CN114042030B - 一种含有脂肪间充质干细胞冻干粉的化妆品及抗炎药物 - Google Patents
一种含有脂肪间充质干细胞冻干粉的化妆品及抗炎药物 Download PDFInfo
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- CN114042030B CN114042030B CN202111434636.3A CN202111434636A CN114042030B CN 114042030 B CN114042030 B CN 114042030B CN 202111434636 A CN202111434636 A CN 202111434636A CN 114042030 B CN114042030 B CN 114042030B
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Abstract
本发明涉及一种含有脂肪间充质干细胞冻干粉的化妆品及抗炎药物。本发明提供了一种特异性的脂肪间充质干细胞冻干粉，所述冻干粉采用含有caspase‑7单抗的冻干保护剂冻干制备获得，所述冻干粉具有促进成纤维迁移以及抑制炎性的技术效果，所述冻干粉可以用于在美容或者药物领域。
Description
技术领域
本发明涉及化妆品或者药物领域，更具体的涉及一种干细胞冻干粉及抗炎老药物。
背景技术
干细胞(stem cells,SC)是身体的起源细胞，是形成人体各组织、器官的始祖细胞，而且它可分化体内所有类型的细胞，例如：组织、器官、骨、软骨、肌腱、韧带等细胞，具有自我复制能力的多潜能细胞，在一定条件下，它可以分化成多种功能细胞，学界称为“万用细胞”；干细胞疗法，通常包括自体脂肪干细胞或干细胞培养基，用于面部抗衰，实现皮肤的新生年轻化。
在美容行业，使用干细胞已经越来越流行。但是干细胞的保存一直是技术难题。目前临床使用的MSCs大多就是经体外培养、低温保存后的。但有研究显示用早期代数的MSCs的治疗效果要比晚期的效果好，这可能与MSCs的分化能力会随细胞的传代而降低有关。因此，MSCs的早期冻存是维持其移植时细胞状态和有效性的必要条件。
冷冻保存的目的是在液氮的低温环境下减缓细胞的代谢活动并维持生命.在细胞悬液的冷冻过程中，冰晶的形成是导致细胞死亡的主要原因之一。避免冰晶的形成就要保持冷却速率缓慢，以使冻存保护剂逐渐进入细胞内部形成浓度梯度。当冷却持续进行时，细胞内的水分在冻存剂的渗透压力下逐步流出细胞，当细胞内的冻存剂浓度达到最大时细胞就会完全脱水皱缩达到最佳冻存状态。如果冻存速率太快就不能让细胞内的水分完全出胞，从而导致细胞内的冰晶形成而对细胞造成损伤。但冻存液对细胞有一定毒性作用，冻存速率过低会导致细胞在冻存液中暴露的时间过长而导致细胞受毒.由冻存保护剂造成的细胞毒性和细胞内冰晶造成细胞损伤这两种冻存伤害已被广泛认同。
除了冷冻速率，冷冻保护剂也是减小冻存损伤需要考虑的重要因素之一。冷冻保护剂根据穿过细胞膜的能力被分为渗透性保护剂和非渗透保护剂。渗透性冷冻保护剂有二甲基亚砜、丙三醇、乙二醇、丙二醇、甲醇、乙醇、丙醇、甲酰胺等，主要作用是防止细胞内冰晶形成、避免高浓度冻存剂的毒性和使细胞在耐受度内脱水。渗透性保护剂的保护能力是由许多小分子构成的高溶解度的水溶性复合物，在高浓度下能够降低水的冰点，因此在冷冻过程中降低了大量冰晶的形成。非渗透性冷冻保护剂有海藻糖、蔗糖、乳糖、葡萄糖、甘露醇、山梨醇、聚合物羟乙基淀粉和聚乙烯吡咯烷酮，它们能够在较低的浓度下保护细胞，但要求更高的冷冻速率.如聚合物羟乙基淀粉不能渗入导致细胞外的浓度增大，在低温下形成反渗透而脱水起到保护作用。换言之，非渗透性冷冻保护剂集中在细胞外导致细胞脱水主要是在冷冻的初始阶段。海藻糖的保护作用是和脂膜之间的相互作用，在冻存和复苏的过程中保持了蛋白质的稳定性，而且能够形成玻璃化基质抑制细胞内冰晶产生。但即便是非渗透性的保护剂，也是能引起细胞损伤的.所以研发最有效的冻存剂也是目前细胞冻存的挑战之一。现已有研究开始关注非渗透性保护剂对细胞的毒性机制，以期使其毒性降至最低。
冻存过程中导致的损伤一般是由细胞在冷冻过程中脱水、氧自由基产生和细胞凋亡酶如caspase的激活所致.因此，有报道通过细胞的存活率、过氧化氢酶(抗氧化酶)和caspase的抑制剂，研究了能够稳定细胞膜的海藻糖对细胞的影响。研究表明用含2.5％、5％和10％的DMSO、60mmol/L海藻糖、100ug/mL过氧化氢酶和30ug/mL zVAD-fmk(caspase抑制剂)的无血清冻存液在液氮中冻存羊水MSCs3周，结果显示，用2.5％DMSO和3种添加剂(海藻糖、过氧化氢酶和caspase抑制剂)的冻存液和含10％DMSO和30％FBS的冻存液，在群体倍增、细胞表面抗原表达和成肌细胞分化能力上表现相似，该结果证实了减少DMSO和无血清冻存对保持MSCs存活率和功能的可行性。
Caspase通常以无活性的酶原形式存在大多数后生动物细胞中,其家族成员在氨基酸序列、结构及酶的特性上均相似。该酶原是一个分子量为30-50ku的单一多肽,由三个结构域构成,分别为N端前域、20ku的大亚基单位和10ku的小亚基单位。酶原分子被激活后,大小亚基解离并重新组装为四聚体形式的活性复合物,两个小亚基单位在中间,两个大亚基单位在外周,每个异二聚体包含一个催化位点。其中最重要的是执行型Caspase(ExecutionerCasepase),包括Caspase-3、Caspase-6、Caspase-7等,其作用在于特异性地裂解底物使细胞发生生化及形态学改变,最终导致细胞凋亡。但是目前，caspase的抑制剂的类型还比较少，为本领域提供的选择还不够多，因此，开发新的caspase的抑制剂从而用于干细胞的冻干粉制备时本领域迫切的需求。
发明内容
本发明一方面，提供一种特异性针对Caspase-7的单克隆抗体。所述抗体的轻重链可变区序列分别如SEQ ID NO:2和3所示。
另外一方面，本发明提供一种脂肪间充质干细胞冻干粉，所述冻干粉采用特定冻干剂来保护脂肪间充质干细胞而获得。
相应的，本发明提供一种冻干保护剂，所述冻干保护剂中含有Caspase-7的单克隆抗体。
另外一方面，本发明的冻存保护剂组成为2％单克隆抗体、20％聚乙烯毗咯烷酮、25％海藻糖、20％丙三醇、5％蔗糖、10％甘露醇和0.5％多聚精氨酸，DMEM/F12培养液中溶解混匀。
另外一方面，本发明提供一种冻干脂肪间充质干细胞的方法，所述方法包括：收集第4代融合度为85％的脂肪间充质干细胞，用胰蛋白酶/EDTA消化液消化细胞，细胞变圆刚脱离培养板面时终止消化，用PBS清洗2遍，1000r/min，离心5min。离心后，取脂肪间充质干细胞与保护剂按体积比2：1混匀。先在4℃下平衡15min，然后快速置入-80度低温冰箱冷冻2h。随后转移到预先冷却至-60℃的冻干机搁板上，维持2h。冻结过程结束后，开启冷阱制冷阀使冷阱温度降至-60℃以下，开启真空泵使冻干室真空度达到10Pa以下，关小搁板制冷阀使搁板温度升至-35℃，维持一次干燥15h。此后，以0.2℃/min的加热速度使搁板温度升高至20℃并维持在此温度直到二次干燥过程结束，这个过程持续约12h。冻干结束后样品呈干饼状，无塌陷，将其严格密封后保存于4℃冰箱内。
进一步的，本发明提供一种上述方法制备的冻干脂肪间充质干细胞在制备美容产品中的用途。其是通过促进成纤维细胞胶原蛋白等的分泌以及抑制凋亡实现的。
另外的一个方面，本发明的美容产品中还添加有抗氧化剂。
本发明所述的抗氧化剂包括：阿斯可比酸及二义盐类及衍生物，如：阿斯可比色氨酸乙酯；阿斯可比色醚；阿斯可比色胺；阿斯可比色氨酸及二义盐类；咖啡因酸及二义盐类等。这些抗氧化剂可用于制造物总重量的0.0001-3重量％。
在本发明中，所述美容产品中还含有会发现溶剂。
可作为挥发性溶剂使用适用于乙醇，异丙醇等化妆品的醇类。并且，本发明的组成物对所述挥发性溶剂对组成物总重量的含量为1-10重量％的量。
本发明的化妆品成分除有效成分外，还包括在化妆品成分中通常使用的成分，如稳定剂，乳化剂，可溶化剂，颜料及香料等通常的助剂。本发明的化妆品组成物可以被制成任何通常制造的剂型，例如溶液、悬浮液、乳状液、面胶、乳液、等，但不限于此。更详细的可以是润肤乳液精华霜眼霜面膜或喷雾剂型。
另外一方面，本发明提供一种上述方法制备的冻干脂肪间充质干细胞在制备抗炎药物中的用途。具体的，所述抗炎是通过降低炎症基因TNF-α的表达来实现的。
本发明的药学组成物可以根据预防和治疗皮肤炎症的通常方法，以片剂，还原剂，散剂，颗粒剂，胶囊剂，悬浮剂，乳剂，糖浆剂，气溶胶，灭菌剂，注射液等形式进行剂型化。
口服给药的固体制剂包括精制环氧制剂，颗粒制剂，胶囊制剂等，这些固体制剂可混合配制成至少一种赋形剂，如淀粉碳酸钙水凝胶。此外，除了单纯的赋形剂外，还可用于镁合金硬脂滑石粉之类的润滑剂。经口给药的液相制剂包括悬浮剂内容液剂乳剂糖浆剂等，除常用的单纯稀释剂--水立方石蜡外，还可用于各种赋形剂如润湿剂甜味剂芳香剂保鲜剂等。
非经口给药的制剂可包括灭菌水溶液非手性溶剂悬浮剂乳胶冻干制剂佐剂等。非手性溶剂和悬浮溶剂可用于可注射的酯类，如丙二醇聚乙二醇橄榄油等。
此外，本发明的药学组成物可进一步包括载体赋形剂或稀释剂。载体赋形剂或稀释剂为乳糖、甲基纤维素、羟甲基纤维素、羟丙基甲基纤维素、微晶质纤维素聚乙烯醇吡咯烷酮；水甲基羟苯甲酰亚胺；邻苯甲酰亚胺；邻苯甲酰亚胺；脱镁；硬脂酰亚胺；可用于二氧化硅等的矿物油等。
本发明所述的药学组成物的具体投药量根据制剂化方法患者的状态及体重患者性别年龄疾病的程度；药物形态投药途径及期间排泄速度反应感应性等因素。可被当业者选择多样，投注量及次数并不在任何方面限制本发明的范围。
有益效果
本发明提供了一种特异性的脂肪间充质干细胞冻干粉，所述冻干粉采用含有caspase-7单抗的冻干保护剂冻干制备获得，所述冻干粉具有促进成纤维迁移以及抑制炎性的技术效果，所述冻干粉可以用于在美容或者药物领域。
附图说明
图1迁移细胞数结果图
图2成纤维细胞个基因表达量结果图
图3细胞炎症因子TNF-α表达量结果图
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅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作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1Caspase-7单克隆抗体的制备
根据Caspase-7的氨基酸序列，通过分析相应的主要活性结合表位的情况，筛选获得了高活性的抗原表位点如SEQ ID NO：1所示，作为免疫原用于相应的单克隆抗体的制备。
rgtelddgiqadsgpindtdanprykipveadflfaystvpgyyswrspgrgsw(SEQ ID NO：1)。
将序列1所示的氨基酸序列对应的编码序列通过原核表达质粒PGEX-4T-1在大肠杆菌BL21中表达带有谷胱甘肽-S-转移酶(glu-tathione-S-transferase，GST)的融合蛋白GST-Caspase-7。融合蛋白经谷胱甘肽琼脂糖凝胶亲和层析纯化，再经凝血酶进行酶切，收集Caspase-7截短肽。经SDS-PAGE电泳检测和Western blot鉴定，确认为目标蛋白Caspase-7截短肽表达正确，将蛋白浓度调整为1mg/mL备用。
按照50μg重组蛋白/只小鼠的剂量将免疫原注射到Balb/c小鼠体内进行第1次免疫。15d后，按照25μg重组蛋白/只小鼠的剂量进行第2次免疫。第25天后进行第3次免疫(同第2次免疫)。第35天后进行第四次免疫(同第3次免疫)，免疫完成后7d用间接ELISA法检测小鼠血清抗体效价，结果显示小鼠均能产生针对Caspase-7的抗体，且抗体效价达到了4×106，复合融合要求。于融合前3d按每只小鼠120μg重组蛋白的剂量，不加佐剂，直接小鼠尾静脉注射进行加强，准备融合。
采用本领域常用的PEG诱导的半固体基培养法进行细胞融合实验，将免疫效价达融合要求的脾细胞与SP2/0细胞按10：1比例进行混合细胞培养。采用间接ELISA法筛选出对重组蛋白呈阳性的细胞株。有限稀释法对筛选出来的阳性杂交瘤细胞进行单细胞分离培养，间接ELISA法进行检测，选取对重组蛋白检测结果是阳性最强的克隆2D5和4F3，进行连续亚克直至细胞阳性率100％，即可定株。选取健康成年F1小鼠，液体石蜡腹腔注射0.5mL/只。一周后，收集生长状态良好的杂交瘤细胞，当细胞浓度为0.5×106-1×106个/mL，每只小鼠腹腔注射0.5mL。12d左右，采集小鼠腹水。通过正辛酸-饱和硫酸铵法纯化腹水。经Bradford法测定纯化后蛋白浓度。再将腹水浓度调整到1mg/ml，纯化后抗体分别用稀释液稀释103、104、105、106和107倍，当OD值大于阴性对照值2.1倍以上的稀释倍数时的值作为腹水的效价。融合小鼠的血清稀释1000倍作阳性对照，未经免疫小鼠血清稀释1000倍作阴性对照，稀释液作空白对照。结果显示，2D5和4F3腹水效价都是在106，二者纯化的蛋白浓度分别是6.52mg/mL和5.14mg/mL。
实施例2 4F3单克隆抗体鉴定
单克隆抗体亚类的鉴定：将无标签SEQ ID NO：1的重组蛋白稀释至0.25ug/mL，加入96孔酶标板中进行抗原的包被。采用ELISA试剂盒，按照说明书操作测定单克隆抗体亚类。结果显示4F3是IgG2b型。
采用非竞争ELISA发测定亲和常数。将无标签SEQ ID NO：1的重组蛋白稀释至1ug/mL，进行倍比稀释，连续稀释11个梯度，最后酶标仪读取OD450数值，选择4个最合适的包被浓度，每个浓度包被2航，重复实验，结果显示，4F3的亲和常数为2.08×1011，具有较好的结合效果。
特异性鉴定：采用Caspase-3、Caspase-7、GST和BSA作为特异性抗原，通过westernblot检测发现，4F3单抗只与Caspase-7形成特异性条带，与其他均无条带产生，表明4F3具有较好的特异性。
采用本领域常规的单抗轻重链可变区序列鉴定方法，鉴定获得了4F3抗体的轻链可变区序列如SEQ ID NO:2所示，重链可变区序列如SEQ ID NO:3所示。轻链可变区(SEQ IDNO:2)
DIVITQRPALMAASPGEKVTITCYHDPAKASILVLWYQQKSGISPKPWIYIGYWECNGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPNAYGWQRGFGAGTKLELK
重链可变区(SEQ ID NO:3)
EVQLEESATELARPGASVKLSCKASGYIFSNEKYAWIKQRPGQGLEWIGEAYSNLCTWAQYFPICGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGSGDMKRRWGLGTTLAVSS
实施例3脂肪间充质干细胞的分离制备
脂肪颗粒15ml，离心1000r/min，5min。取上层脂肪组织及下层细胞。向脂肪组织中加入终浓度0.1％的胰蛋白酶和胶原酶。放入37℃的水浴摇床中消化30min。离心700g/min，5min。留取下层细胞，将两细胞混匀，加PBS缓冲液重悬细胞，再次离心700g/min，5min。原代培养液重悬细胞后接种至培养瓶，37℃、5％的二氧化碳培养箱中培养。记为P0代。观察细胞生长情况。首次第3天半量换液，以后每3d换液一次，细胞达90％融合时传代培养，用0.05％胰蛋白酶消化，按照1∶4的比例传代。传至第3代做免疫表型鉴定，通过流式细胞仪测定表面标记物CD13、CD29、CD31、CD34、CD45、CD44、CD73、CD90、CD105和HLA-DR的表达情况。结果显示脂肪间充质干细胞的CD90、CD105表达呈阳性，阳性率＞96％；CD34、CD45和HLA-DR表达阴性，阳性率＜2％。
实施例4脂肪间充质干细胞的冷冻实验
冻存保护剂：2％单克隆抗体、20％聚乙烯毗咯烷酮、25％海藻糖、20％丙三醇、5％蔗糖、10％甘露醇和0.5％多聚精氨酸，DMEM/F12培养液中溶解混匀。
对照的冻存保护剂：20％聚乙烯毗咯烷酮、25％海藻糖、20％丙三醇、5％蔗糖、10％甘露醇和0.5％多聚精氨酸，DMEM/F12培养液中溶解混匀。
收集第4代融合度为85％的脂肪间充质干细胞，用胰蛋白酶/EDTA消化液消化细胞，细胞变圆刚脱离培养板面时终止消化，用PBS清洗2遍，1000r/min，离心5min。离心后，取脂肪间充质干细胞与保护剂按体积比2：1混匀。先在4℃下平衡15min，然后快速置入-80度低温冰箱冷冻2h。随后转移到预先冷却至-60℃的冻干机搁板上，维持2h。冻结过程结束后，开启冷阱制冷阀使冷阱温度降至-60℃以下，开启真空泵使冻干室真空度达到10Pa以下，关小搁板制冷阀使搁板温度升至-35℃，维持一次干燥15h。此后，以0.2℃/min的加热速度使搁板温度升高至20℃并维持在此温度直到二次干燥过程结束，这个过程持续约12h。冻干结束后样品呈干饼状，无塌陷，将其严格密封后保存于4℃冰箱内。
冻干细胞再水化及复苏：冻干的干细胞由再水化液(含20％胎牛血清的DMEM/F12培养基37℃下培养)再水化复苏。用台盼蓝拒染法(ScienCeH，USA)计算活细胞比例，细胞恢复率R计算公式如下：R＝N1/N2XE1×100％，其中，N1为冻干再水化后的细胞数，N2为冻干前的细胞数，E1为冻干再水化后细胞的台盼蓝拒染率。以孵化12h的结果定义为复苏率。结果如表1所示。
表1细胞恢复率
| 组别 | 细胞恢复率(％) |
| 对照的冻存保护剂冻干的干细胞 | 59.0±4.8 |
| 冻存保护剂冻干的干细胞 | 94.8±9.1* |
冻于保存后的干细胞经12h复苏，采用对照的冻干保护剂冻干的干细胞的平均复苏率为(59.0±4.8)％，而本发明的冻存保护剂冻干的干细胞的复苏率达到(94.8±9.1)％，具有显著的提升效果，这充分说明本发明的Caspase单克隆抗体能够有效的抑制细胞在冻干过程中因为环境变化而激发的凋亡刺激，实现了细胞存活率提高的效果，而且光学显微镜下观察细胞梭状形态保持较好，*表示P＜0.05，差异显著。
实施例5冻干前后脂肪干细胞对成纤维细胞迁徙实验的影响
成纤维细胞迁徙实验在加细胞之前先将RadiusTM迁移板加预制胶溶液处理20min，然后加洗涤液洗涤，洗涤后HFF-1按2×105/ml，每孔添加500μl，培养24h，待细胞长满后，去除凝胶，吸掉培养基，对照组加入完全培养基，实验组1添加含20％实施例4冻存保护剂冻干的干细胞粉再水化液复苏的DMEM/F12培养基，实验组2添加含20％实施例4对照冻存保护剂冻干的干细胞粉再水化液复苏的DMEM/F12培养基，放入活细胞工作站中培养24h，观察细胞迁移情况并统计细胞迁移面积。细胞迁移实验详见
RadiusTM24-WellCellMigrationAssay试剂盒说明书。Transwell观察细胞迁徙情况：24孔板加入含20％实施例4冻干粉再水化液复苏的DMEM/F12培养基600μl(下层小室)，HFF-1细胞按3×104/200μl无血清DMEM培养基加入transwell的上层小室，置于37℃，5％CO2培养箱中培养24h，取出transwell小室，用棉签将小室内膜上未迁移细胞擦除，然后将朝下一侧的膜反转，置于新的24孔培养板中，加入600μl4％多聚甲醛固定20min，用0.1％结晶紫染色液染色3min。PBS漂洗3次，除去未结合的结晶紫，棉签轻轻擦去小室侧壁的结晶紫染料，适当风干。将每个小室置于新的24孔板中，置于倒置显微镜下(10×)，随机拍摄6个视野，并统计分析其迁移细胞数。结果如图1所示。
从图1的结果可以看出，HFF-1长满后划痕，发现2个实验组成纤维细胞迁移能力均高于对照组，且死细胞少，划痕大部分已经修复。特别是实验组1在采用本发明的冻存保护剂冻干的干细胞具有最好的促进成纤维细胞迁移的效果，其迁移细胞达到了(257±10)的效果，而且实验组1和实验组2相对于对照组PP＜0.01，差异显著，具有统计学意义。
实施例6不同冻干剂处理对成纤维细胞基因表达的影响
采用qRT-PCR检测细胞基因mRNA表达情况。将成纤维细胞按5×104个/cm2接种六孔板，空白对照组每孔加2ml完全培养基。实验组每孔加2ml含15％冻干脂肪间充质干细胞冻干粉的完全培养基，每组设置3个平行，置于37℃，5％CO2培养箱中培养48h。提取细胞总RNA，反转录为cDNA，–20℃保存。qRT-PCR测定细胞外基质相关蛋白I型胶原蛋白(collagenI)、MMP-1、弹性蛋白(elastin)、纤维粘连蛋白(fibronectin)、Caspase-7的mRNA表达。PCR扩增条件：50℃保持2min，95℃预变性10min，95℃变性15s，60℃退火1min，循环40次。
具体的引物序列如下(5'-3')：
β-actin内参：
上游Forward AGAAAATCTGGCACCACACC
下游Reverse AGAGGGTACAGGGATAGCA
Collagen I：
上游Forward CACAGAGGTTTCAGTGGTTTGG
下游Reverse GCACCAGTAGCACCATCATTTC
Elastin：
上游Forward GGGTTGTGTCACCAGAAGCA
下游Reverse CAACCCCGTAAGTAGGAATGC
Fibronectin：
上游Forward AAGATTGGAGAGAAGTGGGACC
下游Reverse GAGCAAATGGCACCGAGATA
MMP-1：
上游Forward TTGAGAAAGCCTTCCAACTCTG
下游Reverse CCGCAACACGATGTAAGTTGTA
Caspase-7：
上游Forward AGTGACAGGTATGGGCGTTCG
下游Reverse GCATCTATCCCCCCTAAAGTGG
结果如图2所示。
从图2结果可以看出，经冻干细胞条件培养基处理后的成纤维细胞纤维粘连蛋白、弹性蛋白、I型胶原蛋白显著升高，MMP-1和caspase-7的mRNA水平表达显著降低。特别是采用了caspase单克隆抗体冻干剂处理的脂肪间充质干细胞具有更高的促进成纤维细胞活性的效果，其中I型胶原蛋白相对提高了3.7倍，而且差异显著具有统计学意义。
实施例7脂肪间充质干细胞冻干粉抗炎效果验证
基于LPS刺激巨噬细胞的抗炎功效评估。以小鼠的Raw264.7巨噬细胞为模型，采用LPS诱导其发生炎症反应，研究人脂肪间充质干细胞冻干粉的抗炎作用。Raw264.7巨噬细胞接种量按1×105个/孔接种于12孔培养板中，每孔加入1ml完全培养基，分五组：control、脂多糖(LPS)、LPS+Dex(地塞米松阳性对照)、LPS+50％干细胞和LPS+50％对照干细胞。加药浓度为LPS(100ng/ml)、Dex(5μg/ml)、50％干细胞(冻干粉用1ml蒸馏水稀释后与完全培养基1:1混匀)，置于37℃，5％CO2培养箱中培养24h；收集细胞上清，用TNF-αELISA试剂盒检测细胞培养上清液中的细胞炎症因子TNF-α水平，使用酶标仪检测450nm吸光度值。结果如图3所示。
结果如图3显示，干细胞冻干粉抗炎效果显著，能明显抑制TNF-α的分泌，抑制效果能达到阳性对照地塞米松水平，差异均具有统计学意义。表明脂肪干细胞冻干粉有很好的抗炎作用。特别是采用本发明单克隆抗体的冻干保存剂处理的脂肪间充质干细胞，其活性较高TNF-α水平只有5089pg/ml，抑制抗炎作用比不加单抗的对照冻干保存剂的效果要显著提高。
从以上说明本发明所属技术领域的当业者将能够理解本发明在不改变其技术思想或必备特征的情况下可以以其他具体形式实施。与此相关，以上所述的实施例在所有方面都是预示性的，必须理解为非限定性的。本发明的范围应解释为，与上述详细说明相比，后述专利请求范围的含义和范围，以及由其等效概念导出的任何变更或变形形式都包括在本发明的范围内。
序列表
<110> 北京戴域生物技术有限公司
<120> 一种含有脂肪间充质干细胞冻干粉的化妆品及抗炎药物
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Claims (7)
1.一种脂肪间充质干细胞冻干粉在制备化妆品中的用途，所述冻干粉能够促进成纤维细胞的活性，延缓细胞衰老；其中，所述脂肪间充质干细胞冻干粉是将脂肪间充质干细胞与冻干保护剂混合后冻干制备得到；所述冻干保护剂组成为2%单克隆抗体、20％聚乙烯毗咯烷酮、25％海藻糖、20％丙三醇、5％蔗糖、10％甘露醇和0.5％多聚精氨酸，在DMEM/F12培养液中溶解混匀获得；所述单克隆抗体是Caspase-7单克隆抗体，其轻链可变区序列如SEQ IDNO:2所示，重链可变区序列如SEQ ID NO:3所示。
2.如权利要求1所述的用途，其特征在于其中化妆品中还添加有抗氧化剂。
3.如权利要求2所述的用途，其特征在于化妆品中还含有稳定剂，乳化剂，颜料及香料。
4.一种脂肪间充质干细胞冻干粉在制备抗炎药物中的用途，所述抗炎是通过降低炎症基因TNF-α的表达来实现；其中，所述脂肪间充质干细胞冻干粉是将脂肪间充质干细胞与冻干保护剂混合后冻干制备得到；所述冻干保护剂组成为2%单克隆抗体、20％聚乙烯毗咯烷酮、25％海藻糖、20％丙三醇、5％蔗糖、10％甘露醇和0.5％多聚精氨酸，在DMEM/F12培养液中溶解混匀获得；所述单克隆抗体是Caspase-7单克隆抗体，其轻链可变区序列如SEQ IDNO:2所示，重链可变区序列如SEQ ID NO:3所示。
5.如权利要求4所述的用途，其特征在于药物的剂型为片剂，散剂，颗粒剂，胶囊剂，悬浮剂，乳剂，糖浆剂，气溶胶或注射液。
6.如权利要求5所述的用途，其特征在于片剂中含有赋形剂，所述赋形剂为淀粉。
7.一种Caspase-7单克隆抗体，其特征在于轻链可变区序列如SEQ ID NO:2所示，重链可变区序列如SEQ ID NO:3所示。
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