CN108853145A - Application of the excretion body of mescenchymal stem cell secretion in biological agent - Google Patents
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Abstract
The invention discloses application of the excretion body of mescenchymal stem cell secretion in biological agent, the biological agent includes the biological agent for treating skin injury or the biological agent for promoting skin wound healing.The present invention significantly raises the expression of surface of a wound keratinocyte growth factor albumen, to achieve the purpose that promote skin wound healing, also solves the problems, such as that clinic skin prognosis damages.
Description
Technical field
The invention belongs to field of biotechnology, more particularly to the application of the excretion body of mescenchymal stem cell secretion.
Background technique
Defect of skin is surgery common diseases, commonplace in each clinical hospitals, and defect skin need to be by inflammatory cell
Chemotactic, the formation of granulation tissue, the proliferation of local fibroblasts, several processes such as migration of keratinocyte, only some
Step, which smoothly completes, could make surface of a wound skin completely be restored, new the study found that keratinocyte growth factor
（KGF）The expression of albumen can promote the healing of damage skin.Clinical common method is mainly dermatoplasty at present, but it is not
It can avoid that patient is allowed to form new damage again.
Mescenchymal stem cell (MSC) is the multipotential cell with self-renewal capacity, a variety of diseases of MSC transplantation treatment
Research report it is more and more, later researcher confirms the trophic factors-excretion body for having MSC to secrete, with endocrine and paracrine
Mode promote tissue repair.
A kind of effective biological active agents that the excretion body of mescenchymal stem cell secretion is secreted as mescenchymal stem cell
More and more attention has been paid to.Excretion body is easier to merge with the cell membrane of adjacent cells, selectively by biological active agents
It is delivered to recipient cell, information transmitting is carried out in different iuntercellulars, adjusts intercellular signal transduction, play various biological function
Energy.It is more bubble endosomes of eukaryocyte with after plasma membrane fusion, and nanometer scale in extracellular environment is discharged into a manner of exocytosis
Film property vesicles, in containing a large amount of different types of protein, lipid components and nucleic acid, wherein being with MicroRNA
It is main.Subsequent research confirms the package action due to excretion body again, so that MicroRNA therein can not only fight ribonucleic acid
Enzyme（RNase）Effect, and different acid or alkali environments, anoxic, high temperature, multigelation etc. dislike mal-condition under be still able to maintain compared with
High stability, current study show that the protein of the excretion body carrying of mescenchymal stem cell secretion, messenger RNA（mRNA）
And miRNA（miRNA）It is closely related with the physiological maladies state of body and cell, release to the excretion outside cell
Body participates in a series of bioprocess, as immune response, apoptosis, angiogenesis, inflammatory reaction, condensation process, antigen are offered and are controlled
Damaged organ etc. is treated, therefore, also receives extensive concern using the research of the excretion body treatment disease of mesenchymal cell secretion, such as
CN201510915717 treats hepatitis C using the excretion body of mescenchymal stem cell secretion, and CN105861430A utilizes optimization
The excretion body of umbilical cord mesenchymal stem cells secretion treats pyemia, but at present about the excretion body treatment of mescenchymal stem cell secretion
Skin injury promotes damage skin healing, has not been reported.
How to make defect skin healing as early as possible, and avoid damage to the skin of complete health, it has also become clinician
Problem in the urgent need to address.
Summary of the invention
The present invention is directed to the prior art when treating skin injury, the defect for the damage for easily causing patient new, between providing
Application of the mesenchymal stem cells in the biological agent for the treatment of skin injury or the biological reagent of promotion skin wound healing.
The purpose of the present invention can be realized by following technical measures：
Application of the excretion body of mescenchymal stem cell secretion in biological agent, the difference is that, the biological agent packet
It includes the biological agent for the treatment of skin injury or promotes the biological agent of skin wound healing.
In above-mentioned technical proposal, the excretion body following methods preparation of the mescenchymal stem cell secretion：
Step（1）Mescenchymal stem cell is subjected to secondary culture, is screened out from it between cell state is good, proliferative capacity is strong and fills
Matter stem cell；
Step（2）By step（1）The mescenchymal stem cell of middle selection is cultivated using empty culture medium, after culture, removes impurity,
Isolate culture supernatant；
Step（3）By step（2）The culture supernatant is incubated for after mixing with excretion body extracting solution, and first is obtained after incubation and is incubated
Change liquid, centrifugal concentrating is carried out to the first incubation fluid, obtains the concentrate of the body containing excretion；
Step（4）By step（3）The progress of excretion body extracting solution is added after sterile PBS dissolution is added in the concentrate of the body containing excretion
It is incubated for, the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom precipitation is collected by centrifugation, obtains mescenchymal stem cell excretion
Body.
In above-mentioned technical proposal, the mescenchymal stem cell be umbilical cord mesenchymal stem cells, mesenchymal stem cell or
One of peripheral blood mescenchymal stem cell.
In above-mentioned technical proposal, the excretion preparation step of the mescenchymal stem cell secretion（2）It is complete by following steps
At：
Step 2A:Step is added in blank stem cell ɑ-MEM culture medium without excretion body serum（1）It is filled between the screening
In matter stem cell, it is conditioned medium that cell culture supernatant is collected after 24 hours to 48 hours；
Step 2B：By conditioned medium described in step 2A at 4 DEG C, it is centrifuged 30min under the conditions of 2000g, discards precipitated impurities, receives
Collect supernatant liquid, i.e. culture supernatant.
In above-mentioned technical proposal, the excretion preparation step of the mescenchymal stem cell secretion（3）It is complete by following steps
At：
Step 3A：By culture supernatant described in step 2B and Total exosome Isolation excretion body extracting solution with body
Product is than being 2:After 1 is mixed, it is incubated overnight under the conditions of 4 DEG C, the first Incubating Solution is obtained after incubation；
Step 3B：By the first Incubating Solution obtained by step 3A at 1 DEG C to 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained naked eyes are not
Visible bottom precipitation, the as concentrate of the body containing excretion.
In above-mentioned technical proposal, the excretion preparation step of the mescenchymal stem cell secretion（4）It is complete by following steps
At：
4A：By the concentrate phosphate buffered saline solution of the body containing excretion described in step 3B（PBS）It is dissolved, obtains excretion body
Salting liquid；
4B:Excretion body extracting solution is added in the excretion body salting liquid described in step 4A, is incubated under the conditions of 4 DEG C 1 hour to 12 small
When, the volume ratio of the excretion body salting liquid and Total exosome Isolation excretion body extracting solution is 2:1, after incubation
Obtain the second Incubating Solution；
4C：By the second Incubating Solution described in step 4B at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained lower layer naked eyes are invisible
Precipitating be mescenchymal stem cell secretion excretion body.
Compared with prior art, the beneficial effects of the present invention are the excretion body secreted by mescenchymal stem cell is in life
Application in object preparation, the significant expression for raising surface of a wound keratinocyte growth factor (KGF) albumen, to reach promotion skin
The purpose of wound healing；Application of the excretion body secreted by mescenchymal stem cell in biological agent, also efficiently solves and faces
The problem of bed skin prognosis damage.
Detailed description of the invention
Fig. 1 is that the Electronic Speculum of mescenchymal stem cell excretion body detects figure；
Fig. 2 is the detection figure of Specific marker CD63, CD81 of flow cytomery mescenchymal stem cell；
Fig. 3 is the high-resolution micro-imaging of mescenchymal stem cell excretion body；
Fig. 4 is HE colored graph；
Fig. 5 is the expression figure that immunohistochemistry SABC method detects KGF albumen.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing and specific implementation
Invention is further described in detail for example.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention,
It is not intended to limit the present invention.
The preparation of the excretion body of mescenchymal stem cell secretion：
Step（1）Mescenchymal stem cell is subjected to secondary culture, is screened out from it between cell state is good, proliferative capacity is strong and fills
Matter stem cell；
Step（2）By step（1）The mescenchymal stem cell of middle selection is cultivated using empty culture medium, after cultivating a period of time,
Impurity is removed, culture supernatant is isolated；
Step（3）By step（2）The culture supernatant is incubated for after mixing with excretion body extracting solution, and first is obtained after incubation and is incubated
Change liquid, centrifugal concentrating is carried out to the first incubation fluid, obtains the concentrate of the body containing excretion；
Step（4）By step（3）The progress of excretion body extracting solution is added after sterile PBS dissolution is added in the concentrate of the body containing excretion
It is incubated for, the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom precipitation is collected by centrifugation, obtains mescenchymal stem cell excretion
Body.
Preferably, the mescenchymal stem cell is between umbilical cord mesenchymal stem cells, mesenchymal stem cell or peripheral blood
One of mesenchymal stem cells.
Preferably, the excretion preparation step of the mescenchymal stem cell secretion（1）It is completed by following steps：
Use aseptic bottle（+ 1% mycillin of 200ml physiological saline）Neonatal fresh umbilical cord is taken, with sterile scissors separating blood vessel week
The jelly of Wharton enclosed（Wharton's jelly）, shred, it is in 2mg/ml Type I collagen enzyme that 15ml concentration, which is added,（About 15ml）；Add
Enter dual anti-and 200 μ l the amphotericin B of 200 μ l, is uniformly mixed and is placed in 37 DEG C of incubators overnight；Take out backsight clostridiopetidase A
Depending on digesting situation, 0.25% pancreatin of 10ml is added, is again placed in 37 DEG C of incubators and digests 30min；It takes out, uses physiological saline
3000rpm is centrifuged 15 ~ 20min after 4 ~ 6 times of dilution；Supernatant is abandoned, is transferred to 2 150mm culture dishes after adding stem cell media to be suspended
In, every ware adds the exclusive additive of 1ml stem cell, sets 5%CO2, 37 DEG C of saturated humidity cultures;The visible mesenchyma after 7 to 10 days
Stem cell climbs out of tissue, removes and carries out secondary culture with trypsin digestion after tissue block, chosen after culture cell state it is good,
The 3rd to 5 strong generation mescenchymal stem cell of proliferative capacity.
Preferably, the excretion preparation step of the mescenchymal stem cell secretion（2）It is completed by following steps：
Step 2A:Step is added in the blank stem cell ɑ-MEM culture medium of Hylone company without excretion body serum（1）Institute
It states in the mescenchymal stem cell of screening, it is conditioned medium that cell culture supernatant is collected after 24 hours to 48 hours；
Step 2B：By conditioned medium described in step 2A at 4 DEG C, it is centrifuged 30min under the conditions of 2000g, discards precipitated impurities, receives
Collect supernatant liquid, i.e. culture supernatant.
Preferably, the mescenchymal stem cell excretion preparation step（3）It is completed by following steps：
Step 3A：By culture supernatant described in step 2B and invitogen company's T otal exosome Isolation excretion
Body extracting solution is with volume ratio for 2:After 1 is mixed, it is incubated overnight under the conditions of 4 DEG C, the first Incubating Solution is obtained after incubation；
Step 3B：By Incubating Solution obtained by step 3A at 1 DEG C to 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained naked eyes are invisible
Bottom precipitation, the as concentrate of the body containing excretion.
Preferably, the excretion preparation step of the mescenchymal stem cell secretion（4）It is completed by following steps：
Step 4A：By the concentrate phosphate buffered saline solution of the body containing excretion described in step 3B（PBS）It is dissolved, is obtained outer
Secrete body salting liquid；
Step 4B:Excretion body extracting solution is added in the excretion body salting liquid described in step 4A, is incubated for 1 hour to 8 under the conditions of 4 DEG C
Hour, the volume of the excretion body salting liquid and invitogen company's T otal exosome Isolation excretion body extracting solution
Than being 2:1, the second Incubating Solution is obtained after incubation；
4C：By the second Incubating Solution described in step 4B at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained lower layer naked eyes are invisible
Precipitating be mescenchymal stem cell secretion excretion body.
The identification of mescenchymal stem cell excretion body：
（1）The Electronic Speculum of mescenchymal stem cell excretion body detects：
Fig. 1 is that the Electronic Speculum of mescenchymal stem cell excretion body detects figure.
（2）The flow cytometer detection of mescenchymal stem cell excretion body：
After the excretion body of collection is dissolved with 200ulpbs；
20ulCD83,5ulCD61 is added, is protected from light and is incubated for half an hour, upper machine testing；
Fig. 2 is the detection figure of Specific marker CD63, CD81 of flow cytomery mescenchymal stem cell, as a result excretion body
The positive high expression of Specific marker CD63, CD81.
（3）The high-resolution confocal of mescenchymal stem cell excretion body is imaged：
Excretion body is diluted 20 times with PBS solution;
Respectively plus 1 ul（Anti- cd -63 and anti-cd -69), 20 times of dilution takes 100 ul mixtures being coated with Poly-L-
The coverslip of Lysine is incubated at room temperature 1.5 hours, and PBS is washed 3 times；
Add secondary antibody anti-rabbit Alexa 647 and anti-mouse Alexa 750, dilution 1000；
Incubation at room temperature 1.5 hours, with Dil adipose membrane dyeing 5 minutes of 0.1mol/L；
It is washed 3 times with PBS；
Fig. 3 is the high-resolution micro-imaging of mescenchymal stem cell excretion body, as a result excretion body Specific marker CD63, CD81
Positive high expression, it was demonstrated that the sample isolated and purified is excretion body.
The excretion body of mescenchymal stem cell secretion repairs the observation of curative effect of skin injury：
(1) guinea pig skin damages modeling：
Cavy is grouped and establishes skin injury model：Knock-out experiment group and control rats back chaeta respectively, chloraldurate
0.5ml/100g dosage intraperitoneal injection of anesthesia cavy；
Preserved skin area routine disinfection, each side one piece, central part makes the square surface of a wound that a diameter is 1cm*1cm, needs
It will be as deep as to skin corium（Skin corium is not cut）, the surface of a wound stops blooding naturally, do not wrap up；
The modeling same day chooses cavy similar in modeling success, weight, and random point 3 groups, every group four, first group for the treatment of time is 3
It, second group for the treatment of time is 7 days, and third day treatment time is 14 days, the extension of main detection time therewith, excretion body reparation
How is the effect of skin injury, and every cavy is androgynous control, and left side skin wound injects the experimental group of excretion body, the right injection
The blank control of physiological saline.
（2）Prepare mescenchymal stem cell excretion body physiological saline suspension：
Prepare the mescenchymal stem cell excretion body physiological saline suspension that density is 300 μ g/ml.
（3）Group experiment：
| Group | Inject reagent |
| Control group | 100 μ l physiological saline |
| Experimental group | 300 μ g/ml100 μ l mescenchymal stem cell excretion body physiological saline suspensions |
By reagent injector in experimental group and control rats surface of a wound centre, animal is commonly raised, normal diet.
（4）Observation of curative effect：
a:HE dyeing and result observation：
Control group and experimental group are taken treated the guinea pig back surface of a wound full thickness skin tissue of 3d, 7d, 14d, one is semifixed
In 40 g/L paraformaldehydes, fixed 12h makes paraffin section;Dimethylbenzene dewaxing；Gradient alcohol dehydration；Haematoxylin dyeing 1
Min originally washes 10 ~ 30s；Hydrochloric acid breaks up liquid and breaks up 1 ~ 3s, originally washes 10 ~ 30s；Return indigo plant（45 DEG C of warm water）15s；80% second
Dehydration of alcohols;1 ~ 2s of eosin stains, four step dehydrated alcohols are dehydrated every 10 ~ 30s of step, drying, dimethylbenzene neutral gum mounting, microscope
Lower observation simultaneously compares two groups of wound healing situations；
Fig. 4 is that HE dyes picture, it can be seen that experimental group sample blood capillary proliferation is obvious, and granulation tissue is formed, and wound healing is bright
It is aobvious to be better than control group.
b:The expression of immunohistochemistry SABC method detection KGF albumen：
Control group and experimental group are taken treated the guinea pig back surface of a wound of 3d, 7d, 14d and the skin of surrounding about normal tissue, it will
It is fixed in formaldehyde, and through dewaxing, aquation, PBS is washed, H2O2Room temperature is closed 5~10 minutes, and normal goats are added dropwise in antigen retrieval afterwards
Serum block room temperature 20 minutes, is added dropwise I and resists, and room temperature 1 hour, PBS was washed three times, dropwise addition biotinylation secondary antibody, and 20 DEG C~37
DEG C 20 minutes, PBC was washed 3 times, was added dropwise reagent SABC, 20 DEG C~37 DEG C 20 minutes, PBS is washed 4 times, DAB colour developing, dehydration, transparent, envelope
Piece, microscopy；
Fig. 5 is the expression picture that immunohistochemistry SABC method detects KGF albumen, as the result is shown
Stratified squamous epithelium basically forms under 7 days or so scabs, and surface of a wound keratinocyte growth factor (KGF) protein content is apparently higher than
Control group, 14 days or so epidermises repair completion substantially, and the scab of formation falls off substantially, hence it is evident that are better than control group.
To sum up, the excretion body of mescenchymal stem cell secretion promotes the speed for repairing skin healing to be faster than blank control.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. application of the excretion body in biological agent of mescenchymal stem cell secretion, which is characterized in that the biological agent includes
It treats the biological agent of skin injury or promotes the biological agent of skin wound healing.
2. application of the excretion body of mescenchymal stem cell secretion in biological agent according to claim 1, which is characterized in that
The excretion body following methods preparation of the mescenchymal stem cell secretion：
Step（1）：Mescenchymal stem cell is subjected to secondary culture, is screened out from it between cell state is good, proliferative capacity is strong
Mesenchymal stem cells；
Step（2）：By step（1）The mescenchymal stem cell of middle selection is cultivated using culture medium, after culture, removes impurity,
Isolate culture supernatant；
Step（3）：By step（2）The culture supernatant is incubated for after mixing with excretion body extracting solution, and first is obtained after incubation
Incubation fluid carries out centrifugal concentrating to the first incubation fluid, obtains the concentrate of the body containing excretion；
Step（4）：By step（3）The concentrate of the body containing excretion be added after sterile PBS dissolution be added excretion body extracting solution into
Row is incubated for, and the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom precipitation is collected by centrifugation, and obtains mescenchymal stem cell secretion
Excretion body.
3. application of the excretion body of mescenchymal stem cell secretion in biological agent according to claim 2, which is characterized in that
The mescenchymal stem cell is one in umbilical cord mesenchymal stem cells, mesenchymal stem cell or peripheral blood mescenchymal stem cell
Kind.
4. application of the excretion body of mescenchymal stem cell secretion in biological agent according to claim 3, which is characterized in that
The excretion preparation step of the mescenchymal stem cell secretion（2）It is completed by following steps：
Step 2A:Step is added in blank stem cell ɑ-MEM culture medium without excretion body serum（1）It is filled between the screening
In matter stem cell, it is conditioned medium that cell culture supernatant is collected after 24 hours to 48 hours；
2B：By conditioned medium described in step 2A at 4 DEG C, it is centrifuged 30min under the conditions of 2000g, discards precipitated impurities, in collection
Layer liquid, i.e. culture supernatant.
5. application of the excretion body of mescenchymal stem cell secretion in biological agent according to claim 4, which is characterized in that
The excretion preparation step of the mescenchymal stem cell secretion（3）It is completed by following steps：
Step 3A：By culture supernatant described in step 2B and Total exosome Isolation excretion body extracting solution with body
Product is than being 2:After 1 is mixed, it is incubated overnight under the conditions of 4 DEG C, the first Incubating Solution is obtained after incubation；
Step 3B：By the first Incubating Solution obtained by step 3A at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained naked eyes are sightless
Bottom precipitation, the as concentrate of the body containing excretion.
6. application of the excretion body of mescenchymal stem cell secretion in biological agent according to claim 5, which is characterized in that
The excretion preparation step of the mescenchymal stem cell secretion（4）It is completed by following steps：
4A：By the concentrate phosphate buffered saline solution of the body containing excretion described in step 3B（PBS）It is dissolved, obtains excretion body
Salting liquid；
4B:Excretion body extracting solution is added in the excretion body salting liquid described in step 4A, is incubated under the conditions of 4 DEG C 1 hour to 12 small
When, the volume ratio of the excretion body salting liquid and Total exosome Isolation excretion body extracting solution is 2:1, after incubation
Obtain the second Incubating Solution；
4C：By the second Incubating Solution described in step 4B at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained lower layer naked eyes are invisible
Precipitating be mescenchymal stem cell secretion excretion body.
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| CN110179679A (en) * | 2019-06-25 | 2019-08-30 | 贵州医科大学 | The biological beauty treatment Face-protecting mask essence of the liquid extract of culture containing umbilical cord mesenchymal stem cells, facial mask and preparation method thereof |
| CN110693912A (en) * | 2019-11-18 | 2020-01-17 | 深圳市人民医院 | Application of stem cell exosome in preparation of product for promoting wound healing |
| CN111632075A (en) * | 2020-05-27 | 2020-09-08 | 广州赛琅生物技术有限公司 | Exosome preparation for promoting skin wound healing and preparation method thereof |
| CN114480270A (en) * | 2022-02-17 | 2022-05-13 | 郑州大学 | Preparation method of MSCs culture supernatant, product and application thereof |
| CN115025034A (en) * | 2022-06-24 | 2022-09-09 | 广州美蔻生物科技有限公司 | Mesenchymal stem cell exosome composition and preparation method and application thereof |
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| BIN ZHANG等: "HucMSC-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing", 《STEM CELLS》 * |
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| CN110693912A (en) * | 2019-11-18 | 2020-01-17 | 深圳市人民医院 | Application of stem cell exosome in preparation of product for promoting wound healing |
| CN111632075A (en) * | 2020-05-27 | 2020-09-08 | 广州赛琅生物技术有限公司 | Exosome preparation for promoting skin wound healing and preparation method thereof |
| CN111632075B (en) * | 2020-05-27 | 2021-09-07 | 广州赛琅生物技术有限公司 | Exosome preparation for promoting skin wound healing and preparation method thereof |
| CN114480270A (en) * | 2022-02-17 | 2022-05-13 | 郑州大学 | Preparation method of MSCs culture supernatant, product and application thereof |
| CN115025034A (en) * | 2022-06-24 | 2022-09-09 | 广州美蔻生物科技有限公司 | Mesenchymal stem cell exosome composition and preparation method and application thereof |
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