垂,皮肤无弹性等问题,也有很好的去皱效果。而且本发明所用注射液的效果远远高于脂肪干细胞与弹性蛋白肽单独使用的效果。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易变化或替换,都属于本发明的保护范围之内。因此本发明的保护范围所述以权利要求的保护范围为准。
Claims (6)
1.一种抗衰老的注射针剂,包括弹性蛋白肽,其特征在于,还包括人脂肪间充质干细胞培养基上清和海藻提取物。
2.根据权利要求1所述的一种抗衰老的注射针剂,其特征在于,包括弹性蛋白肽的冻干粉与脂肪干细胞培养基上清混合,其中,脂肪干细胞培养基上清1000份,弹性蛋白肽0.04~0.06份、海藻提取物的浓度为19~21份、维生素A 49~51份、维生素C 8~10 份。
3.根据权利要求1或2所述的一种抗衰老的注射针剂,其特征在于,弹性蛋白肽的制备方法是:将弹性蛋白分散在去离子水中,调节pH值至 pH8.0,然后进行酶解;将酶解所得混合物在 55 ℃下振荡温浴 10 h,酶灭活后进行离心,随后,将收集的上清液冻干并储存在-18℃直至使用。
4.根据权利要求1或2所述的一种抗衰老的注射针剂,其特征在于,人脂肪间充质干细胞培养基上清的获取过程是:
1)将人体脂肪组织移入无菌大培养皿中,用无菌组织剪反复剪切至组织成糊状,移入离心管,加入等体积的0.075%的I型胶原酶,置于摇床上消化45min,得到消化后的脂肪颗粒;
2)消化后的脂肪颗粒加入等体积的含10%胎牛血清的间充质干细胞完全培养基终止消化,100目滤网过滤,滤液离心3min,弃上清后所得沉淀;
3)对步骤2)获得的沉淀用5mL间充质干细胞完全培养基重悬,细胞计数后稀释,以1×105/cm2的密度接种于培养皿中,置于37℃含5%CO2的恒温培养箱中培养,48 h后首次换液去除未贴壁细胞,此后每72h换液一次;
4)待细胞长至80%--90%融合时PBS漂洗两次,0.25%胰酶1mL,培养箱孵育2-3min,低倍镜下观察细胞缩小变圆,细胞脱壁后加入等体积完培终止消化,得到细胞悬液;
5)对步骤4)得到的细胞悬液离心3min后弃上清,以间充质干细胞完全培养基重悬后1:3传代,同法传代培养至P2代;采用无血清培养基第三代培养48小时后离心获取上清,保存于4度环境中待用。
5.根据权利要求4所述的一种抗衰老的注射针剂,其特征在于,所述的无血清培养基配方如下:按照重量份数,包含:a-MEM基础培养基1.5×104份、人血白蛋白4份、腺苷17份、胞苷15份、鸟苷15份、尿苷15份、2-脱氧腺苷16份、2′-脱氧胞苷盐酸16份、2-脱氧鸟苷16份、胸腺嘧啶15份、氢化可的松15份、维生素C 15份、碱性成纤维细胞生长因子0.03份、表皮生长因子0.02份、TGF-β0.03份、促红素0.02份和0.01份褪黑素。
6.如权利要求1~5中任一所述抗衰老的注射针剂在受伤后皮肤修复方向的应用。
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CN115161274A (zh) * | 2022-06-08 | 2022-10-11 | 菲尔生物工程技术有限公司 | 一种脂肪干细胞提取物的抗皱精华液及其制备方法 |
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