CN109010523A - A kind of canker sore ointment machin preparation method prepared with umbilical cord mesenchymal stem cells secrete cytokines - Google Patents
A kind of canker sore ointment machin preparation method prepared with umbilical cord mesenchymal stem cells secrete cytokines Download PDFInfo
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Abstract
The invention discloses a kind of canker sore ointment machin preparation methods prepared with umbilical cord mesenchymal stem cells secrete cytokines, which can significantly improve the speed of canker sore patient recovery from illness, and effectively prevent recurrence rate.The specific steps are that: it is allowed to secrete more immunoregulation type cytokines with the stimulation umbilical cord mesenchymal stem cells such as calcium chloride and gamma interferon, it collects culture supernatant and freeze-dried powder is made, the ingredients such as Mixed Silver/Gold flower, Radix Isatidis, dandelion, folium isatidis, polyethylene glycol, maltodextrin and xanthan gum, canker sore ointment is made in stirring under cryogenic conditions.Canker sore ointment of the invention is applied to canker sore patient part, counts cure time and recurrence rate, the results showed that the canker sore ointment can effectively promote the cure time of canker sore patient and effectively prevent from recurring.
Description
Technical field
It is specifically a kind of to be prepared with umbilical cord mesenchymal stem cells secrete cytokines the present invention relates to stem cells technology field
Canker sore ointment machin preparation method.
Background technique
Recurrent oral ulceration (recurrent oral ulcer, ROU) is that one kind is often sent out in lip, tongue, cheek and soft palate etc.
The oral keritonocytes at position, are often accompanied by spontaneous pain, and easy recurrent exerbation seriously affects patients ' life quality.Recurrent mouth
The pathogenic factor of chamber ulcer mainly includes the factors such as autoimmune disorders, local damage, stress, diet.It there is no root at present
The effective ways of recurrent oral ulceration are controlled, can only be cured by reducing recurrent number, extending breaking-out gap, mitigation pain and promotion
The methods of conjunction is treated.The cause of disease of canker sore passes through research discovery recurrence mainly include the following types: 1) autoimmune response
Property canker sore be to occur due to the cellular immune function of patient abnormal, immunoloregulation function is in imbalance state, normally exempts from
Epidemic disease regulatory function disorder is a kind of autoimmune disease, and pathologic process is directed not only to inherent immunity, is also lost with Th1/Th2
Weighing apparatus, CD4+CD25+Foxp3+Treg cell function lower quantity reductions, cytokine secretion disorder, mucoantibody formation, mouth
A series of immunologic derangements such as chamber mucous membrane part sIgA low expression and allergic reaction react related, concurrent current situation portion microcirculation change
And the immunologic derangement of Mediated by Free Radicals has also assisted in the morbidity of recurrent oral ulceration；2) iron in human serum, zinc, copper,
The shortage that the shortage of the microelements such as calcium, manganese, tin can lead to recurrent oral ulceration, especially ferro element can lead to immunocyte
Activity imbalance, so that whole body and the immunoregulatory disorder of part, oral epithelium is thinning, and ulcer is formed after locally necrosing；
3) endocrine alteration, the moon, premenstrual stress can induce recurrent oral ulceration, and women Blood of Patients with Recurrent Oral is in menstruation
The reason of preceding generation is that the content of progesterone in serum is relatively low；4) local microcirculation obstacle causes canker sore, telangiectasis
And blood viscosity enhancing blood flow velocity slows down, leukocytosis in capillary, to lead to the generation of ulcer；5) oral cavity flora
Change, existing research confirm recurrent oral ulceration morbidity there are the variation of oral cavity flora, streptococcus intermedius and Wei Rong Shi
Bacterium substantially reduces；6) other allergy, gastrointestinal disease, bacterium or virus infection, neural malnutrition, hypovitaminosis, essence
The reasons such as refreshing factor, local trauma, smoking, advanced age and heredity can also cause the generation of canker sore.
The common method for the treatment of of canker sore includes: 1. chemical drugs treatment, mainly disease-resistant including medicine for digestive system, antibacterial
Poison and Cidex-7 object, vitamin medicaments and immunoregulation medicament etc..Medicine for digestive system is mainly montmorillonite powder, is day
Right hectorite microparticle pulvis has fixed and inhibits to make to toxin, the gas etc. of virus in the digestive tract, germ and its generation
With, so that it is lost pathogenic effects, to gastrointestinal mucosal have covering protection ability, can repair, improve mucosal barrier to attack because
The defense function of son；Antibacterial and antiviral drug can have stronger killing effect to a variety of pathogens, may also suppress in inflammatory process
Capillary permeability is eliminated to become silted up around ulcer wound surface and be swollen, inhibits the medium of inflammatory process to discharge, play the effect of analgesic；Dimension
Raw element class drug can also effectively shorten the course of disease, promote ulcer wound surface healing；Immunological regulation and immunosuppressant drug are such as
Glucocorticoid commonly uses prednisone.In addition, having the cytotoxic drug of non-specific anti-inflammatory effect often to join with glucocorticoid
For treating canker sore.Because cytotoxic drug has stronger toxicity only for active lymphocyte is proliferated, but it is right
It is then unobvious in well differentiated small lymphocyte effect, therefore therapeutic effect is limited；The drug for improving immune function turns
Molecule is moved, the immune function of body, such as levamisol can be improved, the immune function of body is adjusted；2. supplementing related micro member
Element, licorzinc treatment recurrent oral ulceration work well, because zinc is the essential microelement of human body, it can enhance wound
The power of regeneration of injured tissue.Copper sulphate treatment canker sore also achieves certain effect, because trace copper may promote
The metabolism of part, accelerates the healing of ulcer wound.Also there is certain curative effect with microelements of calcium treatment canker sore, it is micro-
Secondary element calcium participates in excitement and the contraction process of muscle in human body, so that glandular secretion, influences to pass through on cell membrane for ion
Knife switch, promote blood clotting to carry out the reparation of tissue；3. treating dysendocrinism, treatment dysendocrinism is for warp
The canker sore induced before phase has certain sedation treatment effect；4. traditional Chinese medicine is treated, traditional Chinese medicine treatment is typically used for contracting
The short course of disease mitigates symptom, promotes the healing of ulcer wound.But because recurrent oral ulceration is caused by self immune system obstacle
Autoimmune pathologies, so the above drug therapy have certain limitation, be difficult fundamentally to be treated,
Also it can cause certain adverse reaction, such as to the irritation of oral mucosa, the patient feels are uncomfortable, are not easily accepted by, and there are also one
A little drugs can also generate the side effect of whole body.
Mescenchymal stem cell (mesenchymal stem cell, MSC) can be from groups such as umbilical cord, marrow, fat and amnions
Separation is knitted, there is immunosupress and immunoloregulation function, immunocyte abnormality proliferation can be inhibited and with anti-inflammatory
Immunoregulation effect.Mescenchymal stem cell is inhibited by secretion soluble factor mediated immunity, mainly includes hepatocyte growth factor
Sub (HGF), IL-4, IL-10, sHLA-G and TGF-β 1 etc..Wherein HGF has the biology for promoting cell mitogen and anti-apoptotic
Activity, and can result in an immunosuppressive microenvironment；IL-4 can reduce the delayed allergy for relying on Th1, can hinder
The INF- γ of anti-Th1 secretion plays biological effectiveness, to play immunosuppressive effect；IL-10 is generally acknowledged Treg growth
The factor, antagonism IL-12 during immune response are immune negative growth factors, can be exempted from partial agonist mescenchymal stem cell
Epidemic disease inhibiting effect；SHLA-G is immune tolerance molecule important in body, by unconventionality expression make autoimmune tolerance lack or
Inflammatory conditions, and then induce immunosupress；TGF-β 1 is important immune negative growth factor, can inhibit other than lymphocyte
Cell, in addition to the above cell factor, mescenchymal stem cell also secretes some small molecule chemicals and participates in mediating exempting from for part
Epidemic disease inhibits, such as cyclooxygenase and BT-H1.
Mescenchymal stem cell can also be secreted excretion body (exosome), and excretion body is one kind by plasma membrane organelle intracellular
Matter net and Golgi complex active synthesis and secretion to extracellular environment diameter 30~120nm or so miniature plasma membrane capsule
Body structure is the important paracrine form in addition to cell soluble cytokine, contains in the excretion body of source for mesenchymal stem cells
Cytokine profiles, heat shock protein and microRNA etc. can protect these protein factors and small molecule nucleic acid not to be degraded, prolong
Long half-lift.Exosome has been found to have multiple Multi-organ dysfunctions such as kidney injury, myocardial infarction, the nervous system disease
There is clear protective effect, can promote tissue cell insult reparation with reverse both apoptosis trend, chronic ulcer tissue can be effectively promoted
Reparation, accelerating wound healing.
In conclusion the cell factor and excretion body of mescenchymal stem cell secretion have significant immunoregulation effect, it can
To significantly improve the immunoloregulation function of body, to play immunosuppressive effect.
Summary of the invention
The purpose of the present invention is to provide a kind of canker sore prepared with umbilical cord mesenchymal stem cells secrete cytokines
Ointment machin preparation method, to solve the problems of the prior art.
To achieve the above object, the invention provides the following technical scheme:
A kind of canker sore ointment prepared with umbilical cord mesenchymal stem cells secrete cytokines, the base of canker sore ointment
Plinth liquid is water, represents umbilical cord mesenchymal stem cells secrete cytokines concentration, the formula packet of canker sore ointment with protein concentration
It is 1%~6% containing umbilical cord mesenchymal stem cells secrete cytokines that concentration is 0.5 μ of μ g/ml~6 g/ml, mass percent
Honeysuckle, mass percent be 1%~6% Radix Isatidis, mass percent be 1%~6% dandelion, mass percent
For 1%~6% folium isatidis, mass percent be 1%~8% polyethylene glycol, mass percent be 1%~8% malt
The xanthan gum that dextrin and mass percent are 1%~8%.
As optimization, the formula of the canker sore ointment is secreted comprising the umbilical cord mesenchymal stem cells that concentration is 3 μ g/ml
The Pu that Radix Isatidis that honeysuckle that cell factor, mass percent are 3%, mass percent are 3%, mass percent are 3%
The malt that polyethylene glycol that folium isatidis that public English, mass percent are 3%, mass percent are 4%, mass percent are 4%
The xanthan gum that dextrin and mass percent are 4%.
A method of canker sore ointment being prepared with umbilical cord mesenchymal stem cells secrete cytokines, this method comprises:
(1) acquisition of umbilical cord mesenchymal stem cells: it is sterile to take umbilical cord and abandon blood vessel, after taking blood vessel week substance to shred, then
After first being digested using IV Collagenase Type, centrifuging and taking lower sediment；Then continue to digest with trypsase-PBS again, be then centrifuged for taking
Precipitating；Deposit is suspended in mescenchymal stem cell culture medium and is cultivated, culture changes liquid after a certain period of time and is further continued for training
It supports；It is trained to seeing into fibrous cell when bottom of bottle adherent growth reaches 80% and converges, then with being passed on after trypsase-PBS digestion
It supports；
(2) preparation of umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder: the human umbilical cord mesenchymal in F2~F6 generation is taken
Stem cell, is added immunoregulation promotor and excretion body activator continues after cultivating 24 hours, collects cell culture fluid, is added sweet
Reveal alcohol after, freeze-drying cell factor freeze-dried powder is made, be stored in -80 DEG C it is spare；
(3) preparation of canker sore ointment: honeysuckle, Radix Isatidis, dandelion, folium isatidis are proportionally weighed, is ground respectively
It is dissolved in water after crossing 300 meshes after mill, polyethylene glycol, maltodextrin, xanthan gum is then proportionally added, after being dissolved in water, according to
Umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder is added in ratio, is stirred continuously that canker sore is made is soft under cryogenic conditions
Cream saves after then dispensing in -2 DEG C~-8 DEG C.
As optimization, a method of canker sore ointment being prepared with umbilical cord mesenchymal stem cells secrete cytokines, it should
Method includes:
(1) acquisition of umbilical cord mesenchymal stem cells: it is sterile to take umbilical cord and abandon blood vessel, after taking blood vessel week substance to shred, then
It is digested at 37 DEG C using 1 ‰ IV Collagenase Type, takes lower sediment after being then centrifuged 15min with 2000r/min；It uses again
0.25% trypsase-PBS continues to digest, and then 2000r/min takes lower sediment after being centrifuged 15min, digests 2 times repeatedly；Again
By deposit with 1 × 106The density suspension of a/ml is cultivated in mescenchymal stem cell culture medium, and culture environment is temperature
Spend 37 DEG C, 100% humidity, 5%CO2And dark surrounds, change within every 3~4 days liquid 1 time, through 2 days it is visible at fibrous cell in bottom of bottle
Adherent growth, fibrous cell is when bottom of bottle adherent growth can reach 80% and converge after about 8~12 days, then with 0.25% tryptose
After enzyme-PBS digestion, then with cell density 5 × 104A/cm2Secondary culture；
(2) preparation of umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder: the human umbilical cord mesenchymal in F2~F6 generation is taken
Stem cell, is added immunoregulation promotor and excretion body activator continues after cultivating 24 hours, collects cell culture fluid, matter is added
Measure percentage be 14% mannitol after, freeze-drying cell factor freeze-dried powder is made, be stored in -80 DEG C it is spare；
(3) preparation of canker sore ointment: honeysuckle, Radix Isatidis, dandelion, folium isatidis are proportionally weighed, is ground respectively
It is dissolved in water after crossing 300 meshes after mill, polyethylene glycol, maltodextrin, xanthan gum is then proportionally added, after being dissolved in water, according to
The ultimate density of 0.5 μ of μ g/ml~6 g/ml is added umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder, under cryogenic conditions not
Canker sore ointment is made in disconnected stirring, saves after then dispensing in -2 DEG C~-8 DEG C.
As optimization, it is 0.005mg/ that step (2) the immunoregulation promotor and excretion body activator, which are according to concentration,
The CaCl of ml~0.1mg/ml2, concentration be the poly IC of 20 μ of μ g/ml~100 g/ml, concentration is 1 μ of μ g/ml~10 g/ml
The vitamin C that lipopolysaccharides, concentration are 0.5 μ of μ g/ml~2 g/ml and the gamma interferon that concentration is 0.05ng/ml~0.2ng/ml
The mixed liquor that (IFN-γ) is equipped with.
As optimization, it is 0.01mg/ml that step (2) the immunoregulation promotor and excretion body activator, which are according to concentration,
CaCl2, concentration be the poly IC of 50 μ g/ml, concentration is the lipopolysaccharides of 5 μ g/ml, the vitamin C that concentration is 1 μ g/ml and dense
The mixed liquor that the gamma interferon (IFN-γ) that degree is 0.01ng/ml is equipped with.
Compared with prior art, the beneficial effects of the present invention are: the present invention provides one kind by umbilical cord mesenchymal stem cells
The cell factor of secretion is main formula, the oral cavity painting type ointment for treating canker sore, in order to increase mescenchymal stem cell point
The ability for secreting immunoregulation type cytokines and excretion body, the present invention provides a kind of addition immunoregulation reinforcing agent and excretion bodies
The method that activator activates umbilical cord mesenchymal stem cells, and using umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder in
Herb extracts are prepared into canker sore ointment, and wherein umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder includes immune adjusts
Control type cytokines and tissue repair type cytokines.Appropriate ointment is squeezed out when use of the present invention, is applied to ulcer surface, 15 minutes
It does not inside drink water or feeds as far as possible, can effectively treat canker sore.
In the preparation process for the canker sore ointment that the present invention is announced, using the thin of umbilical cord mesenchymal stem cells secretion
Intracellular cytokine freeze-dried powder additive can be adjusted directly and be immunized under the mucous membrane of mouth of disorder, improved to immune system under mucous membrane, and
Ingredient hormone-free, securely and reliably.And it is aided with honeysuckle, Radix Isatidis, dandelion, folium isatidis etc. and canker sore is treated with significant
The traditional Chinese medicine ingredients of effect, so that preparation nourishing yin and promoting blood circulation, clearing heat and detoxicating, convergence pain-relieving functions, which are more imitated, concentrates one's efforts, and maltodextrin can be in mouth
Chamber ulcer wound surface forms layer protecting film, effectively prevents contact friction of the surface of a wound with other positions, reduces feeling of pain, and make
Obtaining drug has the effect of sustained release in the surface of a wound, can more play lasting drug effect than general drug.
The canker sore ointment that the present invention is announced, the cell factor as secreted by human umbilical cord mesenchymal stem cells is as master
Functional component is wanted, cell factor synthesized by the Escherichia coli than genetic engineering transformation has better protein folding structure and sugar
Baseization modification, thus biological activity is more preferable than gene engineering product cell factor.
Method of the preparation of umbilical cord mesenchymal stem cells cell factor of the present invention oral cavity ointment using directly mixing concentration, work
Skill is simple, can satisfy the needs of most domestic producer production status, and production craft step is simple, and energy consumption is few, almost without dirt
Object discharge is contaminated, the requirement in national new environmental law about energy-saving and environment-friendly green ecological clean manufacturing is met.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
Embodiment 1:
The acquisition of umbilical cord mesenchymal stem cells: it is sterile that umbilical cord is taken to be cut into 3cm or so segment, it takes out and abandons blood vessel, take blood vessel week object
After matter shreds, first using 1 ‰ IV Collagenase Type in digestion 2h, then 2000r/min is centrifuged 10min, and lower sediment is taken to use again
0.25% trypsase-PBS continues to digest 25min at 37 DEG C, and then 2000r/min is centrifuged 10min, and cell precipitation is taken to use
After the digestion of 0.25% trypsin solution, then 800r/min is centrifuged 10min, takes cell precipitation with 1 × 106The density suspension of a/ml
In culture medium, medium component is the commercially available mesenchymal stem cell serum-free culture medium of commercialization.Set 37 DEG C, it is 100% wet
Degree, 5%CO2With cultivated under dark surrounds, change within every 3 days liquid 1 time, through 2 days it is visible at fibrous cell bottom of bottle grow, about 10 days
After can reach 80% when converging with after 0.25% trypsase-PBS digestion, then passed on and trained with 5 × 104/cm2 of cell density
It supports.The cell in F4 generation is for harvesting cell factor.
The human umbilical cord mesenchymal stem cells in F2~F6 generation are taken, it is 50 μ that CaCl2, concentration that concentration is 0.01mg/ml, which is added,
The poly IC of g/ml, the vitamin C and final concentration of 0.01ng/ml that concentration is the lipopolysaccharides of 5 μ g/ml, final concentration of 1 μ g/ml
IFN- γ, continue culture 24 hours after, collect cell culture fluid, be added mass percent be 14% mannitol, freezing
Drying cell factor freeze-dried powder is made, be stored in -80 DEG C it is spare；
The cell culture fluid (Activate group) of collection is measured after being filtered with 0.22 micron membrane filter with ELISA kit
Kynurenin (Kynurenine), prostaglandin E2 (Prostaglandin E, PGE2), sHLA-G (soluble HLA-G),
The content of IL10 and TGF-β 1.The MSC cell culture fluid conduct pair of immunoregulation promotor and excretion body activator is not added
According to group (Ctrl group).As a result the sHLA-G of the MSC cell secretion after the visible activation using method provided by the present invention, dog urinate
Propylhomoserin (Kynurenine), IL10, PGE2 and 1 amount of TGF-β significantly improve, and the results are shown in Table 1.
Cytokine content ELISA testing result statistical form (n=6) in 1 cell culture fluid of table
The preparation of the canker sore ointment of factor-containing: honeysuckle, Radix Isatidis, dandelion, folium isatidis are ground respectively
300 meshes are crossed afterwards, and polyethylene glycol, maltodextrin, xanthan gum and cell factor freeze-dried powder are added according to a certain percentage, is dissolved in
Be made canker sore ointment after water under cryogenic conditions, honeysuckle, Radix Isatidis, dandelion, folium isatidis final mass ratio be 3%,
Polyethylene glycol final mass percentage 4%, maltodextrin final mass percentage are 4%, xanthan gum final mass percentage is
4%, umbilical cord mesenchymal stem cells secrete cytokines ultimate density is 3 μ g/ml.Be sub-packed in aluminium leather hose, be stored in -2 DEG C~-
8℃。
Therapeutic process: 40 canker sore patients are randomly selected as experimental subjects, with mouth obtained in aforesaid operations
Chamber ointment is as experimental example 1 and experimental example 2, with common commercial oral ulcer buccal patch and common commercial oral ulcer lozenge point
It Zuo Wei not reference examples 1 and reference examples 2.At random by one group of every 10 people of 40 experimental subjects, 4 groups are divided into, implements experimental example respectively
1~2 and reference examples 1~2, each experimental subjects canker sore wound situation is observed daily, is continued 30 days, is recorded the time of recovery from illness
(average value) continues to keep observation after patient's recovery from illness, records the number that every group of appearance is recurred, the results are shown in Table 2.As it can be seen that with compareing
It compares, mescenchymal stem cell secrete cytokines canker sore ointment made from the embodiment of the present invention 1~2 can speed up oral cavity and burst
The speed of ulcer recovery from illness, and effectively prevent from recurring.
Canker sore recovery from illness speed statistical form (n=10/ group) after table 2 is treated
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
Claims (6)
1. a kind of canker sore ointment prepared with umbilical cord mesenchymal stem cells secrete cytokines, the basis of canker sore ointment
Liquid is water, represents umbilical cord mesenchymal stem cells secrete cytokines concentration with protein concentration, it is characterised in that: canker sore ointment
Formula comprising concentration be the umbilical cord mesenchymal stem cells secrete cytokines of 0.5 μ of μ g/ml~6 g/ml, mass percent is
Dandelion that Radix Isatidis that 1%~6% honeysuckle, mass percent are 1%~6%, mass percent are 1%~6%, matter
Polyethylene glycol that folium isatidis that amount percentage is 1%~6%, mass percent are 1%~8%, mass percent be 1%~
The xanthan gum that 8% maltodextrin and mass percent is 1%~8%.
2. a kind of canker sore prepared with umbilical cord mesenchymal stem cells secrete cytokines according to claim 1 is soft
Cream, it is characterised in that: the formula of the canker sore ointment is thin comprising the umbilical cord mesenchymal stem cells secretion that concentration is 3 μ g/ml
The Pu that Radix Isatidis that honeysuckle that intracellular cytokine, mass percent are 3%, mass percent are 3%, mass percent are 3% is public
The malt paste that polyethylene glycol that folium isatidis that English, mass percent are 3%, mass percent are 4%, mass percent are 4%
The xanthan gum that essence and mass percent are 4%.
3. according to claim 1 or 2 a kind of soft with umbilical cord mesenchymal stem cells secrete cytokines preparation canker sore
The method of cream, which is characterized in that this method comprises:
(1) acquisition of umbilical cord mesenchymal stem cells: it is sterile to take umbilical cord and abandon blood vessel, after taking blood vessel week substance to shred, then use
After IV Collagenase Type first digests, centrifuging and taking lower sediment；Then continue to digest with trypsase-PBS again, be then centrifuged for taking cell
Precipitating；Cell precipitation is suspended in mescenchymal stem cell culture medium and is cultivated, culture changes liquid after a certain period of time and is further continued for training
It supports；To seeing into fibrous cell when bottom of bottle adherent growth reaches 80% and converges, then with being passed on after trypsase-PBS digestion
Culture；
(2) preparation of umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder: take the human umbilical cord mesenchymal in F2~F6 generation dry thin
Born of the same parents, are added immunoregulation promotor and excretion body activator continues after cultivating 24 hours, collect cell culture fluid, mannitol is added
Afterwards, freeze-drying cell factor freeze-dried powder is made, be stored in -80 DEG C it is spare；
(3) preparation of canker sore ointment: proportionally weighing honeysuckle, Radix Isatidis, dandelion, folium isatidis, after grinding respectively
It is dissolved in water after crossing 300 meshes, polyethylene glycol, maltodextrin, xanthan gum is then proportionally added, after being dissolved in water, according to 0.5 μ
Umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder is added in the ultimate density of the μ of g/ml~6 g/ml, constantly stirs under cryogenic conditions
Mixing saves after then dispensing in -2 DEG C~-8 DEG C at canker sore ointment.
4. a kind of umbilical cord mesenchymal stem cells secrete cytokines preparation canker sore ointment according to claim 3
Method, which is characterized in that this method comprises:
(1) acquisition of umbilical cord mesenchymal stem cells: it is sterile to take umbilical cord and abandon blood vessel, after taking blood vessel week substance to shred, then use
1 ‰ IV Collagenase Type digests at 37 DEG C, takes lower sediment after being then centrifuged 15min with 2000r/min；Again with 0.25% pancreas
Protease-PBS continues to digest, and then 2000r/min removes confluent monolayer cells precipitating after being centrifuged 15min, digests 2 times repeatedly；It again will be thin
Born of the same parents are precipitated with 1 × 106The density suspension of a/ml is cultivated in mescenchymal stem cell culture medium, and culture environment is temperature 37
DEG C, 100% humidity, 5%CO2And dark surrounds, it changes within every 3~4 days liquid 1 time, it is visible adherent in bottom of bottle at fibrous cell through 2 days
Growth, fibrous cell is when bottom of bottle adherent growth can reach 80% and converge after about 8~12 days, then with 0.25% trypsase-
After PBS digestion, then with cell density 5 × 104A/cm2Secondary culture；
(2) preparation of umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder: take the human umbilical cord mesenchymal in F2~F6 generation dry thin
Born of the same parents, are added immunoregulation promotor and excretion body activator continues after cultivating 24 hours, collect cell culture fluid, quality hundred is added
Point than for 14% mannitol after, freeze-drying cell factor freeze-dried powder is made, be stored in -80 DEG C it is spare；
(3) preparation of canker sore ointment: proportionally weighing honeysuckle, Radix Isatidis, dandelion, folium isatidis, after grinding respectively
It is dissolved in water after crossing 300 meshes, polyethylene glycol, maltodextrin, xanthan gum is then proportionally added, after being dissolved in water, according to 0.5 μ
Umbilical cord mesenchymal stem cells secrete cytokines freeze-dried powder is added in the ultimate density of the μ of g/ml~6 g/ml, constantly stirs under cryogenic conditions
Mixing saves after then dispensing in -2 DEG C~-8 DEG C at canker sore ointment.
5. a kind of umbilical cord mesenchymal stem cells secrete cytokines preparation canker sore ointment according to claim 4
Method, it is characterised in that: it is 0.005mg/ml that step (2) the immunoregulation promotor and excretion body activator, which are according to concentration,
The CaCl of~0.1mg/ml2, concentration is the poly IC of 20 μ of μ g/ml~100 g/ml, concentration is 1 μ of μ g/ml~10 g/ml rouge it is more
Sugar, the vitamin C that concentration is 0.5 μ of μ g/ml~2 g/ml and the gamma interferon that concentration is 0.05ng/ml~0.2ng/ml
The mixed liquor that (IFN-γ) is equipped with.
6. a kind of umbilical cord mesenchymal stem cells secrete cytokines preparation canker sore ointment according to claim 5
Method, it is characterised in that: it is 0.01mg/ml that step (2) the immunoregulation promotor and excretion body activator, which are according to concentration,
CaCl2, concentration be the poly IC of 50 μ g/ml, concentration is the lipopolysaccharides of 5 μ g/ml, the vitamin C that concentration is 1 μ g/ml and dense
The mixed liquor that the gamma interferon (IFN-γ) that degree is 0.01ng/ml is equipped with.
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| CN110075146A (en) * | 2019-06-13 | 2019-08-02 | 广州赛琅生物技术有限公司 | A kind of canker sore spray and preparation method thereof |
| CN110201024A (en) * | 2019-06-26 | 2019-09-06 | 广州南医大生物工程有限公司 | A kind of composition of the extract containing stem cell of wound healing and application thereof |
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| CN115400055A (en) * | 2022-09-06 | 2022-11-29 | 北京化工大学 | Multifunctional mouthwash and preparation method thereof |
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| CN110201024A (en) * | 2019-06-26 | 2019-09-06 | 广州南医大生物工程有限公司 | A kind of composition of the extract containing stem cell of wound healing and application thereof |
| CN112891295A (en) * | 2021-01-29 | 2021-06-04 | 陕西中鸿科瑞再生医学研究院有限公司 | Exosome mouthwash as well as preparation method and application thereof |
| CN113398160A (en) * | 2021-06-24 | 2021-09-17 | 云南农业大学 | Stem cell oral spray for cats and preparation method thereof |
| CN114712399A (en) * | 2022-05-11 | 2022-07-08 | 国典(北京)医药科技有限公司 | Exosome ointment composition, exosome ointment and preparation method and application thereof |
| CN115400055A (en) * | 2022-09-06 | 2022-11-29 | 北京化工大学 | Multifunctional mouthwash and preparation method thereof |
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