WO2021077812A1 - 脐带间充质干细胞因子冻干粉、制备方法及其应用 - Google Patents

脐带间充质干细胞因子冻干粉、制备方法及其应用 Download PDF

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WO2021077812A1
WO2021077812A1 PCT/CN2020/102209 CN2020102209W WO2021077812A1 WO 2021077812 A1 WO2021077812 A1 WO 2021077812A1 CN 2020102209 W CN2020102209 W CN 2020102209W WO 2021077812 A1 WO2021077812 A1 WO 2021077812A1
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freeze
umbilical cord
mesenchymal stem
stem cell
cord mesenchymal
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French (fr)
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乐龙
尹鸿萍
刘永军
杨美家
徐红星
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江苏艾洛特医药研究院有限公司
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Publication of WO2021077812A1 publication Critical patent/WO2021077812A1/zh

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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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

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  • the invention belongs to the field of biotechnology, and particularly relates to a freeze-dried powder of umbilical cord mesenchymal stem cell factor, a preparation method and application thereof.
  • MSC Mesenchymal stem cells secrete many kinds of cytokines during their stable growth.
  • MSC secreted TGF- ⁇ (transforming growth factor- ⁇ ), VEGF (vascular endothelial growth factor), FGF, HGF (liver growth factor), SOD (superoxide dismutase), IL-6 (interleukin-6) , EGF (Epidermal Growth Factor), Collagen (COL I, COL III), Fibronectin (FN), PDGF (Platelet Derived Factor), etc.
  • TGF- ⁇ transforming growth factor- ⁇
  • VEGF vascular endothelial growth factor
  • FGF vascular endothelial growth factor
  • HGF liver growth factor
  • SOD superoxide dismutase
  • IL-6 interleukin-6
  • EGF Epidermal Growth Factor
  • Collagen COL I, COL III
  • Fibronectin FN
  • PDGF Platinum Derived Factor
  • TGF- ⁇ 1 secreted by MSC plays a whitening role by inhibiting the activity of TYR (TYR), reducing the expression of tyrosinase-related proteins, and inhibiting the synthesis of melanin.
  • TYR TYR
  • MSC-CM supernatant
  • MSC-CM in the liquid state has a short storage time at room temperature, which is not conducive to transportation. It is freeze-dried to make freeze-dried powder that can retain the activity of cytokines and is convenient for storage and transportation.
  • the purpose of the present invention is to provide a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, which can quickly prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder that can maintain long-term activity and has good anti-aging effects.
  • the present invention provides the following technical solutions:
  • a method for preparing umbilical cord mesenchymal stem cell factor optionally, includes the following steps:
  • the MSC culture medium used for passage in step (2) The MSC culture medium used for passage in step (2).
  • the centrifugation described in step (4) is: centrifuging the supernatant at 800-2000 rpm/min for 5-15 minutes at 2-6°C, and then taking the supernatant.
  • the dialysis described in step (4) is: placing the supernatant in a 3-50KD dialysis bag, placing the dialysis bag in a PBS solution, performing dialysis at 0-6°C for 24 to 48 hours, and collecting the dialysate.
  • the freeze-drying conditions of step (4) are adding a freeze-drying protective agent and freeze-drying to obtain it.
  • the freeze-dried protective agent is: for every 100 mL of the umbilical cord mesenchymal stem cell factor, 0-15 g of mannitol is added. For example, 0.5-6g mannitol is added.
  • the freeze-drying process is: (1) adding the sample to a 5ml vial, 1.5ml/bottle (2) reducing the temperature of cold hydrazine to -80°C, and the temperature of the box is lowered To -40°C; (3) Place the vial half-press stopper in a vacuum freeze dryer and maintain freezing for 4 hours; (4) Reduce the pressure to 25Pa; (5) Increase the temperature to -20°C at a rate of 0.5°C/min, Maintain for 16.5h; (6) Reduce the pressure to 5Pa; (7) Increase the temperature to 25°C at a rate of 0.5°C/min, maintain for 6.5h, and end the freeze-drying; (8) Stopper the vial and take it out.
  • the present invention also provides a freeze-dried powder of umbilical cord mesenchymal stem cell factor.
  • the specific technical scheme is as follows:
  • the present invention also provides an application of the freeze-dried powder of umbilical cord mesenchymal stem cell factor.
  • the specific technical scheme is as follows:
  • the present invention has the following beneficial effects:
  • the present invention prepares the umbilical cord mesenchymal stem cell factor by collecting the conditioned medium of the umbilical cord mesenchymal stem cells.
  • the use of umbilical cord mesenchymal stem cells avoids social ethical disputes, proliferates faster, and has low immune rejection.
  • the present invention adopts the above-mentioned method to cultivate stem cell factors prepared by culturing umbilical cord mesenchymal stem cells and a suitable freeze-dried powder protective agent to prepare umbilical cord mesenchymal stem cell factor freeze-dried powder, which is conducive to the long-term stable storage of stem cell factors and has anti-aging properties effect.
  • Figure 1 is a graph showing the influence of different concentrations of freeze-dried protective agent on the morphology of freeze-dried samples
  • Figure 2 is a DSC detection chart of a freeze-dried sample (MSC-CM+6% mannitol);
  • Figure 3 is a graph showing the influence of each sample group on the moisture content in the skin of mice
  • Figure 4 is a graph showing the effect of each sample group on the content of hydroxyproline in mouse skin
  • Figure 5 is a graph showing the effect of each sample group on SOD activity in mouse skin
  • Figure 6 is a HE staining image of mouse skin
  • Figure 7 is a graph showing the influence of each sample on the thickness of mouse skin dermis
  • Figure 8 is a MASSON staining image of mouse skin
  • Figure 9 shows the effect of each sample on the collagen content of mouse skin.
  • the present invention provides a method for preparing freeze-dried powder of umbilical cord mesenchymal stem cell factor.
  • the present invention will be further described in detail below with reference to specific examples. It should be understood that the specific embodiments described here are only for explaining the present invention and are not limited to the present invention.
  • Various common reagents used in the examples are all commercially available products
  • This embodiment provides a method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor, and the specific steps are as follows:
  • the supernatant was placed in a 3KD dialysis bag, the dialysis bag was placed in PBS solution, dialysis was performed at 4°C for 36 hours, and the dialysate was collected to obtain umbilical cord mesenchymal stem cell factor.
  • This embodiment provides a method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor. Compared with Example 1, in step (2), for every 100 mL of umbilical cord mesenchymal stem cell factor, 0 g of mannitol is added.
  • This embodiment provides a method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor. Compared with Example 1, in step (2), 0.5 g of mannitol is added for every 100 mL of umbilical cord mesenchymal stem cell factor.
  • This embodiment provides a method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor. Compared with Example 1, in step (2), 1 g of mannitol is added for every 100 mL of umbilical cord mesenchymal stem cell factor.
  • This embodiment provides a method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor. Compared with Example 1, in step (2), for every 100 mL of umbilical cord mesenchymal stem cell factor, 2 g of mannitol is added.
  • Example 2 For every 100 mL of Example 2, the lyophilized powder of umbilical cord mesenchymal stem cell factor was obtained, and 6 g of mannitol was added. Place the crucible on a one-hundred-thousandth scale balance, reset the balance to zero, place a small amount of sample solution in the crucible, accurately weigh the sample, and place the crucible at the detection position of the differential scanning calorimeter. Set the detection conditions, the system temperature is gradually reduced to -50°C, and then the temperature is programmed to 50°C, and the detection is over; the result is shown in Figure 2, the sample has a eutectic point of -17°C.
  • the ICR mice were randomly divided into five groups: blank group, model group, MSC-CM group, freeze-dried preparation group, and positive drug group, with 8 mice in each group. Animals are fed with normal feed for a week to adapt to the environment.
  • the skin and hair on the back of the mouse were shaved with electric clippers, and the hair was removed with depilatory cream.
  • D-Gal PBS solution 1000mg/kg was injected subcutaneously into the back of the mouse neck once a day for 42 days of continuous injection, the volume of injection At about 200 ⁇ L, weigh once every six days, and adjust the dosage according to your body weight. 24 hours after the last intervention, the mice were killed by neck removal, and the skin of the depilated area on the back of the mice was quickly taken.
  • the preparations used in the experiment were all aseptically prepared, and the instruments were all sterilized in advance.
  • Blank group daily injection of normal saline, 100 ⁇ L/10 g/mouse, and then 0.5 mL of normal saline.
  • Model group Daily injection of 10% D-Gal, 100 ⁇ L/10 g/mouse, and 0.5 mL of normal saline on the back skin of mice.
  • MSC-CM group daily injection of 10% D-Gal, 100 ⁇ L/10g/mouse, smear 0.5mL MSC-CM on the back skin of mice.
  • Freeze-dried sample group daily injection of 10% D-Gal, 100 ⁇ L/10 g/mouse, the freeze-dried sample was dissolved in 1.5 mL of single-distilled water, and 0.5 mL of the freeze-dried solution was smeared on the back skin of the mouse.
  • Positive control group daily injection of 10% D-Gal, 100 ⁇ L/10 g/mouse, smear 0.5 mL 10 g/L aminoguanidine hydrochloride (AG) solution on the back skin of mice. Drying method is used to detect skin moisture content, and a kit is used to detect skin SOD activity and hydroxyproline content. HE staining and MASSON staining were performed on skin sections.

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Abstract

提供一种脐带间充质干细胞因子冻干粉、制备方法及其应用，该方法收集脐带间充质干细胞条件培养基，同时采用甘露醇作为冻干粉保护剂，通过冷冻干燥工艺进行冻干，制备得到的脐带间充质干细胞因子冻干粉有利于长期稳定保存，且具有良好的抗衰老功效。

Description

脐带间充质干细胞因子冻干粉、制备方法及其应用 技术领域
本发明属于生物技术领域，特别涉及一种脐带间充质干细胞因子冻干粉、制备方法及其应用。
背景技术
间充质干细胞(MSC)在稳定生长过程中，会分泌许多种细胞因子。MSC分泌的TGF-β(转化生长因子-β)、VEGF(血管内皮生长因子)、FGF、HGF(肝脏生长因子)、SOD(过氧化物歧化酶)、IL-6(白细胞介素-6)、EGF(表皮生长因子)、胶原蛋白(COL I，COL III)、纤维粘连蛋白(FN)、PDGF(血小板衍生因子)等可以有效防止氧化损伤，在皮肤抗老化、抗皱、皮肤美白、伤口愈合等方面有显著功效。MSC分泌的TGF-β1通过抑制TYR(TYR)活性，减少酪氨酸酶相关蛋白的表达，抑制黑色素的合成等过程起到美白作用。通过收集处理培养干细胞之后的上清液(MSC-CM)，可以获得大量细胞因子。
液体状态下的MSC-CM在室温保存时间短，不利于运输。对其进行冻干操作，制成冻干粉可以保留细胞因子的活性，便于储存于运输。
发明内容
本发明的目的在于提供一种脐带间充质干细胞因子冻干粉的制备方法，快速制备可长期保持活性且具有良好抗衰老功效的脐带间充质干细胞因子冻干粉。为实现上述目的，本发明提供了如下技术方案：
一种脐带间充质干细胞因子的制备方法，可选地，包括以下步骤：
(1)取脐带上的华通氏胶，离体培养得脐带间充质干细胞，记为P0代；
(2)传代3～5次，即至P3～P5代后，更换无血清空白培养基；
(3)继续培养24h～96h；
(4)收集细胞上清液，离心，透析，收集截留液，即得脐带间充 质干细胞因子，冻干。
步骤(2)中传代用的MSC培养基。
步骤(4)所述的离心为：将上清液于2～6℃条件下，800～2000rpm/min离心5～15min，取上清。
步骤(4)所述的透析为：将上清液置于3～50KD的透析袋内，透析袋置于PBS溶液中，0～6℃条件下进行透析24～48h，收集透析液。
步骤(4)的冻干条件为加入冻干保护剂，冻干，即得。
所述冻干保护剂为：每100mL所述脐带间充质干细胞因子，加入0～15g甘露醇。例如，加入0.5～6g甘露醇。
在其中一些实施例中，可选地，所述冻干的过程为：(1)样品加入5ml西林瓶中，1.5ml/瓶(2)将冷肼温度降至-80℃，箱体温度降至-40℃；(3)西林瓶半压塞置于真空冷冻干燥机内，维持冷冻4h；(4)将压强降至25Pa；(5)以0.5℃/min的速度升温至-20℃，维持16.5h；(6)将压强降至5Pa；(7)以0.5℃/min的速度升温至25℃，维持6.5h，结束冻干；(8)西林瓶压塞，取出。
本发明还提供了一种脐带间充质干细胞因子冻干粉，具体技术方案如下：
可选地，由如上所述的制备方法制备得到。
本发明还提供了一种脐带间充质干细胞因子冻干粉的应用，具体技术方案如下：
脐带间充质干细胞因子冻干粉在皮肤美白、抗衰老、脱发中的应用。
基于上述技术方案，本发明具有以下有益效果：
本发明通过收集脐带间充质干细胞条件培养基，制备得到脐带间充质干细胞因子。其中，选用脐带间充质干细胞避开了社会伦理争议，增殖较快、免疫排斥低。本发明采用上述方法培养脐带间充质干细胞制备得到的干细胞因子与合适的冻干粉保护剂制备得到脐带间充质干细胞因子冻干粉，其有利于干细胞因子的长期稳定保存，具有抗衰老的功效。
附图说明
图1为不同浓度冻干保护剂对冻干样品形态的影响图；
图2为冻干样品(MSC-CM+6％甘露醇)的DSC检测图；
图3为各样品组对小鼠皮肤中水分含量的影响图；
图4为各样品组对小鼠皮肤中羟脯氨酸含量的影响图；
图5为各样品组对小鼠皮肤中SOD活性的影响图；
图6为小鼠皮肤HE染色图；
图7为各样品对小鼠皮肤真皮厚度的影响图；
图8为小鼠皮肤MASSON染色图；
图9为各样品对小鼠皮肤胶原含量的影响。
具体实施方式
本发明提供了一种脐带间充质干细胞因子冻干粉的制备方法，为使本发明的目的、技术方案和效果更加清楚，以下结合具体实施例对本发明进行进一步的详细说明。应当理解的是，此处所描述的具体实施例仅仅为了解释本发明，并不限定于本发明。实施例中所用到的各种常用试剂，均为市售产品
下面结合具体实施例对本发明进行更全面的说明：
实施例1
本实施例提供了一种脐带间充质干细胞因子冻干粉的制备方法，具体步骤如下：
(1)脐带间充质干细胞因子的制备
取脐带上的华通氏胶，离体培养得脐带间充质干细胞，记为P0代。传代5次，即至P5代后，细胞长至75～85％时，更换无血清空白培养基，继续培养72h，收集细胞上清液。将上清液于4℃条件下，1000rpm/min离心5min，取上清，0.22μm滤膜过滤。将上清液置于3KD的透析袋内，透析袋置于PBS溶液中，4℃条件下进行透析36h，收集透析液，即得脐带间充质干细胞因子。
(2)脐带间充质干细胞因子冻干粉
每100mL脐带间充质干细胞因子，加入6g甘露醇。依以下步骤进行冻干：(1)样品加入5ml西林瓶中，1.5ml/瓶；(2)将冷肼温度降至-80℃， 箱体温度降至-40℃；(3)西林瓶半压塞置于真空冷冻干燥机内，维持冷冻4h；(4)将压强降至25Pa；(5)以0.5℃/min的速度升温至-20℃，维持16.5h；(6)将压强降至5Pa；(7)以0.5℃/min的速度升温至25℃，维持6.5h，结束冻干；(8)西林瓶压塞，取出。
实施例2
本实施例提供了一种脐带间充质干细胞因子冻干粉的制备方法，与实施例1相比，步骤(2)中，每100mL脐带间充质干细胞因子，加入0g甘露醇。
其余步骤与实施例1相同。
实施例3
本实施例提供了一种脐带间充质干细胞因子冻干粉的制备方法，与实施例1相比，步骤(2)中，每100mL脐带间充质干细胞因子，加入0.5g甘露醇。
其余步骤与实施例1相同。
实施例4
本实施例提供了一种脐带间充质干细胞因子冻干粉的制备方法，与实施例1相比，步骤(2)中，每100mL脐带间充质干细胞因子，加入1g甘露醇。
其余步骤与实施例1相同。
实施例5
本实施例提供了一种脐带间充质干细胞因子冻干粉的制备方法，与实施例1相比，步骤(2)中，每100mL脐带间充质干细胞因子，加入2g甘露醇。
其余步骤与实施例1相同。
实施例6不同浓度甘露醇效果评价
对实施例1～5中的脐带间充质干细胞因子冻干粉的外观、色泽、复水溶性按表1标准进行评价。
表1脐带间充质干细胞因子冻干粉的外观、色泽、复水溶性标准
Figure PCTCN2020102209-appb-000001
如图1与表2所示，冻干保护剂甘露醇浓度为0、0.5％、1％时，冻干产品形态不佳，出现分层现象，样品萎缩，不饱满，复水溶时间均在120s以上，样品复水溶后呈无色、澄清、透明状，评分低，分别为2、2、3分。甘露醇浓度为2％与6％时，冻干产品形态良好，冻干粉末饱满、无分层现象，样品复水溶时间较快，约为60s，样品复水溶后呈无色、澄清、透明状，评分高，分别为16、16分。
表2不同甘露醇含量对外观与色泽，复水性、综合评价的结果
Figure PCTCN2020102209-appb-000002
实施例7冻干样品共熔点检测
每100mL实施例2获得脐带间充质干细胞因子冻干粉，加入6g甘露醇。将坩埚置于十万分之一天平上，天平归零，将少量样品溶液置于坩埚内，精确称量样品重量，坩埚置于差式扫描量热仪器的检测处。设置检测条件，体系温度逐渐降温至-50℃，再进行程序升温，升温至50℃，检测结束；结果如图2所示，样品共熔点为-17℃。
实施例8脐带间充质干细胞因子与脐带间充质干细胞因子冻干粉抗衰 老功效研究
将ICR小鼠随机分为五组：空白组、模型组、MSC-CM组、冻干制剂组、阳性药组，每组8只。动物用普通饲料饲养一周以适应环境。
小鼠背部皮肤毛发用电推剪剃掉，用脱毛膏将毛脱净，在小鼠后颈部皮下注射D-Gal的PBS溶液(1000mg/kg)，每日一次，连续注射42d，注射体积在200μL左右，每六日称体重一次，根据体重调整用药量。末次干预24h后，脱颈处死小鼠，迅速取小鼠背部脱毛区域皮肤。实验中所用制剂均采用无菌配制，器械均预先经消毒处理。(1)空白组：每日注射生理盐水，100μL/10g/只，再涂抹0.5mL生理盐水。(2)模型组：每日注射10％D-Gal，100μL/10g/只，小鼠背部皮肤涂抹0.5mL生理盐水。(3)MSC-CM组：每日注射10％D-Gal，100μL/10g/只，小鼠背部皮肤涂抹0.5mL MSC-CM。(4)冻干样品组：每日注射10％D-Gal，100μL/10g/只，冻干样品加1.5mL单蒸水溶解，小鼠背部皮肤涂抹0.5mL冻干溶液。(5)阳性对照组：每日注射10％D-Gal，100μL/10g/只，小鼠背部皮肤涂抹0.5mL 10g/L盐酸氨基胍(AG)溶液。烘干法检测皮肤含水量，利用试剂盒检测皮肤SOD活性与羟脯氨酸含量。对皮肤切片进行HE染色与MASSON染色。
检测结果如图3～9所示。模型组中，小鼠皮肤含水量、SOD活性与HYP含量均极显著下降。给药后，MSC-CM与冻干制剂组对模型组的损伤均有改善作用。模型组真皮厚度极显著减小(p<0.001)，胶原含量显著减小(p<0.05)。相对于模型组，MSC-CM与冻干制剂组极显著增加了小鼠的真皮厚度与胶原含量(p<0.01)。小鼠体内实验显示脐带间充质干细胞因子与脐带间充质干细胞因子冻干粉具有一定的抗衰老作用。

Claims (10)

  1. 一种脐带间充质干细胞因子冻干粉的制备方法，其特征在于，包括以下步骤：
    (1)取脐带上的华通氏胶，离体培养得脐带间充质干细胞，记为P0代；
    (2)传代3～5次，即至P3～P5代后，更换无血清空白培养基；
    (3)继续培养24h～96h；
    (4)收集细胞上清液，离心，透析，收集截留液，冻干，获得冻干粉。
  2. 根据权利要求1所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于步骤(2)中传代用的MSC培养基。
  3. 根据权利要求1所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于，步骤(4)所述的离心条件为：将上清液于2～6℃条件下，800～2000rpm/min离心5～15min，取上清。
  4. 根据权利要求1所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于，步骤(4)所述的透析为：将上清液置于3～50KD的透析袋内，透析袋置于PBS溶液中，0～6℃条件下进行透析24～48h，收集透析液。
  5. 根据权利要求1所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于步骤(4)的冻干条件为加入冻干保护剂，冻干，即得。
  6. 根据权利要求5所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于，步骤(4)所述冻干保护剂为：每100mL所述脐带间充质干细胞因子，加入0～15g甘露醇。
  7. 根据权利要求6所述的脐带间充质干细胞因子冻干粉的制备方法， 其特征在于，步骤(4)所述冻干保护剂为：每100mL所述脐带间充质干细胞因子，加入0.5～6g甘露醇。
  8. 根据权利要求5所述的脐带间充质干细胞因子冻干粉的制备方法，其特征在于，步骤(4)所述冻干的过程为：
    A、样品加入1～10ml西林瓶中，1～5ml/瓶
    B、将冷肼温度降至-80～-60℃，箱体温度降至-60～-40℃；
    C、西林瓶半压塞置于真空冷冻干燥机内，维持冷冻1～6h；
    D、将压强降至10～50Pa；
    E、以0.5～5℃/min的速度升温至-25～-17℃，维持10-～20h；
    F、将压强降至0～30Pa；
    G、以0.5～5℃/min的速度升温至0～25℃，维持1～10h，结束冻干；
    H、西林瓶压塞，取出。
  9. 脐带间充质干细胞因子冻干粉在制备皮肤美白、抗衰老、脱发药物中的应用，其特征在于，所述脐带间充质干细胞因子冻干粉按权利要求1-8任意一种方法制备获得。
  10. 一种脐带间充质干细胞因子冻干粉，其特征在于，按权利要求1-8任意一种方法制备获得。
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