CN117482034A - 一种人体脂肪干细胞制备剂与应用 - Google Patents
一种人体脂肪干细胞制备剂与应用 Download PDFInfo
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- CN117482034A CN117482034A CN202311839781.9A CN202311839781A CN117482034A CN 117482034 A CN117482034 A CN 117482034A CN 202311839781 A CN202311839781 A CN 202311839781A CN 117482034 A CN117482034 A CN 117482034A
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- stem cell
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- derived stem
- stem cells
- human adipose
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Abstract
本发明属于生物技术领域,具体公开了一种人体脂肪干细胞制备剂与应用,采用来源于人体废弃脂肪组织分离培养的脂肪干细胞,采用含有生物活性物质的复合水凝胶进行三维培养,通过模拟人体环境进行培养,纯化干细胞和外泌体的扩增,脂肪干细胞外泌体的提取,并制备改性石墨烯量子点负载干细胞外泌体,得到脂肪干细胞制剂,并提供了应用。将得到的脂肪干细胞制剂涂抹到皮肤上,通过可见光照引发,同时该制剂具有极好的生物相容性。
Description
技术领域
本领域属于生物技术领域,具体涉及一种人体脂肪干细胞制备剂与应用。
背景技术
随着现代社会对美丽外观的日益重视,面部肌肤美容已成为重要议题。面部肌肤外观的变化受多种因素影响,包括环境因素(如紫外线照射、污染)、生活方式(比如饮食习惯、睡眠模式)、遗传因素以及随着年龄增长所引发的自然衰老过程。年龄的增长往往伴随着面部肌肤出现皱纹、松弛、色素沉着以及弹性减退等衰老迹象。
为了有效应对这些肌肤衰老的迹象,科学家和美容专家不断探索新技术。其中一种广泛使用的面部年轻化方法是自体脂肪与富含血小板的血浆(PRP)的联合注射,自体脂肪在填充减少的皮下脂肪组织方面发挥作用,而PRP则富含促进皮肤愈合和再生的生长因子。然而,该方法也存在一些局限性和不足。例如,自体脂肪的吸收率可能较高,导致注射后部分脂肪组织被身体吸收,减少了填充效果的持久性。同时,脂肪和PRP的生物活性以及生存能力在一定程度上是有限的,这限制了其修复效果的持续时间。
基于上述问题,迫切需要开发新的面部年轻化策略,这些策略不仅要提供更持久有效的抗衰老效果,还应具备更好的生物相容性。
发明内容
为解决以上技术问题,根据本发明的一个方面,提供一种人体脂肪干细胞制备剂。
本发明解决上述技术问题所提供的技术方案如下:
一种人体脂肪干细胞制备剂,其制备方法包括如下步骤:
S1:人体脂肪干细胞外泌体的提取;
S2:改性石墨烯量子点的制备;
S3:人体脂肪干细胞外泌体的负载。
优选地,步骤S1中所述人体脂肪干细胞外泌体的提取步骤如下:
A1.将提取得到的人体脂肪干细胞加入DMEM/F12培养基中悬浮,然后滴加到复合水凝胶上,在温度37℃、5%CO2的条件下培养,每隔1~2天更换一次DMEM/F12培养基,连续消化传代三次,获得培育脂肪干细胞;
A2.将步骤A1中获得的培育脂肪干细胞3000~5000rpm低速离心10min,取上清液,将上清液以120~180W/L功率超声处理20~30min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,100~1000kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入冻干保护剂,冷冻干燥,获得脂肪干细胞外泌体。
优选地,步骤A1中所述人体脂肪干细胞在加入培养基前需利用含有1wt%青霉素的PBS缓冲液洗涤,且青霉素浓度为90U/mL;所述人体脂肪干细胞浓度为1×105~3×105个/mL。
优选地,步骤A1中所述复合水凝胶是通过将海藻酸钠和胶原蛋白按1:1.4的质量比例加入去离子水中混合,并加入0.6wt%的透明质酸和2.5ppm的表皮生长因子,使用转氨酶进行温和交联,离心后加入溶液体积0.05%的纳米银颗粒,混合均匀,冷冻干燥后制备获得。
优选地,步骤A1中所述更换DMEM/F12培养基步骤为:每次更换DMEM/F12培养基时对人体脂肪干细胞进行一次电击刺激,参数为电压2V,频率52Hz,时间30min;在最后一次更换DMEM/F12培养基时,调整培养温度为32℃。
优选地,步骤A1中所述消化传代即脂肪干细胞融合度超过75%时,需向培养基中加入0.25wt%的胰蛋白酶-EDTA溶液以降低细胞融合度进行消化传代。
优选地,步骤A2中所述冻干保护剂是蔗糖、海藻糖、甘露醇、多聚乙二醇、甘氨酸和丙氨酸中的一种,冻干保护剂的加入量为溶液体积的5%。
优选地,步骤S2中所述改性石墨烯量子点的制备步骤如下:
B1.将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将石墨烯粉末加入浓硫酸和浓硝酸中,120~150W/L密封超声处理4h,加入高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
B2.将步骤B1中获得的混合溶液冷却至室温,继续120~150W/L超声处理4h,30min持续缓慢加入去离子水,控制温度30℃,静置30min,再分三批次加入30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用透析袋透析24h,冷冻干燥,获得石墨烯量子点;
B3.将步骤B2中获得的石墨烯量子点加入PBS缓冲液中,混合均匀,加入1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入偶氮苯,提高温度至40℃,继续上述转速搅拌14~24h,8000rpm离心处理10min,冷冻干燥,获得改性石墨烯量子点。
优选地,步骤B1中所述石墨烯粉末、浓硫酸和浓硝酸的用量之比为0.05g:8~12mL:5~6mL;所述高锰酸钾与石墨烯粉末的质量之比为1:1~5。
优选地,步骤B2中所述去离子水与石墨烯粉末的用量之比为40mL:0.05g;所述过氧化氢用量为去离子水的3~5%;所述透析袋的分子截留量为3kDa。
优选地,步骤B3中所述石墨烯量子点与PBS缓冲液的用量之比为0.1g:20~25mL;所述1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的用量与石墨烯量子点之比为1mM:0.8mM:0.1g;所述偶氮苯和石墨烯量子点的用量之比为0.05mM:0.1g。
优选地,步骤S3中所述人体脂肪干细胞外泌体的负载步骤如下:
C1.将海藻酸钠、透明质酸和明胶加入PBS缓冲液中,温度60℃下,500rpm搅拌30min,加入N-乙酰半胱氨酸,继续搅拌10min,再加入步骤A2制备的脂肪干细胞外泌体和步骤B3中制备的改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,冷冻干燥,获得干细胞制剂。
优选地,步骤C1中所述海藻酸钠、透明质酸、明胶和PBS缓冲液的用量之比为2g:0.6g:5~7g:50mL;所述N-乙酰半胱氨酸与PBS缓冲液用量之比为1g:50mL;所述脂肪干细胞外泌体、改性石墨烯量子点和PBS缓冲液的用量之比为10mg:30~40mg:5mL。
优选地,本发明的另一方面在于提供一种人体脂肪干细胞制备剂的应用,所述人体脂肪干细胞制剂溶作为涂抹凝胶,涂抹于肌肤上,并利用450~550nm之间的可见光引发使其发挥效果。
本发明中所用方法如无特殊说明,均为常规方法,所用材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明的有益效果如下:
本发明直接从人体脂肪干细胞中获得其外泌体,均为人体成分,可以直接被皮肤吸收,避免过敏反应,无副作用;为了培育这些外泌体,本发明采用了一种创新的方法。具体而言,我们将细胞悬液滴加至特制的复合水凝胶中,并在培养过程中应用电刺激。这种独特的组合为细胞提供了一个模拟自然细胞外基质的环境,促进细胞的生长和活性,为了保持培养环境的无菌状态,我们加入了纳米银颗粒,其抗菌效果显著,同时低浓度的纳米银颗粒不会对脂肪干细胞的生长产生影响;更重要的是,电刺激的应用模拟了体内的电生理环境,有助于提高干细胞的外泌体生产。总体而言,通过优化培养环境和刺激方法,本发明有效提高了外泌体的产量和质量。
本发明利用的石墨烯量子点(GQDs)经过特殊改性,能够在450-550nm波长范围内的可见光照射下引发脂肪干细胞外泌体的释放。与传统的紫外光引发系统相比,本发明的石墨烯量子点提供了更高的皮肤安全性,减少了光毒性和皮肤损伤的风险。此外,由于可见光源比紫外光源更普遍和易于控制,无需特殊设备,因此本发明的石墨烯量子点在日常使用中更为方便。此外,本发明的石墨烯量子点能够在可见光范围内实现精确的外泌体释放控制,有效优化了光敏感效果。
本发明利用石墨烯量子点精确控制脂肪干细胞外泌体的释放位置,同时通过光照控制,可以精确地决定何时释放外泌体,可以更好的促进效果的发挥;此外,石墨烯量子点可以保护外泌体免受环境因素(如氧化、酶降解)的影响,有效避免外泌体在达到目标皮肤层之前就被降解或失活,增强其稳定性和保质期。石墨烯量子点可以促进外泌体更深入地渗透到皮肤中,增强其生物利用率,有助于将外泌体输送到皮肤的更深层次,发挥更好的抗衰老和修复效果,石墨烯量子点的物理和化学特性可能与外泌体的生物活性产生协同效应,从而增强抗衰老效果。
附图说明
图1是本发明制备剂对于细胞炎症因子TNF-α表达量图;
图2是本发明制备剂对于成纤维细胞增殖图;
图3是本发明细胞制备剂的保湿性能图。
具体实施方式
下面将结合本发明的实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例;基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:一种干细胞制剂的制备方法,包括如下步骤:
S1:人体脂肪干细胞外泌体的提取;
无菌条件下获取人体腹部废弃脂肪组织,使用无菌盐水冲洗三次后,将其浸入加有1wt%链霉素的PBS缓冲液中,反复冲洗去除血液和其他可见杂质,同时去除脂肪组织中的大块组织和纤维,用手术剪将清洗后的脂肪组织剪切成约1mm³的小块,将30mg脂肪组织加入100mL的PBS缓冲液中,并加入2mL胶原酶,控制温度37℃,并以40rpm的速度轻轻摇晃12h,6000rpm离心处理10min,去掉上清液,获得脂肪干细胞;将脂肪干细胞用含有1wt%青霉素的PBS缓冲液洗涤,移入DMEM/F12培养基中悬浮,配制细胞浓度为1×105个/mL,滴加5mL到复合水凝胶上,在温度37℃、5%CO2的条件下培养,每隔2天更换一次DMEM/F12培养基,并进行一次电击刺激,参数为电压2V,频率52Hz,时间30min,最后一次更换DMEM/F12培养基时,调整培养温度为32℃,其他参数不变,细胞融合度超过75%时,加入0.013mL的胰蛋白酶-EDTA溶液进行消化传代,连续传代三次后,获得培育脂肪干细胞;
S11:复合水凝胶的制备,称取2g海藻酸钠和2.8g胶原蛋白加入100mL去离子水中,混合均匀,加入0.6g透明质酸和2.5μg的表皮生长因子,加入2mL转氨酶,常温下500rpm搅拌反应60min,5000rpm离心处理10min,加入0.05g纳米银,混合均匀,-60℃冷冻干燥,获得复合水凝胶。
取1mL上述培育脂肪干细胞制备成10mL的悬液,加入2mL生理盐水,在3000rpm下离心10min,取上清液,120W/L功率超声处理20min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,1000kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入0.05g海藻糖,-80°C预冷冻4h,升温至-20℃,干燥20h,获得脂肪干细胞外泌体。
S2:改性石墨烯量子点的制备;
将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将1g石墨烯粉末加入160mL浓硫酸和100mL浓硝酸中,120W/L密封超声处理4h,加入1g高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
将混合溶液冷却至室温,继续120W/L超声处理4h,30min持续缓慢加入800mL去离子水,控制温度30℃,静置30min,再分三批次加入24mL的30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用3kDa透析袋透析24h,冷冻干燥24h,获得石墨烯量子点;
将0.8g石墨烯量子点加入160mLPBS缓冲液中,混合均匀,加入8mM的1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和6.4mM的N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入0.4mM的偶氮苯,提高温度至40℃,继续上述转速搅拌14h,8000rpm离心处理10min,-80°C预冷冻4h,温度-20℃,压力0.2mBar,干燥24h,升温至20℃后继续干燥3h,获得改性石墨烯量子点。
S3:人体脂肪干细胞外泌体的负载;
将0.4g海藻酸钠、0.12g透明质酸和1g明胶加入10mLPBS缓冲液中,温度60℃下,500rpm搅拌30min,加入0.2g的N-乙酰半胱氨酸,继续搅拌10min,再加入0.02g脂肪干细胞外泌体和0.06g改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,-80℃冷冻干燥,获得干细胞制剂。
实施例2:一种干细胞制剂的制备方法,包括如下步骤:
S1:人体脂肪干细胞外泌体的提取;
无菌条件下获取人体大腿部废弃脂肪组织,使用无菌盐水冲洗三次后,将其浸入加有1wt%青霉素的PBS缓冲液中,反复冲洗去除血液和其他可见杂质,同时去除脂肪组织中的大块组织和纤维,用手术剪将清洗后的脂肪组织剪切成约1mm³的小块,将30mg脂肪组织加入100mL的PBS缓冲液中,并加入2mL胶原酶,控制温度37℃,并以40rpm的速度轻轻摇晃12h,6000rpm离心处理10min,去掉上清液,获得脂肪干细胞;将脂肪干细胞用含有1wt%青霉素的PBS缓冲液洗涤,移入DMEM/F12培养基中悬浮,细胞浓度为2×105个/mL,滴加到复合水凝胶上,在温度37℃、5%CO2的条件下培养,每隔1天更换一次DMEM/F12培养基,并进行一次电击刺激,参数为电压2V,频率52Hz,时间30min,最后一次更换DMEM/F12培养基时,调整培养温度为32℃,其他参数不变,细胞融合度超过75%时,加入10mL的0.25wt%的胰蛋白酶-EDTA溶液进行消化传代,连续传代三次后,将P4以后的脂肪干细胞作制备剂制备用,获得培育脂肪干细胞;
S11:复合水凝胶的制备,称取2g海藻酸钠和2.8g胶原蛋白加入100mL去离子水中,混合均匀,加入0.6g透明质酸和2.5μg的表皮生长因子,加入2mL转氨酶,常温下500rpm搅拌反应60min,5000rpm离心处理10min,加入0.05g纳米银,混合均匀,-60℃冷冻干燥,获得复合水凝胶。
取1mL上述培育脂肪干细胞制备成10mL的悬液,加入2mL生理盐水,在4000rpm下离心10min,取上清液,150W/L功率超声处理25min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,500kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入0.05g丙氨酸,-60°C预冷冻3h,升温至-20℃,干燥24h,获得脂肪干细胞外泌体。
S2:改性石墨烯量子点的制备;
将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将1g石墨烯粉末加入200mL浓硫酸和110mL浓硝酸中,140W/L密封超声处理4h,加入0.4g高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
将混合溶液冷却至室温,继续130W/L超声处理4h,30min持续缓慢加入800mL去离子水,控制温度30℃,静置30min,再分三批次加入32mL的30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用3kDa透析袋透析24h,冷冻干燥24h,获得石墨烯量子点;
将0.8g石墨烯量子点加入180mLPBS缓冲液中,混合均匀,加入8mM的1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和6.4mM的N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入0.4mM的偶氮苯,提高温度至40℃,继续上述转速搅拌18h,8000rpm离心处理10min,-80°C预冷冻2h,温度-20℃,压力0.2mBar,干燥24h,升温至20℃后继续干燥3h,获得改性石墨烯量子点。
S3:人体脂肪干细胞外泌体的负载;
将0.8g海藻酸钠、0.24g透明质酸和2.5g明胶加入20mLPBS缓冲液中,温度60℃下,500rpm搅拌30min,加入0.4g的N-乙酰半胱氨酸,继续搅拌10min,再加入0.04g脂肪干细胞外泌体和0.14g改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,-80℃冷冻干燥,获得干细胞制剂。
实施例3:一种干细胞制剂的制备方法,包括如下步骤:
在无菌条件下获取人体臀部的废弃脂肪组织。首先,使用无菌生理盐水彻底冲洗脂肪组织三次,以去除血液和其他表面污染物,接着将脂肪组织浸泡在含有1wt%链霉素和0.5wt%链霉亲和素的PBS缓冲液中,用手术剪将清洗后的脂肪组织剪切成约1mm³的小块;将30mg脂肪组织加入到含有1mL胶原酶和0.5mL透明质酸酶混合液的50mLPBS缓冲液中。在37℃下以50rpm的速度轻轻摇晃6h,以8000rpm的速度离心5min,去除上清液,收集沉淀的细胞;将收集到的细胞用含有1wt%青霉素和1wt%链霉素的PBS缓冲液洗涤两次,去除残余的酶和杂质。之后,将细胞转移到DMEM/F12培养基中,细胞浓度为2×105个/mL;将细胞悬液滴加到复合水凝胶上,在37℃和5%CO2的条件下培养细胞,并每天更换一次DMEM/F12培养基。为了促进细胞的生长和分化,每次更换培养基后对细胞进行电刺激,参数设置为电压3V,频率60Hz,持续时间20分钟。在细胞培养的最后阶段,将培养温度调整至30℃,以增强细胞的适应性和稳定性。当细胞融合度达到80%以上时,使用10mL的0.2wt%的胰蛋白酶-EDTA溶液进行细胞分离和消化。细胞分离后,继续在DMEM/F12培养基中进行连续传代,连续传代三次后,获得培育脂肪干细胞;
S11:复合水凝胶的制备,称取2g海藻酸钠和2.8g胶原蛋白加入100mL去离子水中,混合均匀,加入0.6g透明质酸和2.5μg的表皮生长因子,加入2mL转氨酶,常温下500rpm搅拌反应60min,5000rpm离心处理10min,加入0.05g纳米银,混合均匀,-60℃冷冻干燥,获得复合水凝胶。
取1mL培育脂肪干细胞制备成10mL的悬液,加入2mL生理盐水,在5000rpm下离心10min,取上清液,180W/L功率超声处理30min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,100kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入0.05g多聚乙二醇,-80°C预冷冻4h,升温至-20℃,干燥20h,获得脂肪干细胞外泌体。
S2:改性石墨烯量子点的制备;
将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将1g石墨烯粉末加入220mL浓硫酸和110mL浓硝酸中,160W/L密封超声处理4h,加入0.2g高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
将混合溶液冷却至室温,继续160W/L超声处理4h,30min持续缓慢加入800mL去离子水,控制温度30℃,静置30min,再分三批次加入35mL的30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用3kDa透析袋透析24h,冷冻干燥24h,获得石墨烯量子点;
将0.8g石墨烯量子点加入200mLPBS缓冲液中,混合均匀,加入8mM的1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和6.4mM的N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入0.4mM的偶氮苯,提高温度至40℃,继续上述转速搅拌24h,8000rpm离心处理10min,-80°C预冷冻4h,温度-20℃,压力0.2mBar,干燥20h,升温至20℃后继续干燥3h,获得改性石墨烯量子点。
S3:人体脂肪干细胞外泌体的负载;
将0.4g海藻酸钠、0.12g透明质酸和1.4g明胶加入10mLPBS缓冲液中,温度60℃下,500rpm搅拌30min,加入0.2g的N-乙酰半胱氨酸,继续搅拌10min,再加入0.02g脂肪干细胞外泌体和0.08g改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,-80℃冷冻干燥,获得干细胞制剂。
实施例4:一种干细胞制剂的制备方法,包括如下步骤:
无菌条件下获取人体腹部废弃脂肪组织,使用无菌盐水冲洗三次后,将其浸入加有1wt%链霉素的PBS缓冲液中,反复冲洗去除血液和其他可见杂质,同时去除脂肪组织中的大块组织和纤维,用手术剪将清洗后的脂肪组织剪切成约1mm³的小块,将30mg脂肪组织加入100mL的PBS缓冲液中,并加入2mL胶原酶,控制温度37℃,并以40rpm的速度轻轻摇晃12h,6000rpm离心处理10min,去掉上清液,获得脂肪干细胞;将脂肪干细胞用含有1wt%青霉素的PBS缓冲液洗涤,移入DMEM/F12培养基中悬浮,细胞浓度为3×105个/mL,滴加到复合水凝胶上,在温度37℃、5%CO2的条件下培养,每隔2天更换一次DMEM/F12培养基,并进行一次电击刺激,参数为电压2V,频率52Hz,时间30min,最后一次更换DMEM/F12培养基时,调整培养温度为32℃,其他参数不变,细胞融合度超过75%时,加入10mL的0.25wt%的胰蛋白酶-EDTA溶液进行消化传代,连续传代三次后,获得培育脂肪干细胞;
S11:复合水凝胶的制备,称取2g海藻酸钠和2.8g胶原蛋白加入100mL去离子水中,混合均匀,加入0.6g透明质酸和2.5μg的表皮生长因子,加入2mL转氨酶,常温下500rpm搅拌反应60min,5000rpm离心处理10min,加入0.05g纳米银,混合均匀,-60℃冷冻干燥,获得复合水凝胶。
取1mL培育脂肪干细胞制备成10mL的悬液,加入2mL生理盐水,在5000rpm下离心10min,取上清液,180W/L功率超声处理30min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,100kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入0.05g多聚乙二醇,-80°C预冷冻4h,升温至-20℃,干燥20h,获得脂肪干细胞外泌体。
S2:改性石墨烯量子点的制备;
将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将1g石墨烯粉末加入240mL浓硫酸和120mL浓硝酸中,150W/L密封超声处理4h,加入0.2g高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
将混合溶液冷却至室温,继续150W/L超声处理4h,30min持续缓慢加入800mL去离子水,控制温度30℃,静置30min,再分三批次加入40mL的30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用3kDa透析袋透析24h,冷冻干燥24h,获得石墨烯量子点;
将0.8g石墨烯量子点加入200mLPBS缓冲液中,混合均匀,加入8mM的1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和6.4mM的N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入0.4mM的偶氮苯,提高温度至40℃,继续上述转速搅拌24h,8000rpm离心处理10min,-80°C预冷冻4h,温度-20℃,压力0.2mBar,干燥20h,升温至20℃后继续干燥3h,获得改性石墨烯量子点。
S3:人体脂肪干细胞外泌体的负载;
将0.4g海藻酸钠、0.12g透明质酸和1.4g明胶加入10mLPBS缓冲液中,温度60℃下,500rpm搅拌30min,加入0.2g的N-乙酰半胱氨酸,继续搅拌10min,再加入0.02g脂肪干细胞外泌体和0.08g改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,-80℃冷冻干燥,获得干细胞制剂。
试验测试:
选择10mL对数生长期状态良好的成纤维细胞,经0.25wt%胰蛋白酶消化和离心后重悬于10mL含有10%胎牛血清的DMEM培养液中并计数;将成纤维细胞接种于96孔培养板中,每孔细胞数约为5×103个,设置未加细胞的空白对照,边缘孔用无菌PBS填充,37℃、体积分数5%的CO2环境中培养过夜;按照各自终浓度分别加入样品(本发明实施例1~4所制备的干细胞制备剂),未处理组作为细胞对照组,每组设3个复孔,37℃、体积分数5%CO2环境中培养48h。培养结束前4h加入5mg/mL的MTT溶液,继续培养4h后终止培养;吸弃孔内液体,加入DMSO溶解结晶,M3读板仪于570nm处测定各孔吸光度,试验结果如图2所示。
细胞存活率(%)=(试验组吸光度-对照组吸光度)/(未处理组吸光度-对照组吸光度)×100%
选取80名年龄在25~45岁的受试者,随机均分入试验组1~4,每组20人,试验组1~4的受试组分别使用实施例1~4中的干细胞制备剂;每天晚上洁面后,使用实施例1~4所述干细胞制备剂,使用方法为取适量涂抹于面部,并照射10min的500nm之间的可见光,每天涂抹一次,每组受试者连续涂抹30天,期间不使用其他任何护肤品,在0d、7d、14d和28d时,利用TEWL(Trans-Epidermal Water Loss)仪器进行测试其角质层失水量,计算其保湿性,并在使用30天后记录皮肤炎症变化状况,试验标准:皮肤无明显红肿、红肿减轻、无效果;保湿性结果如图3所示,炎症结果如表1所示。
保湿性=(使用前失水量-使用后失水量)/使用前失水量×100%
表1.干细胞制剂对皮肤炎症有益效果。
由表1和图1可知,本发明制备的脂肪干细胞制备剂对皮肤炎症有明显的有益效果,可以有效减轻和缓解炎症的发生,图1中的对照组为未添加外泌体和石墨烯量子点的制备剂;由图2结果可知,本发明制备的干细胞制备剂能有效促进成纤维细胞的增殖,具备较好的抗衰老效果;由图3结果可知,本发明制备的干细胞制备剂对皮肤保湿性也有极好的效果。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.一种人体脂肪干细胞制备剂,其特征在于,所述人体脂肪干细胞制备剂的制备方法包括以下步骤:
S1:人体脂肪干细胞外泌体的提取;
S2:改性石墨烯量子点的制备;
S3:人体脂肪干细胞外泌体的负载;
步骤S1中所述人体脂肪干细胞外泌体的提取步骤如下:
A1.将提取得到的人体脂肪干细胞加入DMEM/F12培养基中悬浮,然后滴加到复合水凝胶上,在温度37℃、5%CO2的条件下培养,每隔1~2天更换一次DMEM/F12培养基,连续消化传代三次,获得培育脂肪干细胞;
A2.将步骤A1中获得的培育脂肪干细胞3000~5000rpm低速离心10min,取上清液,将上清液以120~180W/L功率超声处理20~30min,0.22μm的滤膜过滤;然后再控制转速10000rpm,温度4℃离心处理30min,100~1000kDa的超滤膜过滤,PBS缓冲液洗涤去除杂质,加入冻干保护剂,冷冻干燥,获得脂肪干细胞外泌体。
2.根据权利要求1所述的人体脂肪干细胞制备剂,其特征在于,步骤A1中所述人体脂肪干细胞在加入培养基前需利用含有1wt%青霉素的PBS缓冲液洗涤,且青霉素浓度为90U/mL;所述人体脂肪干细胞浓度为1×105~3×105个/mL;所述消化传代即脂肪干细胞融合度超过75%时,需向培养基中加入0.25wt%的胰蛋白酶-EDTA溶液以降低细胞融合度进行消化传代;步骤A2中所述冻干保护剂是蔗糖、海藻糖、甘露醇、多聚乙二醇、甘氨酸和丙氨酸中的一种,冻干保护剂的加入量为溶液体积的5%。
3.根据权利要求2所述的人体脂肪干细胞制备剂,其特征在于,步骤A1中所述复合水凝胶是通过将海藻酸钠和胶原蛋白按1:1.4的质量比例加入去离子水中混合,并加入0.6wt%的透明质酸和2.5ppm的表皮生长因子,使用转氨酶进行温和交联,离心后加入溶液体积0.05%的纳米银颗粒,混合均匀,冷冻干燥后制备获得。
4.根据权利要求3所述的人体脂肪干细胞制备剂,其特征在于,步骤A1中所述更换DMEM/F12培养基步骤为:每次更换DMEM/F12培养基时对人体脂肪干细胞进行一次电击刺激,参数为电压2V,频率52Hz,时间30min;在最后一次更换DMEM/F12培养基时,调整培养温度为32℃。
5.根据权利要求4所述的人体脂肪干细胞制备剂,其特征在于,步骤S2中所述改性石墨烯量子点的制备步骤如下:
B1.将石墨烯研磨过200目筛筛选,获得石墨烯粉末,将石墨烯粉末加入浓硫酸和浓硝酸中,120~150W/L密封超声处理4h,加入高锰酸钾,控制温度在35℃,1.5℃/min缓慢提升温度至120℃,反应24h,获得混合溶液;
B2.将步骤B1中获得的混合溶液冷却至室温,继续120~150W/L超声处理4h,30min持续缓慢加入去离子水,控制温度30℃,静置30min,再分三批次加入30%的过氧化氢溶液,利用NaOH溶液将pH调至7.8,室温下,100rpm搅拌反应30min,5000rpm离心处理5min,去离子水洗涤,利用透析袋透析24h,冷冻干燥,获得石墨烯量子点;
B3.将步骤B2中获得的石墨烯量子点加入PBS缓冲液中,混合均匀,加入1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,调节pH为7.0,室温下500rpm搅拌30min,加入偶氮苯,提高温度至40℃,继续上述转速搅拌14~24h,8000rpm离心处理10min,冷冻干燥,获得改性石墨烯量子点。
6.根据权利要求5所述的人体脂肪干细胞制备剂,其特征在于,步骤B1中所述石墨烯粉末、浓硫酸和浓硝酸的用量之比为0.05g:8~12mL:5~6mL;所述高锰酸钾与石墨烯粉末的质量之比为1:1~5;步骤B2中所述去离子水与石墨烯粉末的用量之比为40mL:0.05g;所述过氧化氢用量为去离子水的3~5%;所述透析袋的分子截留量为3kDa;步骤B3中所述石墨烯量子点与PBS缓冲液的用量之比为0.1g:20~25mL;所述1-乙基-3-二甲基氨基丙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的用量与石墨烯量子点之比为1mM:0.8mM:0.1g;所述偶氮苯和石墨烯量子点的用量之比为0.05mM:0.1g。
7.根据权利要求6所述的人体脂肪干细胞制备剂,其特征在于,步骤S3中所述人体脂肪干细胞外泌体的负载步骤如下:
C1.将海藻酸钠、透明质酸和明胶加入PBS缓冲液中,温度60℃下,500rpm搅拌30min,加入N-乙酰半胱氨酸,继续搅拌10min,再加入步骤A2制备的脂肪干细胞外泌体和步骤B3中制备的改性石墨烯量子点,调节pH为7.4,室温下200rpm搅拌1h,静置12h,10000rpm离心处理15min,取沉淀,PBS缓冲液洗涤,冷冻干燥,获得干细胞制剂。
8.根据权利要求7所述的人体脂肪干细胞制备剂,其特征在于,步骤C1中所述海藻酸钠、透明质酸、明胶和PBS缓冲液的用量之比为2g:0.6g:5~7g:50mL;所述N-乙酰半胱氨酸与PBS缓冲液用量之比为1g:50mL;所述脂肪干细胞外泌体、改性石墨烯量子点和PBS缓冲液的用量之比为10mg:30~40mg:5mL。
9.一种根据权利要求1-8任一项所述的人体脂肪干细胞制备剂的应用,其特征在于,所述人体脂肪干细胞制剂作为涂抹凝胶,涂抹于肌肤上,并利用450~550nm之间的可见光引发使其发挥效果。
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