CN109295001A - A kind of compound excretion body of umbilical cord mesenchymal stem cells and preparation method thereof - Google Patents
A kind of compound excretion body of umbilical cord mesenchymal stem cells and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to stem cells technology fields, more particularly, to compound excretion body of a kind of umbilical cord mesenchymal stem cells and preparation method thereof.The compound excretion body is the umbilical cord mesenchymal stem cells excretion body that inside contains mir-181b, and preparation method includes the following steps: the culture of (1) human umbilical cord mesenchymal stem cells；(2) human umbilical cord mesenchymal stem cells excretion body is extracted, is purified；(3) mir-181b is synthesized；(4) electroporation mir-181b contains the umbilical cord mesenchymal stem cells excretion body purified in step (2) and obtains compound excretion body sample；(5) compound excretion body sample carries out film reparation；(6) extraction and purifying of compound excretion body.This method, which will have, promotes the mir-181b of human vascular endothelial proliferation, migration and angiogenesis function contain in forming compound excretion body in human umbilical cord mesenchymal stem cells excretion body, keep the treatment specific aim for the compound excretion body to be formed stronger, this method is simple and easy, contains success rate height.
Description
Technical field
The present invention relates to stem cells technology fields, more particularly, to a kind of compound excretion body of umbilical cord mesenchymal stem cells
And preparation method thereof.
Background technique
Umbilical cord mesenchymal stem cells are located away from the postnatal discarded tissue-umbilical cord of newborn.Because its is from a wealth of sources, easily obtain
Take, without moral check dispute, immunogenicity it is low, to advantages such as donor fanout free regions, become between most Transformation Application prospect
Mesenchymal stem cells.Umbilical cord mesenchymal stem cells can be used for treating diabetes, central nervous system injury, rheumatic arthritis, erythema
The diseases such as lupus.The multiple biological function that mescenchymal stem cell has clinically has obtained extensive concern, therapy apparatus
It is made for the hot spot of stem cell field correlative study in recent years.Excretion body is considered as that mescenchymal stem cell plays paracrine action
Carrier, by the way that molecule to be wrapped in film, excretion body can protect enzyme or RNA from degradation, and pass through endocytosis
To promote cellular uptake.In addition, excretion body is a kind of particle of nanosized, it is easy in blood and other body fluid transfers
It moves.
A large number of studies show that, the excretion body of source for mesenchymal stem cells can albumen, RNA ingredient by its content at present
Intercellular transfer plays the tissue repair similar with mescenchymal stem cell, immunosupress, adjusts the effects of immune function.However
Compared with mescenchymal stem cell, mescenchymal stem cell excretion body is also had the advantage that
1, mescenchymal stem cell excretion body does not have the activity of cell, and there is no the risks for forming tumour.Further, since
Embrane-associated protein content is lower, and the immunogenicity of excretion body is lower than mescenchymal stem cell.
2, mescenchymal stem cell excretion body is important to maintaining cell homeostasis and reacting to environmental stimuli to play
Effect.When tissue microenvironment stable state due to disease or injury by destroying when, this effect just seems more important.
3, mescenchymal stem cell excretion body is rich in bioactive molecule, such as protein and RNA.It is present in excretion body and is permitted
Polyprotein belongs to enzyme, has catalytic activity, and these activity are related with locating environment, such as concentration of substrate and pH.
Thus, if perhaps the enzyme in excretion body can alleviate drug dose problem insufficient or excessive for treating.
In addition to this, the excretion volume property of source for mesenchymal stem cells is more stable, saves convenient transportation, therefore mesenchyma is dry
Cell excretion body is expected to become a kind of new skin repair therapeutic strategy.However existing umbilical cord mesenchymal stem cells excretion body is being repaired
Ineffective when multiple dermal layer of the skin, the specific aim in disease treatment field is not strong.
Summary of the invention
For the technical problems in the prior art, the invention proposes a kind of compound excretions of umbilical cord mesenchymal stem cells
Body and preparation method thereof, this method will have the mir- for promoting human vascular endothelial proliferation, migration and angiogenesis function
181b contains the medical needle for making the compound excretion body to be formed in forming compound excretion body in human umbilical cord mesenchymal stem cells excretion body
Stronger to property, this method is simple and easy, contains success rate height.
To achieve the above object, the present invention is achieved by the following technical solutions:
The first purpose of this invention is to propose a kind of compound excretion body of umbilical cord mesenchymal stem cells, the compound excretion
Body is the umbilical cord mesenchymal stem cells excretion body that inside contains mir-181b.
Second object of the present invention is to propose a kind of preparation method of compound excretion body of umbilical cord mesenchymal stem cells, be somebody's turn to do
Preparation method is used to prepare above-mentioned compound excretion body, includes the following steps:
(1) culture of human umbilical cord mesenchymal stem cells；
(2) human umbilical cord mesenchymal stem cells excretion body is extracted, is purified；
(3) mir-181b is synthesized；
(4) it is compound to contain the umbilical cord mesenchymal stem cells excretion body acquisition purified in step (1) by electroporation mir-181b
Excretion body sample；
(5) compound excretion body sample carries out film reparation；
(6) extraction, purifying and the preservation of compound excretion body.
Further, the culture of step (1) human umbilical cord mesenchymal stem cells, concrete operations are: taking well-grown
Human umbilical cord mesenchymal stem cells carry out cell count with DMEM/F12 culture solution, reach originally culture after 106/ml, by 5 ×
106cells/ml is inoculated into T75 Tissue Culture Flask, is placed in 37 DEG C of incubators that CO2 volumetric concentration is 5% and cultivates, cell reaches
It when 80% fusion, is digested at 37 DEG C with 0.25% pancreatin of mass concentration, is passed on and expanded with 1 × 104cells/ml, take P2~P5
The human umbilical cord mesenchymal stem cells in generation；The human umbilical cord mesenchymal stem cells in P2~P5 generation are placed in 37 that O2 volume by volume concentration is 21%
It in DEG C incubator, is cultivated with DMEM/F12 culture solution, until cell is up to the hungry culture for when 70% fusion, using serum-free instead
Liquid cultivates 12h, and the hungry culture solution of serum-free is changed to the culture of the DMEM/F12 containing 10ng/ml epithelical cell growth factor
Liquid, culture to cell are in logarithmic growth phase, collect cell culture supernatant.
Further, the method for the step (4) are as follows: respectively into the phosphate buffered saline solution of 1ml trehalose containing 50mM
The umbilical cord mesenchymal stem cells excretion body of step (2) purifying and the mir-181b of step (3) synthesis is added, is uniformly mixed to form mixed
Reaction solution is closed, mixed reaction solution is added in perforation ware and carries out electroporation；The mixed reaction solution includes umbilical cord mesenchymal stem cells
100 μ g~300ug of excretion body, 5~20 μ l of mir-181b.
Further, in the step (4) electroporation perforation condition are as follows: voltage 100V, 125 μ F of capacitor, electric discharge when
Between 1ms, discharge time be 1 time.
Further, the method for the step (6) are as follows: compound excretion body sample is super through 100,000~140,000 g (RCF) centrifugal force
Twice, 70~90min, abandons supernatant to the greatest extent, sediment is while loading the compound excretion body of mir-181b, compound every time for speed centrifugation
- 80 DEG C of excretion body saves backup.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention contains mir-181b in human umbilical cord mesenchymal stem cells excretion body, sufficiently combines between people's umbilical cord
The proliferation for the promotion human vascular endothelial that the tissue repair and mir-181 that mesenchymal stem cells excretion body has the function of have is moved
It moves and angiogenesis function, is worn by electricity and contain mir-181 in human umbilical cord mesenchymal stem cells excretion body, the compound excretion
The treatment specific aim of body is stronger.This method is simple and easy, and it is simple and easy to contain the high this method of success rate, and success rate is high.
(2) umbilical cord mesenchymal stem cells materials are convenient, and amoral dispute of ethic, retrievable cell quantity is more, energetic,
Convenient for expanding and passing on, at the same again without distribution type, rejection the problems such as, be excellently suitable for clinical research and application.
(3) compound excretion body prepared by the present invention provides on the basis of playing the effect for the treatment of for clinical application research
Reliable experimental study data and theoretical basis.
Detailed description of the invention
Fig. 1 is the electromicroscopic photograph of the umbilical cord mesenchymal stem cells excretion body before 1 middle punch of embodiment.
Fig. 2 is the electromicroscopic photograph of the umbilical cord mesenchymal stem cells excretion body after 1 middle punch of embodiment.
Fig. 3 is that Western Blot CD81, CD63 identify compound excretion body protein in embodiment 3.
Specific embodiment
It shows that example illustrates certain embodiments of the present invention, and should not be construed as limiting model of the invention
It encloses.Present disclosure can be improved from material, method and reaction condition simultaneously, all these improvement should all
It falls within spirit and scope of the invention.The reagents and materials used in the present invention are commercially available or can be by literature method system
It is standby.In the following examples, the experimental methods for specific conditions are not specified, usually (writes [beauty] J. Sha's nurse Brooker, Huang according to reference book
Training hall is translated, " Molecular Cloning:A Laboratory guide ", Science Press) described in condition, or according to the normal condition proposed by manufacturer.
Embodiment 1: the preparation of compound excretion body
One, the preparation of human umbilical cord mesenchymal stem cells excretion body
Prepare DMEM/F12 culture solution, culture solution 20%FBS containing mass concentration, 100U/ml penicillin and 100 μ g/ml chains
Mycin.
75% ethyl alcohol of umbilical cord is impregnated into 4min, PBS soaking and washing 3 times, is cut into 1cm after rejecting blood vessel2Fritter is immersed in
II Collagenase Type of mass concentration 0.1%, 37 DEG C digest 50 minutes, and 0.25% pancreatin is added, and 37 DEG C digest 10 minutes, and blood is added
It is clear to terminate pancreatin digestion, aforementioned cells tissue fluid is filtered, is centrifuged, is discarded supernatant liquor, remove confluent monolayer cells.
Add DMEM/F12 culture solution to carry out cell count lower confluent monolayer cells, reaches 106Originally culture after/ml, by 5 ×
106Cells/ml is inoculated into T75 Tissue Culture Flask, and DMEM/F12 nutrient solution volume is 20ml in each Tissue Culture Flask, is placed in
CO2It is cultivated in 37 DEG C of incubators of volume by volume concentration 5%, liquid removal non-adherent cell is changed after 3d, later every 4d half, which is measured, changes liquid 1
Secondary, cell is digested at 37 DEG C, with 1 × 10 up to when 80% fusion with 0.25% pancreatin of mass concentration when 10d4Cells/ml is passed
Generation amplification, takes the human umbilical cord mesenchymal stem cells in P2~P5 generation.
The human umbilical cord mesenchymal stem cells in P2 generation are placed in O2In 37 DEG C of incubators that volume by volume concentration is 21%, DMEM/ is used
F12 culture solution is cultivated, and until cell is up to the hungry culture solution of serum-free when 70% fusion, is used instead, culture 12h will be without blood
Clear hungry culture solution is changed to the DMEM/F12 culture solution containing 10ng/ml epithelical cell growth factor, and culture to cell is in
Logarithmic growth phase collects cell culture supernatant 300g~10000g gradient centrifugation 60-120min, the excretion weight sunk to the bottom
It is suspended from the PBS solution of the trehalose containing 50mM, -80 DEG C save backup.
Two, mir-181b is synthesized
Search sequence information
> hsa-miR-181b-5p MIMAT0000257 AACAUUCAUUGCUGUCGGUGGGU,
>hsa-miR-181b-3p MIMAT0022692 CUCACUGAACAAUGAAUGCAA。
Synthesis mir-181b and PCR enrichment are synthesized by Shanghai JiMa pharmacy Technology Co., Ltd.
Three, electroporation obtains compound excretion body sample
In following ratio, filled being separately added between the umbilical cord of purifying in the phosphate buffered saline solution of 1ml trehalose containing 50mM
The mir-181b of matter stem cell excretion body and synthesis, is uniformly mixed to form mixed reaction solution, and mixed reaction solution is added in perforation ware
Using Invitrogen company Neon electroporation apparatus, 1 subpulse completion electroporation is contained under the conditions of 100V, 125 μ F of capacitor, 1ms；
The mixed reaction solution includes 100 μ g~300ug of umbilical cord mesenchymal stem cells excretion body, 5~20 μ l of mir-181b.After perforation
Sample immediately, light absorption value of the test sample at 260nm on ultraviolet-visible spectrophotometer, and with puncherless sample pair
Than judging whether perforation succeeds.The electromicroscopic photograph of umbilical cord mesenchymal stem cells excretion body before perforation and after perforation is respectively such as Fig. 1
With shown in Fig. 2, it can be seen from the figure that method of the invention is applied to, preparing for compound excretion body is simple and easy, and success rate is high.
Four, compound excretion body sample carries out film reparation
Sample is immediately placed in 1 hour of incubation in cell incubator after perforation, to promote excretion body film reparation.
Five, the extraction and purifying of compound excretion body
Twice through 100,000~140,000 g (RCF) centrifugal force ultracentrifugations, 70~90min, is abandoned to the greatest extent compound excretion body sample every time
Supernatant, sediment are while loading the compound excretion body of mir-181b, and compound -80 DEG C of excretion body save backup.
Embodiment 2:PBS is resuspended compound excretion body and surveys excretion body content with BCA method (with protein refractometer)
1, reagent A include: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjust pH value to 11.25.
2, reagent B:4% copper sulphate.
3, BCA working solution: reagent A 100ml+ reagent B 2ml mixing.
4, protein standard liquid: with crystallization bovine serum albumin(BSA) according to its purity normal saline at 1.5mg/ml's
Protein standard liquid.(purity can be determined through Kjeldahl nitrogen determination protein content)
5, it sample to be tested: is diluted with the sample of biuret measuring method.
6, it is 0.5ug/ul that PBS, which is resuspended compound excretion body and surveys excretion body content with BCA method,.
Embodiment 3:western-blot detects the expression of excretion body film surface characteristic protein
1, it collects protein sample to be cracked using cell pyrolysis liquid sample, the albumen for then measuring each protein sample is dense
Degree.
2, electrophoresis, transferring film.
3, room temperature in 5%BSA confining liquid is immersed in slowly to sway two hours.It is incubated for 4 DEG C of primary antibody overnight.
4, suitable secondary antibody is selected according to primary antibody source, dilutes (1:1000~1:10000) by corresponding proportion, room temperature jog
Two hours.
5, it after TBS washing, is developed the color using ECL luminescence reagent.As a result excretion body Specific marker CD81 and CD63 are clear
It can be seen that (as shown in Figure 3).
Embodiment 4: cytology research of the compound excretion body to fibroblast proliferation and differentiation ability of regulation and control
With its influence to fibroblastic ability of cell proliferation of fluidic cell cycle detection
1, fibroblast is laid in six orifice plates, when cell density is 70%, TGF β cell factor is added in a hole
While compound excretion body (100ug/ml) (experimental group), the hole addition TGF β cell factor of the preparation of embodiment 1 are added simultaneously
TGF β cell factor (positive controls) is only added in addition NC excretion body, a hole, and a hole is that any intervention is not added.
2, fluidic cell cycle experimental is carried out with 0.25% trypsin digestion cell after 48h.
3, result is visible:
It is 20.85% that ratio of the fibroblast of the compound excretion body of embodiment 1 in the G2 phase, which is added, and NC excretion body is added
Ratio 20.69% of the fibroblast in the G2 phase, and the positive controls G2 phase is 14.84%, and blank control group is
14.08%.Show that the fibroblast proliferation of the compound excretion body of umbilical cord mesenchymal stem cells containing embodiment 1 is most fast.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (6)
1. a kind of compound excretion body of umbilical cord mesenchymal stem cells, which is characterized in that the compound excretion body is that inside has contained
The umbilical cord mesenchymal stem cells excretion body of mir-181b.
2. a kind of preparation method of the compound excretion body of umbilical cord mesenchymal stem cells, which is characterized in that the preparation method is used to prepare
Compound excretion body described in claim 1, includes the following steps:
(1) culture of human umbilical cord mesenchymal stem cells；
(2) human umbilical cord mesenchymal stem cells excretion body is extracted, is purified；
(3) mir-181b is synthesized；
(4) electroporation mir-181b contains the umbilical cord mesenchymal stem cells excretion body purified in step (2), obtains compound excretion
Body sample；
(5) compound excretion body sample carries out film reparation；
(6) extraction, purifying and the preservation of compound excretion body.
3. a kind of preparation method of the compound excretion body of umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that
The culture of step (1) human umbilical cord mesenchymal stem cells, concrete operations are: taking well-grown human umbilical cord mesenchymal dry thin
Born of the same parents carry out cell count with DMEM/F12 culture solution, reach 106Originally culture after/ml, by 5 × 106Cells/ml is inoculated into T75
Tissue Culture Flask is placed in CO2It is cultivated in 37 DEG C of incubators that volumetric concentration is 5%, cell uses mass concentration up to when 80% fusion
0.25% pancreatin digests at 37 DEG C, with 1 × 104Cells/ml passage amplification takes the human umbilical cord mesenchymal in P2~P5 generation dry thin
Born of the same parents；The human umbilical cord mesenchymal stem cells in P2~P5 generation are placed in O2In 37 DEG C of incubators that volume by volume concentration is 21%, DMEM/ is used
F12 culture solution is cultivated, and until cell is up to the hungry culture solution of serum-free when 70% fusion, is used instead, culture 12h will be without blood
Clear hungry culture solution is changed to the DMEM/F12 culture solution containing 10ng/ml epithelical cell growth factor, and culture to cell is in
Logarithmic growth phase collects cell culture supernatant.
4. a kind of preparation method of the compound excretion body of umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that
The method of the step (4) are as follows: step (2) purifying is separately added into the phosphate buffered saline solution of 1ml trehalose containing 50mM
The mir-181b of umbilical cord mesenchymal stem cells excretion body and step (3) synthesis, is uniformly mixed to form mixed reaction solution, hybrid reaction
Liquid is added in perforation ware and carries out electroporation；The mixed reaction solution includes 100 μ of μ g~300 of umbilical cord mesenchymal stem cells excretion body
G, 5~20 μ l of mir-181b.
5. a kind of preparation method of the compound excretion body of umbilical cord mesenchymal stem cells according to claim 4, which is characterized in that
The perforation condition of electroporation in the step (4) are as follows: voltage 100V, 125 μ F of capacitor, discharge time 1ms, discharge time are 1 time.
6. a kind of preparation method of the compound excretion body of umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that
The method of the step (6) are as follows: compound excretion body sample through 100,000~140,000 g (RCF) centrifugal force ultracentrifugations twice, every time 70
~90min abandons supernatant to the greatest extent, and sediment is while loading the compound excretion body of mir-181b, and compound -80 DEG C of excretion body preservations are standby
With.
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Cited By (5)
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| CN110037979A (en) * | 2019-04-25 | 2019-07-23 | 广州优理氏生物科技有限公司 | Excretion body freeze-dried powder of mescenchymal stem cell containing sheep and preparation method thereof |
| CN110193027A (en) * | 2019-05-08 | 2019-09-03 | 广州市番禺区中心医院(广州市番禺区人民医院、广州市番禺区心血管疾病研究所) | A kind of preparation method and new opplication of stem cell excretion body |
| CN111281889A (en) * | 2020-04-16 | 2020-06-16 | 博生众康(厦门)医药生物技术股份有限公司 | Application of human umbilical cord mesenchymal stem cell exosome |
| WO2022105156A1 (en) * | 2020-11-17 | 2022-05-27 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method for and application of mesenchymal stem cell exosome |
| CN114540296A (en) * | 2022-03-22 | 2022-05-27 | 和携科技有限公司 | Preparation method of composite exosome and application of composite exosome in directionally enhancing angiogenesis capacity |
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