CN106511384A - Freeze-dried powder used for promoting healing of diabetic wound and preparing method thereof - Google Patents
Freeze-dried powder used for promoting healing of diabetic wound and preparing method thereof Download PDFInfo
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- CN106511384A CN106511384A CN201610979822.8A CN201610979822A CN106511384A CN 106511384 A CN106511384 A CN 106511384A CN 201610979822 A CN201610979822 A CN 201610979822A CN 106511384 A CN106511384 A CN 106511384A
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention discloses freeze-dried powder used for promoting healing of a diabetic wound and a preparing method thereof. The freeze-dried powder is made by freeze-drying a serum-free nutrient solution in an endothelial progenitor cell. The preparing method specifically comprises the steps of separating cells with mononuclear cell layers, inoculating the cells in a culture flask to be subjected to preliminary cultivation, changing a culture solution in a whole-content mode in the fourth day, and afterwards changing the culture solution once every 2-3 days; conducting continuous cell culture after the cells grows until 70-80% of the cells fuse; culturing the cells to the third to fifth generation, and changing the culture solution to be subjected to hypoxia inducing; conducting reoxygenation cultivation on the inducing cultured cells after changing the culture solution into the serum-free culture solution; conducting centrifugal filtration on the culture solution, collecting the culture solution, and conducing concentration and freeze-drying on the culture solution. The preparing method is simple, convenient and easy to operate, low in cost, high in efficiency and good in wound treating effect of diabetes, and the freeze-dried powder can be stored for a long time.
Description
Technical field
The present invention relates to a kind of preparation for promoting union of wounded skin, is related specifically to a kind of promotion diabetic wound healing
Freeze-dried powder preparation.
Background technology
Diabetes have become the serious chronic disease for threatening human health after tumor, cardiovascular pathological changes.Diabetes
To the maximum threat of patient from its complication, wherein, Tissue of Diabetic Wound (diabetic wound) has become serious and has threatened sugar
Urine patient existence and quality of life severe complication, heal with refractory, easy infection, high amputation rate the characteristics of, its pathology machine
System is complicated.At present, Tissue of Diabetic Wound is mainly limited to conventional process, and clinical therapeutic efficacy is undesirable, and some newly developed are single
The effect of drugs such as somatomedin class are still relatively limited, lack easy economy, safely and efficiently newtype drug and Biotherapeutics means.Cause
This, how to effectively facilitate diabetic wound healing has become the problem of multidisciplinary common concern, captures the treatment of Tissue of Diabetic Wound
Need new strategy and break-through point.
As cell therapy (cell therapy) is in the rise in clinical application research field, the cell based on stem cell
Treatment shows good using value in clinical treatment, and obtains certain effect, day by day becomes one and promising controls
Treat and select.At present, cell transplantation and paracrine mechanism be stem cell be clinical application research two main paths.With to dry
Cell research deepens continuously, and the clinical application research of cell transplantation runs into some problems.Research shows transplanting stem cell to body
Inside be divided into that histiocytic probability is very low and the time-to-live is short after transplanting, be not suitable for it is external a large amount of produce, using inconvenience, may
It is related to some disputes that stem cell uses, these problems become the obstacle of clinical practice.At the same time, stem cell is produced by which
Paracrine factor network improve Pathologic niche, the thinking for regeneration and restoration is gradually received, become stem cell rush
Enter the important channel of wound repair.Stem cell paracrine mechanism is of increasing concern, and then formation one kind with paracrine mechanism is
New " acellular " therapeutic strategy (cell-free strategy) on basis.
At present, conventional Therapeutic Method be stem cell is cultivated after treat Tissue of Diabetic Wound preparation, but it is this
Method slowly effect, it is relatively costly to be unfavorable for popularization and application on a large scale.Therefore invent a kind of safe and efficient, easy easy-to-use promotion
The preparation of diabetic wound healing is significant.
The content of the invention
It is an object of the invention to provide a kind of be used to promote diabetic wound healing lyophilized powder and preparation method thereof
The freeze-dried powder preparation of the present invention is formed by human endothelial cell Disordered hopping model lyophilizing, its preparation method
For：
1) mononuclear cell confluent monolayer cells are separated, and is tentatively cultivated in being inoculated in culture bottle, full dose changes culture fluid within the 4th day, with
It is every afterwards
Culture fluid was changed once every 2-3 days；
2) Secondary Culture is carried out when cell growth to 70-80% merges；
3) to 3-5 generations, change culture fluid carries out hypoxia inducible to cultured cells；
4) by the cell after inducing culture, after changing serum-free medium, carry out reoxygenation culture；
5) centrifugal filtration, collects culture fluid, concentration and lyophilizing.
Preferably, tentatively cultivate under saturated humidity, 5% gas concentration lwevel, 37 DEG C of constant temperatures.
Preferably, in 0.5-2% oxygen concentrations, carry out inducing 24-72 hours under saturated humidity, 5% gas concentration lwevel
Under the conditions of induced.
Preferably, the culture fluid of replacing is serum-free basic culture solution.
Preferably, in 21% oxygen concentration, reoxygenation culture under saturated humidity, 5% gas concentration lwevel.
Preferably, reoxygenation incubation time is 24～72h.
The invention has the beneficial effects as follows：
1) with angiogenesis and neurotrophy dual-use function is promoted, angiogenesiss and the god of Tissue of Diabetic Wound can be promoted
Jing regenerates, and accelerates diabetic wound healing, promotes wound repair, reduces wound surface and further deteriorates and its complication brought and not
Good consequence.
2) people's endothelial progenitor cells are obtained from umbilical blood, abundance is easy to operation, low cost, efficiency high, for glycosuria
Sick Wound treating effect is good.Lyophilized powder is prepared, under certain preservation condition, active factorses can preserve maximum activity, and play
Maximum therapeutical effect
Specific embodiment
With reference to specific embodiment, the claim of the present invention is described in further detail, but do not constitute it is right
Any restriction of the present invention, the modification of anyone limited number of time for being made within the scope of the invention as claimed, still the present invention's
In claims.
1 EPC cell culture fluid lyophilized powders (I) of embodiment
1) extraction of people's umbilical cord blood endothelial progenitor cells：Syringe extraction 60ml healthy newborn Cord blood, heparin sodium anticoagulant, plus
Volume ratio 1:1PBS dilutes, and is slowly added to containing 2:In Ficoll (1.077) the lymphocyte separation medium centrifuge tube of 1 volume ratio,
By density－gradient centrifuga－tion method, horizontal centrifuge 2000rpm, be centrifuged within 25 minutes, mononuclearcell layer carefully drawn with suction pipe.So
Equal-volume PBS cell, horizontal centrifuge 2000rpm is used to be centrifuged within 8 minutes, cell is inoculated in people's fibronectin afterwards
Coated 25cm2In culture bottle, plus appropriate culture fluid of endothelial cell culture (containing 10% hyclone), saturated humidity, 5% 2
Oxidation concentration of carbon, cultivates under 37 DEG C of constant temperature.
2) amplification cultivation of people's umbilical cord blood endothelial progenitor cells：Full dose changes culture fluid within 4th day, changes training every 2-3 days later
Nutrient solution is once.People's umbilical cord blood endothelial progenitor cells are adhere-wall culture cell, as incubation time, cell shape and arrangement mode have become
Change, Secondary Culture is carried out when cell growth to 70-80% merges.
3) hypoxic preconditioning of people's umbilical cord blood endothelial progenitor cells：Cultured cells treats cell growth to 80% fusion to the 3rd generation
When, cell is placed in into 2% oxygen concentration, induction 48 hours under saturated humidity, 5% gas concentration lwevel, is carried out；
4) the reoxygenation culture of people's umbilical cord blood endothelial progenitor cells：Replacing cell culture fluid is serum-free basic culture solution, is recovered
21% oxygen concentration, cultivates 24h under saturated humidity, 5% gas concentration lwevel；
5) centrifugal filtration of people's umbilical cord blood endothelial progenitor cells conditioned medium, concentration and lyophilizing.
2 EPC cell culture fluid lyophilized powders (II) of embodiment
According in embodiment 1 1), 2) the step of Secondary Culture endothelial progenitor cells.In cultured cells to the 3rd generation, treat that cell is given birth to
It is long to 70-80% merge when, cell is placed in into 0.5% oxygen concentration, hypoxia under the conditions of saturated humidity, 5% gas concentration lwevel
Induction 36h, afterwards, replacings cell culture fluid is serum-free basic culture solution, 21% oxygen concentration of recovery, saturated humidity, 5% 2
72h is cultivated under oxidation concentration of carbon.By the centrifugal filtration of culture medium, concentration and lyophilizing.
3 EPC cell culture fluid lyophilized powders (III) of embodiment
According in embodiment 1 1), 2) the step of Secondary Culture endothelial progenitor cells.In cultured cells to the 5th generation, treat that cell is given birth to
It is long to 70-80% merge when, cell is placed in into 1.5% oxygen concentration, hypoxia under the conditions of saturated humidity, 5% gas concentration lwevel
Induction 24h, afterwards, replacings cell culture fluid is serum-free basic culture solution, 21% oxygen concentration of recovery, saturated humidity, 5% 2
48h is cultivated under oxidation concentration of carbon.By the centrifugal filtration of culture medium, concentration and lyophilizing.
4 EPC cell culture fluid lyophilized powders (IV) of embodiment
According in embodiment 1 1), 2) the step of Secondary Culture endothelial progenitor cells.In cultured cells to the 4th generation, treat that cell is given birth to
It is long to 70-80% merge when, cell is placed in into 1% oxygen concentration, under the conditions of saturated humidity, 5% gas concentration lwevel, hypoxia is lured
72h is led, afterwards, replacing cell culture fluid is serum-free basic culture solution, recovers 21% oxygen concentration, saturated humidity, 5% dioxy
Change 24h is cultivated under concentration of carbon.By the centrifugal filtration of culture medium, concentration and lyophilizing.
Comparative example EPC cell culture fluid lyophilized powder (O)
According in embodiment 1 1), 2) the step of Secondary Culture endothelial progenitor cells.In cultured cells to the 3rd generation, treat that cell is given birth to
It is long to 70-80% merge when, cell is placed in into 2% oxygen concentration, under the conditions of saturated humidity, 5% gas concentration lwevel, hypoxia is lured
48h is led, afterwards, by the centrifugal filtration of culture medium, concentration and lyophilizing.
Experimental example
1) time of wound healing
The freeze-dried powder preparation of embodiment 1 and comparative example is carried out into wound healing experiment：Wound surface records wound after being formed daily
Wound surface area after formation, records wound healing time.Using method：Directly shoot little by the unified parameters for arranging with digital camera
Mus wound surface region (containing scale).Wound surface part in picture is surveyed using IPP (Image-Pro Plus) image processing software
Measure its area.As a result show, according to lyophilized powder prepared by the method for embodiment 1, its wound healing time is 12.8 ± 0.789
My god, and its wound healing time of lyophilized powder prepared by the method for comparative example is 14.1 ± 0.738 days.Further, wound surface is healed
The IOD values for closing CD31 in the 10th day detection wound healing tissue are detected：Specimen Jing is fixed embedding, section, the aquation that dewaxes, is resisted
Former hot repair is multiple, microscopy after one AntiCD3 McAb 1 of rabbit anti-mouse and two anti-incubations, under the microscope per 5 visuals field of section random acquisition
(l0x10 times), measures its average integral optical density (mean using Image analysis system (Image-pro plus 6.0)
Of IOD)), as a result show：Lyophilized powder prepared by the method for embodiment 1, in the 10th day detection wound healing tissue of wound healing
The IOD values of CD31 are 15903.85 ± 1729.30.And lyophilized powder prepared by the method for comparative example, IOD values for 13399.19 ±
752.23。
2) in EPC-CM active factorses content
With in protein chip half-quantitative detection EPC cell culture fluid lyophilized powder (O) and EPC cell culture fluid lyophilized powders (I)
Promote the content of wound healing correlation factor, its result is as shown in the table：
The rush wound healing correlation factor content of EPC cell culture fluid lyophilized powders (O)
| Active factorses | Signal value | Active factorses | Signal value | Active factorses | Signal value |
| PDGF-BB | 395,269 | Angiopoietin-2 | 278,013 | Angiogenin | 247,908 |
| MCP-3 | 200,966 | uPAR | 105,039 | EGF | 73,550 |
| IL-6 | 82,900 | RANTES | 29,276 | I-TAC | 33,456 |
| GRO | 46,711 | MMP-1 | 33,457 | PECAM-1 | 16,160 |
| VEGF R2 | 5,790 | ENA-78 | 5,740 | PLGF | 4,097 |
| IL-1alpha | 2,841 | Tie-2 | 2,545 | IL-4 | 2,878 |
| TGF-beta1 | 2,108 | Thrombopoietin | 1,669 | VEGF R3 | 1,630 |
| IFN-gamma | 1,078 | GRO-alpha | 1,193 | IL-10 | 1,212 |
The rush wound healing correlation factor content of EPC cell culture fluid lyophilized powders (I)
The above results show, in the lyophilized powder that the culture medium lyophilized powder in embodiment 1 is prepared compared to comparative example method, promote
The content of wound healing correlation factor is higher.
In sum, compared to comparative example, healing time is shorter, contains for the lyophilized powder that prepared by the method for the present patent application
Related conglutinant protein factor is more, with more good promoting healing effect.
Claims (8)
1. a kind of preparation for promoting diabetic wound healing, it is characterised in that：The preparation is cell non-serum culture fluid
Lyophilized powder.
2. preparation according to claim 1, it is characterised in that：Cell culture fluid lyophilized powder is that derived from cord blood people endothelium ancestral is thin
Born of the same parents' culture fluid lyophilized powder.
3. according to claim 1 ~ 3 any one claim preparation preparation method, comprise the following steps：
1）Mononuclear cell confluent monolayer cells are separated, and is tentatively cultivated in being inoculated in culture bottle, full dose changes culture fluid within the 4th day, often later
Culture fluid was changed once every 2-3 days；
2）Secondary Culture is carried out when cell growth to 70-80% merges；
3）To 3-5 generations, change culture fluid carries out hypoxia inducible to cultured cells；
4) by the cell after inducing culture, after changing serum-free medium, carry out reoxygenation culture；
5）Centrifugal filtration, collects culture fluid, concentration and lyophilizing.
4. preparation method according to claim 4, it is characterised in that：In saturated humidity, 5% gas concentration lwevel, 37 DEG C permanent
Tentatively cultivate under the conditions of temperature.
5. preparation method according to claim 4, it is characterised in that：In 0.5-2% oxygen concentrations, saturated humidity, 5% dioxy
Change and induced under the conditions of carrying out inducing 24-72 hours under concentration of carbon.
6. preparation method according to claim 4, it is characterised in that：The culture fluid changed after hypoxia inducible culture is depletion of blood
Clear basic culture solution.
7. preparation method according to claim 4, it is characterised in that：In 21% oxygen concentration, saturated humidity, 5% titanium dioxide
Reoxygenation culture under concentration of carbon.
8. preparation method according to claim 4, it is characterised in that：Reoxygenation incubation time is 24～72h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610979822.8A CN106511384A (en) | 2016-11-08 | 2016-11-08 | Freeze-dried powder used for promoting healing of diabetic wound and preparing method thereof |
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|---|---|---|---|
| CN201610979822.8A CN106511384A (en) | 2016-11-08 | 2016-11-08 | Freeze-dried powder used for promoting healing of diabetic wound and preparing method thereof |
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| CN101985052A (en) * | 2010-08-31 | 2011-03-16 | 中国人民解放军第二军医大学 | Skin substitute for automatically capturing endothelial progenitor cells and promoting vascularization and construction method thereof |
| CN102482698A (en) * | 2009-07-10 | 2012-05-30 | 希斯托金公司 | Conditioned Medium And Extracellular Matrix Compositions From Cells Cultured Under Hypoxic Conditions |
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| CN104971382A (en) * | 2014-04-01 | 2015-10-14 | 上海合锐生物技术有限公司 | Adhesive bandage type artificial active tissue constructed by using culture solution without serum or bovine pituitary extracts and construction method of adhesive bandage type artificial active tissue |
| CN105073980A (en) * | 2012-09-28 | 2015-11-18 | 公益财团法人先端医疗振兴财团 | Method for in vitro proliferation of cell population containing cells suitable for treatment of ischemic disease |
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2016
- 2016-11-08 CN CN201610979822.8A patent/CN106511384A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101151362A (en) * | 2004-06-01 | 2008-03-26 | 运作投资有限公司 | In vitro techniques for use with stem cells |
| CN101153317A (en) * | 2007-10-19 | 2008-04-02 | 北京航空航天大学 | Method for continuously detecting amount of viable cell on tissue engineering bracket and tissue implant article |
| CN102482698A (en) * | 2009-07-10 | 2012-05-30 | 希斯托金公司 | Conditioned Medium And Extracellular Matrix Compositions From Cells Cultured Under Hypoxic Conditions |
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| CN101985052A (en) * | 2010-08-31 | 2011-03-16 | 中国人民解放军第二军医大学 | Skin substitute for automatically capturing endothelial progenitor cells and promoting vascularization and construction method thereof |
| CN105073980A (en) * | 2012-09-28 | 2015-11-18 | 公益财团法人先端医疗振兴财团 | Method for in vitro proliferation of cell population containing cells suitable for treatment of ischemic disease |
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| CN104971382A (en) * | 2014-04-01 | 2015-10-14 | 上海合锐生物技术有限公司 | Adhesive bandage type artificial active tissue constructed by using culture solution without serum or bovine pituitary extracts and construction method of adhesive bandage type artificial active tissue |
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