CN106377544A - Application of supernatant fluid of umbilical-cord mesenchymal stem cell to preparation of medicine for treating oral ulcer as well as gargle and preparation method thereof - Google Patents
Application of supernatant fluid of umbilical-cord mesenchymal stem cell to preparation of medicine for treating oral ulcer as well as gargle and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of stem cells, and particularly relates to application of a supernatant fluid of an umbilical-cord mesenchymal stem cell to the preparation of a medicine for treating an oral ulcer as well as a gargle and a preparation method thereof. The gargle comprises the supernatant fluid of the umbilical-cord mesenchymal stem cell. The total effective rate of the oral ulcer treated by the gargle provided by the invention can be up to 90 percent or above; the curative effect of the gargle on the oral ulcer is obviously better than the curative effect of a supernatant fluid of a bone-marrow mesenchymal stem cell; the gargle provided by the invention has no stimulation to an oral mucosa, cannot cause any adverse reaction, and is easily accepted by a patient.
Description
Technical field
The present invention relates to stem cells technology field, particularly to umbilical cord mesenchymal stem cells supernatant in preparation treatment oral cavity
Application in medicine for ulcer and collutory and its preparation method.
Background technology
Oral ulcer is commonly called as " aphtha ", is a kind of common ulcerative damages disease betiding oral mucosa, is more common in
Inside lip, the position such as tongue, tongue abdomen, cheek mucosa, vestibule ditch, soft palate, the mucosa at these positions lacks keratinization layer or keratinization relatively
Difference.Tongue ulcer refers to betide tongue, the oral ulcer of tongue abdominal part position.Sharp ache during oral ulcer outbreak, local causalgia is bright
Aobvious, severe patient also can affect diet, speak, and daily life is caused with very big inconvenience；Can concurrently halitosis, chronic pharyngitiss, constipation, head
The General Symptomies such as bitterly, dizzy, nauseous, weak, irritated, heating, lymphadenectasis.
The cause of disease of oral ulcer is at present not yet clearly it is generally recognized that oral ulcer is many factors comprehensive function
Result.Immunity, h and E are probably " three factors " of oral ulcer morbidity, i.e. genetic background and suitable environmental factorss
Can the immunoreation of exception throw and oral ulcer feature lesions occur.
Existing Aphthasol thing mainly has：Local antibacterial medicine, topical corticosteroid, local analgesia agent,
The antimicrobial antiphlogistic medication of local anti-inflammatory preparation or whole body.Although above medicine can produce different degrees of to oral ulcer
Therapeutic effect, but also can cause certain untoward reaction, the such as zest to oral mucosa, patient feels uncomfortable, is difficult to connect
By etc., also have some medicines can produce the side effect of whole body.In the therapeutic process of oral ulcer, generally all first adopt peace and quiet water
Or normal saline gargles, make oral cavity keep cleaning, then repaste and smear medicine, it is to avoid in oral cavity, affected part is unholiness and affect medicine
Effect.But gargled using peace and quiet water or normal saline and be only capable of making oral cavity keep cleaning, single function, still lack at present and can coordinate medicine
The suitable collutory of thing treatment.
Xu Na delivered in 2015《Rinsing the mouth human marrow mesenchymal stem cell supernatant is to Post kidney transplantation oral ulcer
Curative effect》In report the mesenchymal stem cells MSCs supernatant of In vitro culture and contain cytokine profiles, there is antiinflammatory, promote blood
Pipe forms and repairs the function of damaged tissues.But applicant's research finds always having of bone mesenchymal stem cells treatment oral ulcer
Efficiency only 83%.Therefore, for the higher stem cell medicine of dental ulcer treatment total effective rate, there is still a demand.
Content of the invention
In view of this, the invention provides umbilical cord mesenchymal stem cells supernatant is in preparation treatment stomatocace medicine
Application and collutory and its preparation method.The total effective rate that this umbilical cord mesenchymal stem cells supernatant treats oral ulcer may be up to
More than 90%.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides application in preparation treatment stomatocace medicine for the umbilical cord mesenchymal stem cells supernatant.
Have been reported that the mesenchymal stem cells MSCs supernatant of display In vitro culture has the effect for the treatment of oral ulcer at present,
Present invention research finds, compared with mesenchymal stem cells MSCs supernatant, the umbilical cord mesenchymal stem cells supernatant of In vitro culture
More notable in the curative effect for the treatment of oral ulcer.Therefore, umbilical cord mesenchymal stem cells supernatant is used for controlling of oral ulcer
Treat or auxiliary treatment has more wide prospect.
Present invention also offers a kind of collutory for treating oral ulcer, including umbilical cord mesenchymal stem cells supernatant
Liquid.
In the embodiment that the present invention provides, the preparation method of umbilical cord mesenchymal stem cells supernatant is：Take and fill between umbilical cord
Matter stem cell carries out the first culture using complete medium, then carries out the second training using the DMEM/F12 culture medium of serum-free
Support, collect the supernatant of cell culture medium, obtain umbilical cord mesenchymal stem cells supernatant.
Preferably, umbilical cord mesenchymal stem cells are the 3rd～5 generation umbilical cord mesenchymal stem cells.
Preferably, the inoculum density of the umbilical cord mesenchymal stem cells of the first culture is (1～10) × 103Individual/cm2.
In the embodiment that the present invention provides, the inoculum density of the umbilical cord mesenchymal stem cells of the first culture is 7 × 103
Individual/cm2.
Preferably, the incubation time of the first culture is 20～30 hours.
In the embodiment that the present invention provides, the incubation time of the first culture is 24 hours.
Preferably, the condition of culture of the first culture is 37 DEG C, 5%CO2.
Preferably, the incubation time of the second culture is 40～60 hours.
In the embodiment that the present invention provides, the incubation time of the second culture is 48 hours.
Preferably, the condition of culture of the second culture is 37 DEG C, 5%CO2.
In the embodiment that the present invention provides, also include centrifugation after collecting the supernatant of cell culture medium, take supernatant, filter
The step of membrane filtration.
In the embodiment that the present invention provides, the rotating speed of centrifugation is 1000rpm, and the time is 5min.
In the embodiment that the present invention provides, the aperture filtering adopted filter membrane is 0.22 μm.
Preferably, also including pharmaceutically acceptable adjuvant for treating the collutory of oral ulcer.
Preferably, the storage temperature of the umbilical cord mesenchymal stem cells supernatant obtaining is -20～-80 DEG C.
Present invention also offers the preparation method of this collutory, including：Take umbilical cord mesenchymal stem cells using cultivating completely
Base carries out the first culture, then carries out the second culture using the DMEM/F12 culture medium of serum-free, collects the upper of cell culture medium
Clear liquid, obtains umbilical cord mesenchymal stem cells supernatant.
Preferably, umbilical cord mesenchymal stem cells are the 3rd～5 generation umbilical cord mesenchymal stem cells.
Preferably, the inoculum density of the umbilical cord mesenchymal stem cells of the first culture is (1～10) × 103Individual/cm2.
In the embodiment that the present invention provides, the inoculum density of the umbilical cord mesenchymal stem cells of the first culture is 7 × 103
Individual/cm2.
Preferably, the incubation time of the first culture is 20～30 hours.
In the embodiment that the present invention provides, the incubation time of the first culture is 24 hours.
Preferably, the condition of culture of the first culture is 37 DEG C, 5%CO2.
Preferably, the incubation time of the second culture is 40～60 hours.
In the embodiment that the present invention provides, the incubation time of the second culture is 48 hours.
Preferably, the condition of culture of the second culture is 37 DEG C, 5%CO2.
In the embodiment that the present invention provides, also include centrifugation after collecting the supernatant of cell culture medium, take supernatant, filter
The step of membrane filtration.
In the embodiment that the present invention provides, the rotating speed of centrifugation is 1000rpm, and the time is 5min.
In the embodiment that the present invention provides, the aperture filtering adopted filter membrane is 0.22 μm.
In the embodiment that the present invention provides, the isolated culture method of umbilical cord mesenchymal stem cells is：
After taking umbilical cord tissue PBS, separate the fertile Dun Shi glue around arteries, after shredding, be inoculated in culture dish
In, add complete medium, place 37 DEG C, 5%CO2In incubator, quiescent culture changes liquid after 7 days, changes liquid within every 3 days later, when
Microscopic observation, to 5～6 cell colonies, removes piece of tissue；After attached cell degree of converging reaches 80%～90%, with 0.1%～
0.25% trypsinization, stops, after digestion, cell suspension to be centrifuged with 1000～1500rpm with the culture medium containing serum
5min, according to (0.5～1) × 104cells/cm2Cell density passes on.
The invention provides umbilical cord mesenchymal stem cells supernatant is treated the application in stomatocace medicine in preparation and is gargled
Oral fluid and its preparation method.This collutory includes umbilical cord mesenchymal stem cells supernatant.The present invention at least has beneficial effect as follows
One of fruit：
1st, present invention research shows that the total effective rate of umbilical cord mesenchymal stem cells supernatant treatment oral ulcer may be up to
More than 90%, result of the test shows, umbilical cord mesenchymal stem cells supernatant will be considerably better than between bone marrow the curative effect of oral ulcer and fill
The curative effect of matter stem cell supernatant；
2nd, the collutory that the present invention provides is non-stimulated to oral mucosa, will not cause any untoward reaction, patient easily connects
It is subject to.
Brief description
Fig. 1 shows the testing result of the umbilical cord mesenchymal stem cells surface antigen of flow cytomery separation and Culture；Wherein,
1-1～1-6 shows the expression of CD73, CD90, CD105, CD34, CD45, HLA-DR respectively；
Fig. 2 shows the testing result of the mesenchymal stem cells MSCs surface antigen of flow cytomery separation and Culture；Wherein,
2-1～2-6 shows the expression of CD73, CD90, CD105, CD34, CD45, HLA-DR respectively.
Specific embodiment
The invention discloses umbilical cord mesenchymal stem cells supernatant is treated the application in stomatocace medicine in preparation and is gargled
Oral fluid and its preparation method, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter and realize.Especially need
It is noted that all similar replacements and change are apparent to those skilled in the art, they are considered as wrapping
Include in the present invention.The method of the present invention and application are described by preferred embodiment, and related personnel substantially can be not
In disengaging present invention, spirit and scope, method described herein and application are modified or suitably change and combine, come
Realize and application the technology of the present invention.
Term is explained：
Umbilical cord mesenchymal stem cells are that the class being present in umbilical cord tissue has self renewal, multiplication capacity by force, and many
Mescenchymal stem cell to differentiation capability.
The umbilical cord mesenchymal stem cells supernatant that the present invention provides is treated the application in stomatocace medicine in preparation and is gargled
In application in oral fluid and its preparation method and medicine, biomaterial used, reagent, instrument etc. all can be buied by market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1：
1st, separation and Culture and identification umbilical cord mesenchymal stem cells
After taking umbilical cord tissue PBS, separate the fertile Dun Shi glue around arteries with tissue shear, inoculate after shredding
In in culture dish, add complete medium, place 37 DEG C, 5%CO2In incubator, quiescent culture changes liquid after 7 days, and later every 3
It changes liquid, when Microscopic observation to 5-6 cell colony, removes piece of tissue；After attached cell degree of converging 80%～90%, use
0.25% trypsinization, stops, after digestion, cell suspension to be centrifuged 5min with 1500rpm, presses with the culture medium containing serum
According to (0.5～1) × 104cells/cm2Cell density passes on, and takes 3-5 and is used for testing for cell.
The identification of the surface marker of umbilical cord mesenchymal stem cells:
Take P3 cell, when cell confluency degree reaches 80%-90%, digest centrifuge cell with 0.25% pancreatin, and after counting
Often pipe adds 2 × 105Cell number, dye solution is washed 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, blown and beaten with dye solution
Mix cell；Add each 2 μ L of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibody, and set a pipe as blank；
At 4 DEG C, lucifuge reacts 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of labelling is abandoned
Clearly, lucifuge adds the sample-loading buffer of 500 μ L, mixes, with 200 eye mesh screen filtration cell samples, flow cytomery cell
Surface antigen.Testing result is shown in Fig. 1.
As seen from Figure 1, the expression of mescenchymal stem cell positive surface label CD73, CD90, CD105 is all higher than
95%, and negative surface marker CD34, CD45, HLA-DR expression is respectively less than 2%, meets mescenchymal stem cell surface markers
Thing feature.Illustrate that the umbilical cord mesenchymal stem cells of separation and Culture keep the characteristic of mescenchymal stem cell.
2nd, the collection of umbilical cord mesenchymal stem cells culture medium supernatant
Take the 3rd generation umbilical cord mesenchymal stem cells, first according to 7 × 103/cm2Cell density inoculated and cultured ware, complete medium
After carrying out cultivating 24 hours, after being cleaned with normal saline, after adding the DMEM/F12 culture medium of serum-free to continue culture 48 hours,
Collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube, 5min is centrifuged with 1000rpm.Remove precipitation, in collection
Clearly.Draw conditioned medium with disposable syringe, with 0.22 μm of membrane filtration, be collected in sterile culture flask.It is placed on -20
DEG C incubator preserves 6 months -12 months about or is placed on -80 DEG C and preserves more than 1 year.
Embodiment 2：
The collection of umbilical cord mesenchymal stem cells culture medium supernatant：
4th generation umbilical cord mesenchymal stem cells in Example 1, first according to 7 × 103/cm2Cell density inoculated and cultured ware, complete
After full culture medium carries out cultivating 24 hours, after being cleaned with normal saline, the DMEM/F12 culture medium of serum-free is added to continue culture
After 48 hours, collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube, 5min is centrifuged with 1000rpm.It is heavy to remove
Form sediment, collect supernatant.Draw conditioned medium with disposable syringe, with 0.22 μm of membrane filtration, be collected in sterile culture flask.
It is placed on -20 DEG C of incubators to preserve 6 months -12 months about or be placed on -80 DEG C of preservations more than 1 year.
Embodiment 3：
The collection of umbilical cord mesenchymal stem cells culture medium supernatant：
5th generation umbilical cord mesenchymal stem cells in Example 1, first according to 7 × 103/cm2Cell density inoculated and cultured ware, complete
After full culture medium carries out cultivating 24 hours, after being cleaned with normal saline, the DMEM/F12 culture medium of serum-free is added to continue culture
After 48 hours, collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube, 5min is centrifuged with 1000rpm.It is heavy to remove
Form sediment, collect supernatant.Draw conditioned medium with disposable syringe, with 0.22 μm of membrane filtration, be collected in sterile culture flask.
It is placed on -20 DEG C of incubators to preserve 6 months -12 months about or be placed on -80 DEG C of preservations more than 1 year.
Comparative example 1：
1st, separation and Culture and identification mesenchymal stem cells MSCs
1) method of mesenchymal stem cells MSCs separation and Culture：
The sample of bone marrow taking normal person from hospital in an aseptic environment, adds isopyknic normal saline, gently in bone marrow
Light mixing, obtains bone marrow mixed liquor A；The isopyknic lymphocyte separation medium of bone marrow mixed liquor (A) is added in new centrifuge tube；
Draw bone marrow mixed liquor (A) with syringe to be slowly added in lymphocyte separation medium along tube wall；10- is centrifuged with 300-400g
15min, after centrifugation, visible liquid layered draws the mononuclear cell layer in intermediate layer with pasteur pipet, transfers to containing 5-10mL
In the centrifuge tube of Hanks buffer solution, gently mix；5-15min is centrifuged with 200-300g, removes supernatant, cleaned again with Hanks
1-2 time；Resuspended with growth of marrow mesenchyme stem cell culture medium (serum-free culture, bFGF containing 10ng/mL, 10ng/mLEGF)
In cell, and culture dish in inoculating；It is placed on 37 DEG C, 5%CO2Cultivate in incubator；After 48 hours, remove culture supernatant, weight
Newly change growth of marrow mesenchyme stem cell culture medium；Change liquid within every 3 days, be within 4-8 days that visible cell confluency degree reaches 80-90%；
Passage operates：With 0.25%-0.125% trypsinization attached cell, 5-10min is centrifuged with 200-300g, with life
Long culture medium re-suspended cell precipitation, according to 5000-10000/cm2In cell density inoculated and cultured ware, it is passaged to P3-P5 generation.
2) identification of the surface marker of mesenchymal stem cells MSCs：
Take P3 cell, when cell confluency degree reaches 80%-90%, digest centrifuge cell with 0.25% pancreatin, and after counting
Often pipe adds 2 × 105Cell number, dye solution is washed 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, blown and beaten with dye solution
Mix cell；Add each 2 μ L of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibody, and set a pipe as blank；
At 4 DEG C, lucifuge reacts 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of labelling is abandoned
Clearly, lucifuge adds the sample-loading buffer of 500 μ L, mixes, with 200 eye mesh screen filtration cell samples, flow cytomery cell
Surface antigen.Testing result is shown in Fig. 2.
From Figure 2 it can be seen that the expression of mescenchymal stem cell positive surface label CD73, CD90, CD105 is all higher than
95%, and negative surface marker CD34, CD45, HLA-DR expression is respectively less than 2%, meets mescenchymal stem cell surface markers
Thing feature.Illustrate that the mesenchymal stem cells MSCs of separation and Culture keep the characteristic of mescenchymal stem cell.
2nd, the collection of mesenchymal stem cells MSCs culture medium supernatant
Take the 4th generation mesenchymal stem cells MSCs, first according to 7 × 103/cm2Cell density inoculated and cultured ware, complete medium
After carrying out cultivating 24 hours, after being cleaned with normal saline, after adding the DMEM/F12 culture medium of serum-free to continue culture 48 hours,
Collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube, 1000rpm is centrifuged 5min.Remove precipitation, in collection
Clearly.Draw conditioned medium with disposable syringe, with 0.22 μm of membrane filtration, be collected in sterile culture flask.It is placed on -20
DEG C incubator preserves 6-12 month about or is placed on -80 DEG C and preserves more than 1 year.
The curative effect to Oral ulcer model mice for the test example stem cell supernatant
With 0.025g/kg pentobarbital sodium anesthetized rat, leaned in bilateral lower lip with NaOH crystal and at bicker mucosa, burn 5～
8s, forms ulcer after normal saline flushing.Choose 60 healthy mices and be randomly divided into experimental group and matched group, every group 30.
Experimental group：Oral ulcer is with, after normal saline cleaning skin surface, being dipped with cotton swab and filling between embodiment 1～3 umbilical cord
Matter stem cell media supernatant is smeared at ulcer.
Matched group：Oral ulcer is with, after normal saline cleaning skin surface, dipping comparative example 1 medulla mesenchyma with cotton swab and doing
Cell culture medium supernatant is smeared at ulcer.
Successive administration 7 days, every day entry ulcer situation of change, judge curative effect by following standard：
(1) effective：Ulcer area is obviously reduced, and pain substantially mitigates, and patient can normally take food；
(2) effective：Ulcer area has earlier above and reduces, and pain mitigates earlier above；
(3) invalid：Infectionss area does not reduce or even expands, and clinical symptoms are no improved or even deteriorated.
Each group efficacy result is as follows：
The therapeutic effect to murine oral ulcer for the table 1 each group stem cell supernatant
As table 1 test data understands, compare matched group, the umbilical cord mesenchymal stem cells supernatant of experimental group is burst in treatment oral cavity
The total effective rate of infectionss is higher.Result of the test shows, umbilical cord mesenchymal stem cells supernatant will be considerably better than to the curative effect of oral ulcer
The curative effect of mesenchymal stem cells MSCs supernatant.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. application in preparation treatment stomatocace medicine for the umbilical cord mesenchymal stem cells supernatant.
2. a kind of collutory for treating oral ulcer is it is characterised in that include umbilical cord mesenchymal stem cells supernatant.
3. collutory according to claim 2 is it is characterised in that described medicine also includes pharmaceutically acceptable adjuvant.
4. the collutory according to Claims 2 or 3 is it is characterised in that described umbilical cord mesenchymal stem cells supernatant is the 3rd
～5 generation umbilical cord mesenchymal stem cells supernatant.
5. as any one of claim 2 to 4 preparation method of collutory it is characterised in that to take umbilical cord mesenchyma to do thin
Born of the same parents carry out the first culture using complete medium, then carry out the second culture using the DMEM/F12 culture medium of serum-free, collect
The supernatant of cell culture medium, obtains umbilical cord mesenchymal stem cells supernatant.
6. preparation method according to claim 5 is it is characterised in that described umbilical cord mesenchymal stem cells are the 3rd～5 generation umbilicuss
Band mescenchymal stem cell.
7. the preparation method according to claim 5 or 6 is it is characterised in that the umbilical cord mesenchyma of described first culture is done carefully
The inoculum density of born of the same parents is (1～10) × 103Individual/cm2.
8. the preparation method according to any one of claim 5 to 7 it is characterised in that described first culture culture when
Between be 20～30 hours, condition of culture be 37 DEG C, 5%CO2.
9. the preparation method according to any one of claim 5 to 8 it is characterised in that described second culture culture when
Between be 40～60 hours, condition of culture be 37 DEG C, 5%CO2.
10. the preparation method according to any one of claim 5 to 9 is it is characterised in that described collection cell culture medium
The step being centrifuged, taking supernatant, membrane filtration is also included after supernatant.
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| CN109010523A (en) * | 2018-05-30 | 2018-12-18 | 北京壹典壹生生物技术有限公司 | A kind of canker sore ointment machin preparation method prepared with umbilical cord mesenchymal stem cells secrete cytokines |
| CN110859853A (en) * | 2019-11-28 | 2020-03-06 | 浙江卫未生物医药科技有限公司 | Medicine preparation method based on stem cell supernatant collection, extraction, identification and analysis |
| 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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109010523A (en) * | 2018-05-30 | 2018-12-18 | 北京壹典壹生生物技术有限公司 | A kind of canker sore ointment machin preparation method prepared with umbilical cord mesenchymal stem cells secrete cytokines |
| CN110859853A (en) * | 2019-11-28 | 2020-03-06 | 浙江卫未生物医药科技有限公司 | Medicine preparation method based on stem cell supernatant collection, extraction, identification and analysis |
| 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 |
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Application publication date: 20170208 |