WO2019144485A1 - 一种皮米级细胞提取物的制备方法 - Google Patents

一种皮米级细胞提取物的制备方法 Download PDF

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WO2019144485A1
WO2019144485A1 PCT/CN2018/079703 CN2018079703W WO2019144485A1 WO 2019144485 A1 WO2019144485 A1 WO 2019144485A1 CN 2018079703 W CN2018079703 W CN 2018079703W WO 2019144485 A1 WO2019144485 A1 WO 2019144485A1
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preparation
cell extract
cell
ultrasonic
intermittent
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马颖
杨阳
阮雯莉
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广州资生生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

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  • the invention belongs to the technical field of stem cells, and in particular relates to a method for preparing a picograde cell extract.
  • Cell extracts generally refer to supernatants and/or cell lysates of cell culture fluids, and cell culture supernatants contain a large number of proteins, polypeptides and cytokines with various biological activities, which are involved in the maintenance of cell structure.
  • Information exchange and tissue repair and regeneration have good anti-photoaging, anti-oxidation, anti-wrinkle, whitening skin, wound healing, cell repair and other effects. Therefore, with the increasing application value of cell extracts, how to obtain a large number of cell extracts with stable and controllable quality has become a problem that people are committed to.
  • the traditional methods for preparing cell extracts are as follows: First, a small number of cells are extracted from a very small amount of tissues from different organs, and then these cells are expanded and cultured, and when the cells are expanded to a certain amount, The extraction of the cell extract is performed. The entire extraction cycle is often more than 30 days, which is time consuming and laborious, and the amount of cell extract obtained is also limited. Moreover, the cell extract obtained by most of the current extraction methods can only achieve nanometer level, which is not conducive to human body absorption, and the effect is unstable.
  • the object of the present invention is to provide a method for preparing a picograde cell extract.
  • a method for preparing a picograde cell extract comprising the steps of:
  • the cell suspension is subjected to intermittent ultrasonication at a temperature of 4 ° C to 10 ° C for 10 to 15 minutes, and centrifuged to obtain a supernatant;
  • the picograde cell extract has a molecular weight of less than 100K.
  • the cell suspension is refrigerated at 4 ° C for 1 h such that the temperature of the intermittent ultrasound is maintained between 4 ° C and 10 ° C.
  • the intermittent ultrasonic wave has an ultrasonic frequency of 15 to 25 kHz and an ultrasonic time of 10 to 15 minutes.
  • the intermittent ultrasound is: first ultrasonicing for 30 to 50 s, then stopping the ultrasound for 50 s, and then continuing the ultrasound, thus repeating 10 cycles.
  • the temperature of the oscillation in the step b) is 3 to 5 ° C, and the time is 1 to 2 h.
  • the intermittent ultrasound is performed in an ultrasonic cell pulverizer
  • the ultrasonic vibrating rod of the ultrasonic cell pulverizer is immersed in the tissue suspension, and a bottom thereof has a preset distance from a bottom of the tissue suspension;
  • the preset distance is 3 to 4 cm.
  • the centrifugation in step c) is: centrifugation at 5500-6500 rpm for 30-40 min;
  • the temperature of the freezing fixation in step a) was -85 ° C for 2 h.
  • the animal tissue is fresh tissue derived from the brain, heart, liver, lung, gastric mucosa, intestinal mucosa, ovary, muscle or bone.
  • the present invention also provides the use of the above preparation method or the picograde cell extract obtained by the preparation method for the preparation of a preparation for promoting skin repair and/or anti-aging.
  • the invention adopts a combination of cryosectioning, shaking and intermittent ultrasound to make the cells break more completely, and can extract the active components in the cells to the maximum extent and enrich the active components in the cells;
  • the cell extract of the present invention does not need to add any chemicals during the extraction process, and the obtained pico-grade cell extract has no chemical residue;
  • the preparation method of the present invention is carried out at a temperature of 4 ° C to 10 ° C to avoid the deactivation of the active material and to ensure good biological activity of the cell extract;
  • the method of the invention has the advantages of process optimization, simple and controllable operation, is convenient for repeated production, and can be completed within 6 hours to ensure good biological activity of the cell extract.
  • Example 1 is a chromatogram of a photometric detection analysis of an amino acid in a brain cell extract of Example 1 at 570 nm;
  • Example 2 is a chromatogram of the photometric detection analysis of amino acids in the brain cell extract of Example 1 at 440 nm;
  • Fig. 3 is a graph showing the results of electrophoresis analysis of the brain cell extract of Example 1.
  • the brain is sent to a GMP-certified sterile laboratory for distilled water cleaning, 100% pure alcohol disinfection, ozone sterilization and the like.
  • the brain was cut into small pieces and placed in a tissue microtome box, and then frozen in a -85 ° C refrigerator for 2 hours to obtain a fixed brain tissue; then, the brain tissue was cut into 2 ⁇ m sheets.
  • the sheet was placed in distilled water, and the cell suspension was prepared by shaking at 200 rpm for 1.5 h in a 4 ° C constant temperature air bath, and then sterilized by ozone to obtain a brain cell suspension.
  • the brain cell suspension was placed in a refrigerator at 4 ° C for 1 h, and then intermittently sonicated in a ultrasonic cell pulverizer at a frequency of 20 khz and a power of 500 w: 400 ml of the brain cell suspension was placed in a 500 ml glass container, and the ultrasonic vibrating rod was placed.
  • the ultrasonic vibrating rod Immerse in the cell suspension and keep the bottom of the ultrasonic vibrating rod at a distance of about 3cm-4cm from the bottom of the container (the distance is too large, the ultrasonic vibrating rod can not penetrate deep into the liquid and break the cells more effectively; the distance is too small, the ultrasonic vibrating rod is easy Touch the bottom surface of the container to generate impurities), turn on the ultrasonic wave for 30-50 seconds, and repeat for 50 seconds and then repeat the ten cycles.
  • dry ice is placed around the bottom of the vessel and the contact turbid liquid to ensure that the temperature of the turbid liquid is not higher than 10 ° C, and the thermostat is monitored. If the temperature of the turbid liquid reaches 10 °C during the degradation process, it is necessary to stop the degradation immediately and transfer it to a refrigerator at 4 °C for 1 hour, and then continue to perform ultrasonic degradation according to the set cycle.
  • the column uses a cation exchange resin chromatography column with a particle size of 4 ⁇ m (125 ⁇ 4 mm ID), post-column derivatization with ninhydrin at 125 ° C and 570 nm (primary amino acid) and 440 nm (secondary amino acid). Simultaneous photometric detection was performed for a sample size of 20 ⁇ L, and the data was recorded using the chromatography software ChromStar 6.0.
  • Fig. 1 is a chromatogram of photometric detection analysis at 570 nm
  • Fig. 2 is a chromatogram of photometric detection analysis at 440 nm.
  • the cell extract prepared by the method of the present invention contains up to 18 kinds of high-concentration amino acids, and these high-concentration amino acids are the cells of the present invention.
  • Active factors of the substance including a variety of biologically active proteins, peptides and cytokines, which are involved in the maintenance of cell structure, information exchange, tissue repair and regeneration, have good anti-photoaging, anti-oxidation, anti-wrinkle, whitening skin, wounds healing, cell repair and other effects.
  • the molecular weight distribution of each active ingredient in the brain cell extract obtained by the method of the present invention was 35-75 KD, demonstrating that the cell extract was picmi size.
  • Example 2 Comparative test of wound healing in rat skin
  • Table 2 is the statistical data of the wound diameter of each group of rats.
  • the data of Table 2 shows that the wound healing rate of the experimental group is significantly faster than that of the blank group, and the healing time is significantly shorter than that of the blank group, indicating that the cell extract obtained by the method of the present invention can be Significantly promote the repair of damaged skin.
  • the cell extract obtained by the preparation method provided by the present invention is a pico-grade cell extract which is more easily absorbed by human skin; and the extract contains various active ingredients. , can promote the repair of skin damaged cells. Therefore, the preparation method provided by the present invention can be widely applied to the field of promoting skin damage repair and anti-aging technology.
  • the acute dermal toxicity test can determine whether the test substance can be toxic through skin absorption and short-term effects. It can provide a basis for the toxicity classification and labeling of cosmetic raw materials and the determination of subchronic and other toxicological test doses.

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Abstract

一种皮米级细胞提取物的制备方法，包括：a) 将动物组织冷冻固定，切片，得到厚度为 2～100μm 的薄片；b) 将所述薄片和水混合，振荡，得到细胞悬液；c) 将所述细胞悬液在 4℃～10℃的温度下进行间歇式超声 10～15min，离心，得到上清液；d) 将所述上清液过滤，收集滤液，得到所述皮米级细胞提取物。通过该方法得到的细胞提取物为皮米级细胞提取物，更易于被人体皮肤吸收；而且，该细胞提取物中含有多种活性成分，可促进皮肤损伤细胞的修复，可广泛应用于促进皮肤损伤修复、抗衰老技术领域。

Description

一种皮米级细胞提取物的制备方法
本申请要求于2018年01月23日提交中国专利局、申请号为2018100782492、发明名称为“一种皮米级细胞提取物的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于干细胞技术领域，具体涉及一种皮米级细胞提取物的制备方法。
背景技术
细胞提取物一般是指细胞培养液的上清液和/或细胞裂解液，其细胞培养液上清液含有大量的具有多种生物活性的蛋白质、多肽及细胞因子，它们参与细胞结构的维持运动信息交流以及组织修复与再生，具有较好的抗光老化、抗氧化、抗皱、美白肌肤、伤口愈合、细胞修复等功效。因此，随着细胞提取物的应用价值越来越受到人们的关注，如何获得大量的质量稳定可控的细胞提取物成为人们致力研究的问题。
目前，传统的细胞提取物的制备方法主要为：首先从不同器官来源的极少量组织中提取出很少的细胞，然后对这些细胞进行扩增培养，当细胞达扩增至一定数量后才可以进行细胞提取物的提取。整个提取周期常在30天以上，耗时费力，得到的细胞提取物数量也比较有限。而且，目前大多数提取方法得到的细胞提取物仅能做到纳米级别，不利于人体吸收，效果不稳定。
发明内容
为了解决现有细胞提取物提取周期长、数量稀少和分子量大等技术问题，本发明的目的在于提供一种皮米级细胞提取物的制备方法。
本发明的具体技术方案如下：
一种皮米级细胞提取物的制备方法，包括以下步骤：
a)将动物组织冷冻固定，切成厚度为2～100μm的薄片；
b)将所述薄片和水混合，振荡，得到细胞悬液；
c)将所述细胞悬液在4℃～10℃的温度下进行间歇式超声10～15min，离心，得到上清液；
d)将所述上清液过滤，收集滤液，得到所述皮米级细胞提取物。
优选的，所述皮米级细胞提取物的分子量在100K以下。
优选的，在步骤b)和步骤c)之间还包括：将所述细胞悬液在4℃下冷藏1h，使得所述间歇超声的温度维持在4℃～10℃。
优选的，所述间歇式超声的超声波频率为15～25kHz，超声时间为10～15min。
优选的，所述间歇式超声为：首先超声30～50s，然后停止超声50s，接着继续超声，如此重复10个循环。
优选的，步骤b)所述振荡的温度为3～5℃，时间为1～2h。
优选的，所述间歇式超声在超声波细胞粉碎仪中进行；
所述超声波细胞粉碎仪的超声波振动棒浸入所述组织悬液中，其底部与所述组织悬液的底部存在预置距离；
所述预置距离为3～4cm。
优选的，步骤c)所述离心为：在5500～6500rpm下离心30～40min；
步骤a)所述冷冻固定的温度为-85℃，时间为2h。
优选的，所述动物组织为来源于大脑、心脏、肝脏、肺、胃粘膜、肠粘膜、卵巢、肌肉或骨骼的新鲜组织。
本发明还提供了上述制备方法或由所述制备方法得到的皮米级细胞提取物在制备促进皮肤修复和/或抗衰老制剂中的应用。
与现有技术相比，本发明技术方案具有以下优点：
1)本发明采用冷冻切片、震荡和间歇式超声相结合的方法，使得细胞破碎得更加完全，能最大限度的将细胞内的活性成分提取出来，富集细胞内的活性成分；
2)本发明的细胞提取物在提取过程中无需添加任何的化学药品，得到的皮米级细胞提取物无化学成分残留；
3)本发明的制备方法在4℃～10℃的温度下进行，可避免活性物质失 活，保证细胞提取物良好的生物活性；
4)本发明方法工艺优化，操作简单可控，利于重复生产，而且可在6小时内完成，保证细胞提取物良好的生物活性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为实施例1中大脑细胞提取物中的氨基酸在570nm下进行光度检测分析的色谱分析图；
图2为实施例1中大脑细胞提取物中的氨基酸在440nm下进行光度检测分析的色谱分析图；
图3为图1为实施例1大脑细胞提取物进行电泳分析的检测结果。
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
1、大脑细胞提取物的提取过程
1)将哺乳动物幼崽被屠宰后一小时内完成(运输在24小时内进行)；用电锯割掉颅骨拱型部分，获取大脑和小脑时需要仔细分离左右眼球的视神经，然后分离脊椎割掉脑干，摘取大脑并将大脑放入生理盐水中4℃保存备用。
2)在距离活体取样30min以内，将大脑送入经过GMP认证的无菌实验室内进行蒸馏水清洗、100％纯酒精消毒、臭氧灭菌等处理。
3)将大脑切成小块后放入组织切片机盒后，置于-85℃冰箱中冷冻2h，得到固定的大脑组织；然后，将大脑组织切成2μm的薄片。
4)将薄片放入蒸馏水中，在4℃恒温气浴锅中以200rpm振荡1.5h制成细胞悬液，再经臭氧灭菌，得到脑细胞悬液。
5)将脑细胞悬液置于4℃冰箱冷藏1h，然后在超声波细胞粉碎仪中，以频率20khz和功率500w进行间歇式超声：将400ml脑细胞悬液放入500ml玻璃容器，将超声波振动棒浸入细胞悬液中，并将超声振动棒的底部保持距离容器底部约3cm-4cm距离(距离太大，超声波振动棒不能深入液体内部进而更有效地打碎细胞；距离太小，超声波振动棒容易碰触容器底面进而产生杂质)，开启超声波30-50秒，停止50秒后再重复十个循环。降解过程中容器底部和接触浊液的周围均放置干冰以确保浊液温度不高于10℃，同时配置恒温器监控。如降解过程中浊液温度达到10℃，需立即停止降解并转移到4℃冰箱内冷藏1个小时，之后再继续按照设定的循环完成超声解降解。
6)将超声波降解后的细胞悬液于离心机中6krpm分离30分钟，收集上清液，经100K道尔顿过滤器过滤，再收集滤液，得到分子量为100K以下的细胞提取物。
2、对大脑细胞提取物的分析测定
1)测定大脑细胞提取物中的氨基酸
取100μL样品溶液，转移至水解管中，干燥，加入1200μL的6当量的HCl混合，在真空(＜20mbar)下熔化并在110℃下水解24h；待水解完全后，将样品转移至真空离心机中干燥，加入缓冲液溶液干燥物；接着，采用氨基酸分析仪LC3000测定大脑细胞提取物中的氨基酸含量。其中，色谱柱采用阳离子交换树脂色谱分离柱，粒径为4μm(125×4mm ID)，在125℃下用茚三酮进行柱后衍生以及在570nm(一级氨基酸)和440nm(二级氨基酸)同时进行光度检测进行分析，样本量为20μL，数据记录借助色谱软件ChromStar 6.0。图1为在570nm下进行光度检测分析的色谱分 析图，图2为在440nm下进行光度检测分析的色谱分析图。
测定表1、图1和图2为测定结果，如结果所示，采用本发明方法制备得到的细胞提取物中含有多达18种高浓度的氨基酸，而这些高浓度氨基酸正是本发明细胞提取物的活性因子，包括多种生物活性的蛋白质、多肽及细胞因子，它们参与细胞结构的维持运动信息交流以及组织修复与再生，具有较好的抗光老化、抗氧化、抗皱、美白肌肤、伤口愈合、细胞修复等功效。
表1
Figure PCTCN2018079703-appb-000001
Figure PCTCN2018079703-appb-000002
2)分析检测大脑细胞提取物中各活性成分的分子量大小
配制12％Tricine-SDS-PAGE聚丙烯酰胺凝胶；然后，取按标示量配制好的大脑细胞提取物溶液原液20μl，加入2×上样缓冲液，混合，并于80℃下加热40min，得到处理好的样品；接着，往电泳仪中加入处理好的样品，每孔加入20μl，之后于8mA电流稳流进行电泳约6h；最后，根据图3的电泳结果，分析大脑细胞提取物中各活性成分的分子量大小。
如图3结果所示，采用本发明方法得到的大脑细胞提取物中各活性成分的分子量分布为35-75KD，证明细胞提取物为皮米级大小。
实施例2大鼠皮损伤口愈合对比试验
1、选取7周龄的SD大鼠10只，随机分为空白组和实验组，每组各半，制备大鼠皮损模型，皮损面积为直径3厘米的伤口。
2、造模成功后，在空白组大鼠的皮损部位涂抹生理盐水，在实验组大鼠的皮损部位涂抹实施例1的细胞提取物，然后在给药后的第1、3、5、7、9、11、13天对伤口直径进行测量，并统计。
3、对统计数据进行分析，分析各组伤口愈合的速度和时间差异。
表2为各组大鼠伤口直径的统计数据，表2数据显示，实验组大鼠伤口的愈合速度明显快于空白组，愈合时间明显短于空白组，说明由本发明方法得到的细胞提取物可明显促进损伤皮肤的修复。和现有常规方法得到的细胞提取物相比，由本发明所提供的制备方法得到的细胞提取物为皮米级细胞提取物，更易于被人体皮肤吸收；而且，提取物中含有多种活性成分， 可促进皮肤损伤细胞的修复。因此，本发明所提供的制备方法可广泛应用于促进皮肤损伤修复、抗衰老技术领域。
表2
Figure PCTCN2018079703-appb-000003
实施例3大鼠急性经皮毒性试验
急性皮肤毒性试验可确定受试物能否经皮肤吸收和短期作用所产生的毒性反应，可为化妆品原料毒性分级和标签标识以及确定亚慢性实验和其他毒理学试验剂量提供依据。
在本实施例中，选取10只SD大鼠(雌雄各半，体重为135.±10.6g)，经皮肤涂抹1800ug/kg体重计量的高剂量的细胞提取物溶液，观察14天，当未引起动物死亡，则不再进行多个计量的急性经皮毒性试验。观察14天后，未发觉试验动物有任何不良反应，也没有出现任何动物死亡。说明本发明细胞提取物的制备方法安全性高，可操作性强。
以上对本发明所提供的一种皮米级细胞提取物的制备方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种皮米级细胞提取物的制备方法，其特征在于，包括以下步骤：
    a)将动物组织冷冻固定，切成厚度为2～100μm的薄片；
    b)将所述薄片和水混合，振荡，得到细胞悬液；
    c)将所述细胞悬液在4℃～10℃的温度下进行间歇式超声10～15min，离心，得到上清液；
    d)将所述上清液过滤，收集滤液，得到所述皮米级细胞提取物。
  2. 根据权利要求1所述的制备方法，其特征在于，所述皮米级细胞提取物的分子量在100K以下。
  3. 根据权利要求1所述的制备方法，其特征在于，在步骤b)和步骤c)之间还包括：将所述细胞悬液在4℃下冷藏1h，使得所述间歇超声的温度维持在4℃～10℃。
  4. 根据权利要求1所述的制备方法，其特征在于，所述间歇式超声的超声波频率为15～25kHz，超声时间为10～15min。
  5. 根据权利要求1所述的制备方法，其特征在于，所述间歇式超声为：首先超声30～50s，然后停止超声50s，接着继续超声，如此重复10个循环。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤b)所述振荡的温度为3～5℃，时间为1～2h。
  7. 根据权利要求1所述的制备方法，其特征在于，所述间歇式超声在超声波细胞粉碎仪中进行；
    所述超声波细胞粉碎仪的超声波振动棒浸入所述组织悬液中，其底部与所述组织悬液的底部存在预置距离；
    所述预置距离为3～4cm。
  8. 根据权利要求1所述的制备方法，其特征在于，步骤c)所述离心为：在5500～6500rpm下离心30～40min。
  9. 根据权利要求1所述的制备方法，其特征在于，所述动物组织为来源于大脑、心脏、肝脏、肺、胃粘膜、肠粘膜、卵巢、肌肉或骨骼的新鲜组织。
  10. 如权利要求1至9任意一项所述制备方法或由所述制备方法得到的皮米级细胞提取物在制备促进皮肤修复和/或抗衰老制剂中的应用。
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EP1382673A1 (en) * 2001-04-23 2004-01-21 Nitta Gelatin Inc. Method of culturing collected biopsy cells
CN101411721A (zh) * 2008-12-01 2009-04-22 郭凌云 鲜鹿茸活性细胞提取液冻干粉的制备方法
CN103776667A (zh) * 2012-10-19 2014-05-07 中国科学院大连化学物理研究所 一种可保持蛋白质活性的软骨组织分级处理方法
CN106389491A (zh) * 2016-09-30 2017-02-15 广州赛莱拉干细胞科技股份有限公司 一种组合物及其在制备改善黄褐斑的产品中的应用
CN107550934A (zh) * 2017-09-11 2018-01-09 上海亚睿生物科技有限公司 一种利用干细胞提取物和中药提取物混合制备的皮肤修复剂及其应用

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EP1382673A1 (en) * 2001-04-23 2004-01-21 Nitta Gelatin Inc. Method of culturing collected biopsy cells
CN101411721A (zh) * 2008-12-01 2009-04-22 郭凌云 鲜鹿茸活性细胞提取液冻干粉的制备方法
CN103776667A (zh) * 2012-10-19 2014-05-07 中国科学院大连化学物理研究所 一种可保持蛋白质活性的软骨组织分级处理方法
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