CN110317784A - A kind of preparation method of umbilical cord mesenchymal stem cells excretion body and stem cell beauty, it is anti-ageing, repair and disease treatment in application - Google Patents
A kind of preparation method of umbilical cord mesenchymal stem cells excretion body and stem cell beauty, it is anti-ageing, repair and disease treatment in application Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses a kind of preparation method of umbilical cord mesenchymal stem cells excretion body and in stem cell beauty, it is anti-ageing, it repairs and the application in disease treatment, preparation method includes: to take complete medium culture of the hUC-MSCs containing soybean lecithin, collect cell, washing, it is resuspended again with serum-free α-MEM culture medium, supernatant is collected after Nature enemy, 10min and 29500 × g centrifugation 20min successively, which is centrifuged, with 300 × g collects supernatant again, use 0.22 μm of membrane filtration, last 120000 × g centrifugation 1.5h collects precipitating, after precipitating is resuspended with appropriate PBS, -80 DEG C of preservations.Preparation method of the present invention is under the premise of maintaining excretion body purity (using the content of excretion body specific transmembrane PROTEIN C D9 and CD81 as index) to be basically unchanged, the secretory volume that umbilical cord mesenchymal stem cells excretion body can be significantly improved can be used for stem cell beauty, anti-ageing, reparation or disease treatment.
Description
Technical field
The invention belongs to stem cell fields, are related to the preparation and application of stem cell excretion body, and in particular between a kind of umbilical cord
The preparation method of mesenchymal stem cells excretion body and the application in stem cell beauty, anti-ageing, reparation and disease treatment.
Background technique
Excretion body is found in the granulophilocyte of rat by Harding etc. first in nineteen eighty-three, then in Pan in 1985
Equal electricity consumptions sem observation sheep granulophilocyte is into mature erythrocyte growth course it is again seen that this folliculus secreted by cell
This vesicles formal definition is " exosomes ", i.e. excretion body by bubble, Johnstone in 1987 etc..Originally excretion body is recognized
For some unwanted cells components for being only cell clearance, until Valadi in 2007 et al. has found for the first time in excretion body
The inhereditary materials such as microRNA and mRNA, so that people have further insight to the composition of excretion body, while people also anticipate
Knowing excretion body may be a kind of new mechanism communicated between mediated cell, therefore people are to the ingredient, Forming Mechanism, work of excretion body
With Mechanism and FunctionsDNA carried out deeper into exploration.Bibliography: source for mesenchymal stem cells excretion body promotes injury tissue to repair
Multiple and regenerated application and progress, Chinese Tissue Engineering Study, 1 phases of volume 22 in 2018.
Stem cell has broad application prospects in terms of tissue repair and regeneration, has been widely used in translational medicine neck
Domain, but the mechanism that stem cell plays a role at present is not also fully aware of.In recent years multiple studies have shown that, stem cell is played
Tissue repair and regeneration function be largely to be realized by its paracrine action, rather than it is in damage location
Proliferation and differentiation.Meanwhile there is certain potential risk always in stem cell transplantation, as transplanted cells cause blood vessel embolism, dry thin
The variation of born of the same parents' inhereditary material promotes the problems such as metastases and tumorigenesis teratogenesis.And excretion body is one of important paracrine
The factor, by more and more experiments have shown that playing a key effect in terms of tissue damage reparation and regeneration.Bibliography: mesenchyma
The promotion injury tissue reparation of source of human stem cell excretion body and regenerated application and progress, Chinese Tissue Engineering Study, 2018 22
Rolled up for 1 phase.
The discovery such as Ling Hao, mescenchymal stem cell source property excretion body are the homogeney membrane vesicles of mescenchymal stem cell secretion, are contained
There are the bioactive substances such as a large amount of and miscellaneous protein, cell factor and Microrna, in myocardial infarction, hypertension, the heart
There is high application prospect in terms of the prevention and treatment of force failure and cardiomyopathy etc..Bibliography: mescenchymal stem cell source property excretion body exists
Progress in cardiovascular disease, international cardiovascular disease magazine, 05 phase in 2018.
The discovery such as Chen Chen, mescenchymal stem cell source property excretion body have low immunogenicity and long circulating half-life period and can take
Band small-molecule substance pass through blood-brain barrier the features such as, nerve growth and neuron differentiation etc. can be promoted, facilitate improve by
The nervous function of damage and enhance nervus vasculairs, becomes the emerging therapeutic means for treating neurodegenerative disease.With reference to
Document: mescenchymal stem cell source property excretion body treats neurodegenerative disease: the problems in application and future prospect, China's tissue
Engineering research.
The discovery such as Xing Fei, mescenchymal stem cell excretion body regulates and controls its cellular activity and then acting on recipient cell, right
The Various Tissues such as bone, cartilage, skin, nerve have good repair function.Bibliography: mescenchymal stem cell excretion body is in bone
The application of section, West China medicine, 12 phases in 2018.
It is repaired as it can be seen that the tissue repair of stem cell excretion body makes it possible to palingenesis for beauty, anti-ageing and skin
Multiple, the pharmacological activity of stem cell excretion body makes it possible to for disease treatment.
But the secretory volume of the MSCs excretion body such as umbilical cord mesenchymal stem cells is less, it is necessary to improve its secretory volume.
Summary of the invention
The present invention is directed to overcome the shortcomings of that the secretory volume of umbilical cord mesenchymal stem cells excretion body in the prior art is less, provide
A kind of preparation method of umbilical cord mesenchymal stem cells excretion body, to improve its secretory volume, for beauty, anti-ageing, reparation and disease
Treatment.
The object of the invention is achieved through the following technical solutions:
A kind of preparation method of umbilical cord mesenchymal stem cells excretion body, comprising: take the good hUC-MSCs of growth conditions, use
Complete medium culture is collected cell, washing, then is resuspended with serum-free α-MEM culture medium, collects supernatant after Nature enemy, according to
It is secondary to collect supernatant again with 300 × g centrifugation 10min and 29500 × g centrifugation 20min, using 0.22 μm of membrane filtration, finally use
120000 × g of ultracentrifuge be centrifuged 1.5h collect precipitating, will precipitating with appropriate PBS resuspension after, -80 DEG C save backup it is spare,
The complete medium contains soybean lecithin.
Preferably, the complete medium is the α-MEM culture medium containing 10% fetal calf serum.
Preferably, soybean lecithin concentration is 100 μ g/mL.
Preferably, with the complete medium culture 48h containing soybean lecithin.
Preferably, being resuspended again with serum-free α-MEM culture medium to cell concentration after cell washing is 5 × 105/mL。
Preferably, supernatant is collected after Nature enemy 72h.
The purposes that soybean lecithin is used to that umbilical cord mesenchymal stem cells excretion body to be promoted to secrete.
The umbilical cord mesenchymal stem cells excretion body that above-mentioned preparation method is prepared.
Application of the above-mentioned umbilical cord mesenchymal stem cells excretion body in stem cell beauty, anti-ageing, reparation or disease treatment.
The utility model has the advantages that
Preparation method provided by the invention is in maintenance excretion body purity (with excretion body specific transmembrane PROTEIN C D9 and CD81
Content be index) be basically unchanged under the premise of, the secretory volume of umbilical cord mesenchymal stem cells excretion body can be significantly improved, can be used
In stem cell beauty, anti-ageing, reparation or disease treatment.
Detailed description of the invention
Fig. 1 is the flow cytometer detection result of hUC-MSCs；
Fig. 2 is that the Electronic Speculum of hUC-MSCs excretion body observes figure, and A is the hUC-MSCs excretion body of soybean lecithin culture group,
B for no soybean lecithin culture group hUC-MSCs excretion body；
Fig. 3 is the Western blot testing result of hUC-MSCs excretion body, and A is the hUC- of soybean lecithin culture group
MSCs excretion body, B for no soybean lecithin culture group hUC-MSCs excretion body, C be soybean lecithin culture group 120000 ×
G is centrifuged the supernatant of 1.5h, and D is centrifuged the supernatant of 1.5h for 120000 × g of no soybean lecithin culture group.
Specific embodiment
Essentiality content of the invention is introduced below with reference to specific embodiment, but protection of the present invention cannot be limited with this
Range.
One, experimental material
Umbilical cord tissue is derived from healthy Cesarean esction pregnant woman, 4 DEG C of preservations, for 24 hours in carry out the separation of umbilical cord mesenchymal stem cells with
Culture.Puerpera's signed informed consent form states that taken umbilical cord can only be used to scientific research.
Cell incubator is Thermo Fisher Scientific product.
Flow cytometer is U.S. BD Biosciences product.
Fetal calf serum is U.S. Gibco Products, and α-MEM culture medium is Hyclone Products, and pancreatin is purchased from green cloud
It.
Streaming identification use fluorescent marker streaming antibody CD14-PE, CD31-FITC, CD34-PE, CD44-FIT,
CD45-FITC, CD90-FITC, CD105-PE, HLA-DR-PE are Beckman product.
BCA kit is purchased from the green skies.
Low temperature refrigerator is Thermo Fisher Scientific product.
Two, experimental method
1, hUC-MSCs is separately cultured, identifies
According to this field conventional practices, umbilical cord is cut into 3-5cm segment in aseptic operating platform, it is abundant with physiological saline
Washing removes umbilical vein and endarterial remaining blood, rejects vein, artery.Umbilical cord jelly of Wharton is shredded into 0.5-1.0mm3
Tissue block, it is evenly laid out in the sterile petri dish of diameter 100mm, be placed in 37 DEG C, 5%CO2, in saturated humidity incubator,
After about 1~2h when tissue block slightly drying can be securely affixed on culture dish wall, it is slowly added to the α-MEM containing 10% fetal calf serum
Culture medium (complete medium) makes culture solution impregnate tissue block, continues to cultivate, change liquid 1 time every 3~4d, long to cell clone
When being merged to 80%-90%, with 0.05% pancreatin had digestive transfer culture into new sterile petri dish.
Streaming identification method is as follows: taking the good 5th generation umbilical cord mesenchymal stem cells of growth conditions, washed, filtering is consolidated
It is fixed, fluorescence antibody label, wash again, flow cytometer loading, detection surface marker CD14, CD31, CD34, CD44,
The expression rate of CD45, CD90, CD105 and HLA-DR.
2, the extraction of hUC-MSCs excretion body, secretion level measurement, Electronic Speculum observation and average particle size determination
Excretion body extracts method particularly includes: the good 5th generation hUC-MSCs of growth conditions is taken, with containing 100 μ g/mL or not
Complete medium culture 48h containing soybean lecithin collects cell, washing, then is resuspended with serum-free α-MEM culture medium to thin
Born of the same parents' concentration is 5 × 105/ mL (for convenient for comparing, two groups respectively take 20mL cell suspension to carry out subsequent processing and the extraction of excretion body, is put down
Three parts of row operation), after Nature enemy 72h, supernatant is collected, successively with 300 × g centrifugation 10min and 29500 × g centrifugation 20min
Supernatant is collected again, using 0.22 μm of membrane filtration, finally collects excretion body with 120000 × g of ultracentrifuge centrifugation 1.5h,
After the excretion body of extraction is resuspended with 500 μ L PBS, -80 DEG C are saved backup.
Protein quantification is carried out to the excretion body isolated and purified with BCA kit, calculates yield, evaluation excretion body secretes water
It is flat.
Appropriate excretion body suspension is taken, no soybean lecithin culture group dilutes 100 times, soybean lecithin culture group dilution 500
Times, it under the microscope and takes a picture after sample preparation in electric according to a conventional method.Using Nanosight-NS500 according on instrument operation instructions
Machine carries out nanoparticle specificity analysis, measures the average grain diameter of excretion body.
3, the purity testing of hUC-MSCs excretion body
The memebrane protein of excretion body is rich in transmembrane protein family CD9 and CD81, therefore we are measured using Western blot
The content of CD9 and CD81 evaluates the purity of hUC-MSCs excretion body, method particularly includes: use BCA kit measurement PBS suspension
Protein concentration, take the PBS suspension containing 30 μ g albumen, nitrogen is blown, be added 5 × Loading buffer, 50 μ L, boiling water bath
5min takes 40 μ L that well is added and is transferred to cellulose nitrate with 10% polyacrylamide gel electrophoresis protein isolate after cooling
Film closes 1h with confining liquid room temperature, the anti-human CD9 and CD81 of primary antibody mouse is added, is incubated overnight in 4 DEG C, washes film 15min × 3 time,
The secondary antibody that HRP label is added is incubated at room temperature 1h, film 15min × 3 time is washed, after ECL enhances luminescence reagent colour developing, in gel imaging system
System exposes, and observes result after developing fixing.Internal reference is β-actin.
4, data processing
Experimental result data is indicated with mean value ± standard deviation, is analyzed using SPSS17.0 software.Between different groups
Comparison using t examine, it is statistically significant that P < 0.05 represents difference.
Three, experimental result
1, the streaming qualification result of hUC-MSCs
Streaming qualification result as shown in Figure 1, CD44, CD90, CD105 positive expression in hUC-MSCs, CD14, CD31,
The characteristics of CD34, CD45 and HLA-DR feminine gender are expressed, and human umbilical cord mesenchymal stem cells are met.
2, the secretion level of hUC-MSCs excretion body
BCA method carries out protein quantification, no soybean lecithin culture group every 1 × 10 to the excretion body isolated and purified7A hUC-
MSCs collects to obtain excretion body to be about (645 ± 58) μ g, soybean lecithin culture group every 1 × 107A hUC-MSCs collects to obtain
Excretion body be about (3593 ± 286) μ g, the secretion level of soybean lecithin culture group hUC-MSCs excretion body improve 5 times it
It is more.
3, the Electronic Speculum observation and average particle size determination result of hUC-MSCs excretion body
Electronic Speculum observes result as shown in Fig. 2, wherein A is the hUC-MSCs excretion body of soybean lecithin culture group, and B is without big
The hUC-MSCs excretion body of beans lecithin culture group, two groups of hUC-MSCs excretion bodies are rounded or oval, and diameter about 30~
100nm has complete capsule structure, includes materials of low density.It is measured through Nanosight-NS500, soybean lecithin culture group
HUC-MSCs excretion body average grain diameter be (75.9 ± 26.5) nm, the hUC-MSCs excretion body of no soybean lecithin culture group is flat
Equal partial size is (76.4 ± 25.2) nm, and average grain diameter is without significant difference.
4, the Western blot measurement result of hUC-MSCs excretion body
Western blot measurement result is as shown in figure 3, wherein A is the hUC-MSCs excretion of soybean lecithin culture group
Body, B are the hUC-MSCs excretion body of no soybean lecithin culture group, and CD9 and CD81 is in sun in two groups of hUC-MSCs excretion bodies
Property expression, expression illustrates that two groups of hUC-MSCs excretion body purity are almost the same without significant difference.
Above-described embodiment shows preparation method of the present invention in maintenance excretion body purity (with excretion body specific transmembrane albumen
The content of CD9 and CD81 be index) be basically unchanged under the premise of, umbilical cord mesenchymal stem cells excretion body can be significantly improved
Secretory volume can be used for stem cell beauty, anti-ageing, reparation or disease treatment.
Above-described embodiment is used to introduce essentiality content of the invention, but cannot limit the scope of the present invention with this.
Claims (9)
1. a kind of preparation method of umbilical cord mesenchymal stem cells excretion body, comprising: take the good hUC-MSCs of growth conditions, be finished
Full culture medium culture is collected cell, washing, then is resuspended with serum-free α-MEM culture medium, collects supernatant after Nature enemy, successively
10min and 29500 × g centrifugation 20min is centrifuged with 300 × g and collects supernatant again, using 0.22 μm of membrane filtration, finally with super
Fast 120000 × g of centrifuge centrifugation 1.5h collects precipitating, will after precipitating appropriate PBS resuspension, -80 DEG C save backup it is spare,
Be characterized in that: the complete medium contains soybean lecithin.
2. preparation method according to claim 1, it is characterised in that: the complete medium is to contain 10% fetal calf serum
α-MEM culture medium.
3. preparation method according to claim 2, it is characterised in that: soybean lecithin concentration is 100 μ g/mL.
4. preparation method according to claim 1, it is characterised in that: with the complete medium culture containing soybean lecithin
48h。
5. preparation method according to claim 1, it is characterised in that: use serum-free α-MEM culture medium again after cell washing
Being resuspended to cell concentration is 5 × 105/mL.
6. preparation method according to claim 1, it is characterised in that: collect supernatant after Nature enemy 72h.
7. the purposes that soybean lecithin is used to that umbilical cord mesenchymal stem cells excretion body to be promoted to secrete.
8. the umbilical cord mesenchymal stem cells excretion body that any preparation method of claim 1-6 is prepared.
9. umbilical cord mesenchymal stem cells excretion body according to any one of claims 8 is in stem cell beauty, anti-ageing, reparation or disease treatment
Application.
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| CN112472621A (en) * | 2020-12-09 | 2021-03-12 | 北京欣颂生物科技有限公司 | Preparation method of stem cell exosome and medicine or cosmetic prepared by stem cell exosome |
| CN112695015A (en) * | 2020-12-18 | 2021-04-23 | 浙江卫未生物医药科技有限公司 | Preparation method of immortalized umbilical cord mesenchymal stem cells and preparation method and application of exosomes thereof |
| CN114832015A (en) * | 2022-04-22 | 2022-08-02 | 西安初源赛尔生物科技有限责任公司 | Facial repair injection based on PRP and stem cell exosome and preparation method thereof |
| CN115261310A (en) * | 2021-04-23 | 2022-11-01 | 广州明宇泰生物科技有限公司 | Starvation treatment method suitable for preparation of mesenchymal stem cell exosomes |
| CN117844747A (en) * | 2024-03-07 | 2024-04-09 | 天津百恩生物科技有限公司 | Conditional medium for promoting secretion of mesenchymal stem cell exosome and application thereof |
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| CN112472621A (en) * | 2020-12-09 | 2021-03-12 | 北京欣颂生物科技有限公司 | Preparation method of stem cell exosome and medicine or cosmetic prepared by stem cell exosome |
| CN112695015A (en) * | 2020-12-18 | 2021-04-23 | 浙江卫未生物医药科技有限公司 | Preparation method of immortalized umbilical cord mesenchymal stem cells and preparation method and application of exosomes thereof |
| CN115261310A (en) * | 2021-04-23 | 2022-11-01 | 广州明宇泰生物科技有限公司 | Starvation treatment method suitable for preparation of mesenchymal stem cell exosomes |
| CN115261310B (en) * | 2021-04-23 | 2024-05-31 | 广州明宇泰生物科技有限公司 | Hunger treatment method suitable for mesenchymal stem cell exosome preparation |
| CN114832015A (en) * | 2022-04-22 | 2022-08-02 | 西安初源赛尔生物科技有限责任公司 | Facial repair injection based on PRP and stem cell exosome and preparation method thereof |
| CN117844747A (en) * | 2024-03-07 | 2024-04-09 | 天津百恩生物科技有限公司 | Conditional medium for promoting secretion of mesenchymal stem cell exosome and application thereof |
| CN117844747B (en) * | 2024-03-07 | 2024-05-07 | 天津百恩生物科技有限公司 | Conditional medium for promoting secretion of mesenchymal stem cell exosome and application thereof |
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