CN104784209B - A kind of stem cell medicine that treating chronic ulcer of skin and preparation method - Google Patents
A kind of stem cell medicine that treating chronic ulcer of skin and preparation method Download PDFInfo
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Abstract
A kind of stem cell medicine that treating chronic ulcer of skin and preparation method are related to a kind of stem cell medicine and preparation method thereof.The present invention provides a kind of stem cell medicine for treating chronic ulcer of skin and preparation method.Said preparation includes mescenchymal stem cell, platelet rich plasma lysate and Fasudil solution.Method: one, isolating and purifying mescenchymal stem cell, secondary culture, takes P3 generation~P5 for mescenchymal stem cell；Two, viral, sterile, immunophenotype, Cell viability, differentiation function detection is carried out for mescenchymal stem cell to P3 generation~P5；Three, platelet rich plasma lysate is prepared；Four, mescenchymal stem cell is resuspended in platelet rich plasma lysate, and Fasudil solution is then added, stem cell medicine can be obtained；The present invention can obviously improve wound healing and shorten wound healing time.The present invention is for treating chronic ulcer of skin.
Description
Technical field
The present invention relates to a kind of stem cell medicine for treating chronic ulcer of skin and preparation methods.
Background technique
Skin is the maximum organ of human body area, is body and extraneous mechanical barrier, have complicated institutional framework and
Multiple physiological function.Creating intractable skin ulcer caused by defect of skin or diabetes chronic diseases after (burnings) hurts is common clinical
One of disease, timely and effectively wound repair is basis and the key problem of such patient's treatment.With the expansion of organizational engineering,
It is expected to provide new means for wound repair using stem cells technology.In recent years, mescenchymal stem cell (Mesenchymal Stem
Cells, MSCs) effect in terms of wound repair is increasingly subject to the extensive concern of scholars.MSCs is that have in mesoderm
The multipotential stem cell of height self-renewing and multi-lineage potential is widely present in whole body Various Tissues, can be cultivated in vitro
Amplification, and it can be divided into the Various Tissues cell such as nerve, sweat gland, bone, cartilage, fat, liver, pancreas under given conditions.Rouge
Fat, bleeding of the umbilicus, umbilical cord, amniotic fluid, the MSCs in placenta source are convenient with materials, and passage capacity is strong, and immune response is low, contaminated general
The advantages that rate is smaller, and no ethics limits gradually is applied to the fields such as wound healing by people.
Wound healing is an extremely complex pathophysiological process, is roughly divided into inflammatory phase, proliferation period and remodeling phase.Damage
When hurting serious, the factors such as tissue continuous anoxic, necrosis, severe infection make 3 periods of wound healing be significantly longer, and heal
It is difficult.It is current studies have shown that MSCs can influence each stage of wound healing to some extent, so that the surface of a wound be accelerated to be cured
It closes.In vivo study shows that, in inflammatory phase, the inflammatory factor and chemotactic factor (CF) of injury tissue and inflammatory cell release can promote MSCs
Migration is gone back to the nest to the surface of a wound, and MSCs is by being divided into wound repair cell and the anti-inflammatory and Anti-G value of paracrine mechanism performance.?
Proliferation period, MSCs can also be played antibacterial by paracrine mechanism and accelerate wound healing the effects of promoting surface of a wound vascularization.
In the remodeling phase, MSCs then passes through the moulding transformation that I, III Collagen Type VI ratio in the regulation surface of a wound realize the surface of a wound.Fasudil, English name
For Fasudil, alias HA1077, entitled -1 (the H) -1,4- azepine of hexahydro -1- (5- sulfonyl isoquinolin) of chemistry, is intracellular
Ca2+ antagonist, kinases inhibitor.HA1077 is Rho kinases specific inhibitor, it, which mainly passes through, inhibits Rho kinases
Activity and play its pharmacological action.Rho albumen is the signal adapter or molecular switch of the Signal Transduction Pathways of cell, Rho activation
Signal transmitting is related with JNK, P38 access, and two signal paths of ERK, p38 and rat bone marrow mesenchymal stem cells are induced to table
Epithelial cell differentiation is related.Studies have shown that HA1077 can promote bone marrow MSCs to enter S phase, the proliferation and differentiation potency of enhanced MSC s
Power.In addition, HAl077 also has facilitation to skin wound healing.HAl077 combined fats, bleeding of the umbilicus, umbilical cord, amniotic fluid, tire
The MSCs treatment refractory wounds in disk source will have good potential applicability in clinical practice.
Platelet rich plasma (PRP) is the concentrate of the blood platelet by obtaining after centrifugation whole blood, after platelet activation,
α particle releases a variety of growth factors, such as platelet derived growth factor, transforming growth factor, insulin-like growth factor, blood
Endothelial tube growth factor etc..Containing the growth factor of higher concentration, about 17 in whole blood times in PRP lysate, activity can
It holds 5-8 days.The concentration of platelet count and growth factor is positively correlated.A variety of growth factor interactions, induce in PRP lysate
Intracellular signals transmission systems can promote cell growth, differentiation, to participate in a series of process of tissue reparation.PRP can promote
The effect of blood coagulation can also stimulate soft tissue regeneration, and promote wound immediate union.Research confirms that these growth factors can also play
Synergy and synergistic effect influence chemotactic, proliferation and the differentiation of cell.
Currently, more stem cell medicine uses merely the solvent of PBS or physiological saline as cell preparation, in complexity
Vivo environment in, largely limit stem cell repair wound healing ability, keep therapeutic effect unsatisfactory.
Summary of the invention
The present invention provides a kind of stem cell medicine for treating chronic ulcer of skin and preparation method.
A kind of stem cell medicine for treating chronic ulcer of skin of the present invention includes that mescenchymal stem cell, platelet rich plasma are split
Liquid and Fasudil solution are solved, the mescenchymal stem cell is umbilical cord mesenchymal stem cells, Amniotic Fluid-derived Mesenchymal Stem Cells or fat
Mescenchymal stem cell.
The preparation method of the stem cell medicine of above-mentioned treatment chronic ulcer of skin sequentially includes the following steps:
One, to mescenchymal stem cell secondary culture, take P3 generation~P5 for mescenchymal stem cell；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function are carried out for mescenchymal stem cell to P3 generation~P5
Detection is used for the preparation of stem cell medicine after indices are qualified；
Three, platelet rich plasma lysate is prepared；
Four, mescenchymal stem cell is resuspended in platelet rich plasma lysate, and the Fasudil that concentration is 20mM is then added
Solution obtains stem cell medicine, that is, completes；The mescenchymal stem cell is umbilical cord mesenchymal stem cells, amniotic fluid mesenchyma is dry thin
Born of the same parents or fat mesenchymal stem cell；(0.5~5) × 10 containing mescenchymal stem cell in platelet rich plasma lysate after resuspension7
A/mL.
Beneficial effects of the present invention:
(1) the invention is characterized in that have drawn HA1077, PRP lysate and fat or umbilical cord or amniotic fluid mesenchyma dry
The strong point of cell and the best compatibility of three, common preparation of completing is to the Regeneration and Repair function of skin injury.
(2) mescenchymal stem cell of the invention is obtained from umbilical cord, fat and sheep water equivalent tisssues.Mescenchymal stem cell is through body
Outer experiment confirms there is the potential for being divided into epidermal cell, fibroblast and vascular endothelial cell, convenient with materials, carefully
The advantages that born of the same parents' quantity is more, and infection risk is small are widely studied, apply；Umbilical cord and Amniotic Fluid-derived Mesenchymal Stem Cells are attached from fetus
The multipotential stem cell isolated in tissue has stronger Proliferation, Differentiation ability, immunogenicity low, therefore has bigger research
Value.
(3) fat stem cell has the characteristics that acquisition is convenient, quantity is more and it is small to injure to donor, and it is dry thin to increase acquisition
Crowd's quantity of born of the same parents can also achieve the effect that beauty treatment weight reducing while obtaining stem cell.
(4) contain high concentration in platelet rich plasma of the invention (Platelet-Rich Plasma, PRP) lysate
Growth factor, these growth factors, which mutually cooperate with, can expand biological effect, and the reparation of wound healing and soft tissue improves wound
Face healing quality.The PRP lysate that this experiment obtains may be from patient itself peripheral blood, be prepared using the method for secondary centrifuging,
The higher active blood platelet of higher concentration can be obtained, and then obtain the growth factor of high concentration, bigger effect is played to wound healing
With.This method materials are simple to operate, small to patient's injury, while avoiding immunological rejection when application, in wound
Reparation field has a good application prospect.
(5) HA1077 is by inhibiting Rho kinases to play pharmacological action, and Rho kinases takes part in various kinds of cell function.
HA1077 can promote skin wound healing, and it is thin to epidermal cell and fat can to enhance it with mesenchyma stem cell combined use
Born of the same parents' Proliferation, Differentiation.Stem cell, HA1077 combine with the various growth factors performance in thrombocyte plasma, act synergistically, and influence thin
Chemotactic, proliferation and the differentiation of born of the same parents makes mescenchymal stem cell play bigger effect.
(6) mescenchymal stem cell preparation prepared by the present invention can by muscle it is dotted injection, subcutaneous tissue injection or directly
It is sprayed on affected part, it is easy to operate, it can obviously improve wound healing and shorten wound healing time, opened for skin ulcer or wound healing
New approach is warded off, and then promotes skin wound healing, is clinically had broad application prospects.
Detailed description of the invention
Fig. 1 is the histogram for testing the rat wound healing index contrast situation for the treatment of group and control group in 1；Wherein
For each group wound healing index after 3d,For each group wound healing index after 7d,It is cured for each group wound after 14d
Hop index；
Fig. 2 is the histogram for testing the rat wound healing index contrast situation for the treatment of group and control group in 2；Wherein
For each group wound healing index after 3d,For each group wound healing index after 7d,It is cured for each group wound after 14d
Hop index；
Fig. 3 is the histogram for testing the rat wound healing index contrast situation for the treatment of group and control group in 3；Wherein
For each group wound healing index after 3d,For each group wound healing index after 7d,It is cured for each group wound after 14d
Hop index.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the stem cell medicine of present embodiment treatment chronic ulcer of skin includes that mesenchyma is dry thin
Born of the same parents, platelet rich plasma lysate and Fasudil solution, the mescenchymal stem cell is between umbilical cord mesenchymal stem cells, amniotic fluid
Mesenchymal stem cells or fat mesenchymal stem cell.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: the Fasudil solution
Concentration is 20mM.It is other same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first and the second embodiment in that: among stem cell medicine
The concentration of mesenchymal stem cells is (0.5~5) × 107A cell/mL.It is other the same as one or two specific embodiments.
Specific embodiment 4: unlike one of present embodiment and specific embodiment one to three: stem cell medicine
The concentration of middle Fasudil solution is 10~40 μm of ol/L.It is other identical as one of specific embodiment one to three.
Specific embodiment 5: the preparation method of the stem cell medicine of present embodiment treatment chronic ulcer of skin is by following
Step carries out:
One, to mescenchymal stem cell secondary culture, take P3 generation~P5 for mescenchymal stem cell；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function are carried out for mescenchymal stem cell to P3 generation~P5
Detection is used for the preparation of stem cell medicine after indices are qualified；
Three, platelet rich plasma lysate is prepared；
Four, mescenchymal stem cell is resuspended in platelet rich plasma lysate, and the Fasudil that concentration is 20mM is then added
Solution obtains stem cell medicine, that is, completes；The mescenchymal stem cell is umbilical cord mesenchymal stem cells, amniotic fluid mesenchyma is dry thin
Born of the same parents or fat mesenchymal stem cell；(0.5~5) × 10 containing mescenchymal stem cell in platelet rich plasma lysate after resuspension7
A/mL.
The preparation method of the umbilical cord mesenchymal stem cells: in aseptic working platform, umbilical cord is disappeared by 75% alcohol
Poison processing, is then cleaned 2 times with physiological saline, then the section for being 2~3cm at length by the umbilical cord scissors after cleaning is placed on plate
It is interior, magnificent general formula glue is removed, then dissect to the bulk of 0.2~2mm, is then cultivated in Tissue Culture Flask, the every 3~5d of originally culture
After carry out changing liquid, after cell fusion degree reaches 80% or more, cell culture fluid is collected, then 1300rpm be centrifuged 6min, take
Supernatant；Twice with normal saline flushing by solid formation, it is then digested at 37 DEG C with the pancreatin that hundred concentration of volume is 0.25%
3min is added supernatant and terminates pancreatin reaction, collect cell, is then centrifuged 6min in 1300rpm, regathers cell, then weigh
It is newly inoculated into another culture bottle and is expanded, finally obtain umbilical cord mesenchymal stem cells.
The preparation method of the umbilical cord mesenchymal stem cells: in aseptic working platform, umbilical cord is disappeared by 75% alcohol
Poison processing, is then cleaned 2 times with physiological saline, then the section for being 2~3cm at length by the umbilical cord scissors after cleaning is placed on plate
It is interior, magnificent general formula glue is removed, then dissect to 0.2~2mm3Bulk, then at 37 DEG C by mass concentration be 0.1%~
0.2% Collagenase I digests 1 hour, obtains single cell suspension, counts and adjusts cell concentration, is then inoculated in sterile culture flask
In, it is placed in 37 DEG C, CO2It is cultivated in the incubator that volumetric concentration is 5%, carries out changing liquid after the every 3~5d of originally culture, melt to cell
It is right reach 80% or more after, cell culture fluid is collected, then 1300rpm be centrifuged 6min, take supernatant；Solid formation is given birth to
It manages salt water to rinse twice, then digests 3min at 37 DEG C with the pancreatin that percent by volume is 0.25%, supernatant is added and terminates
Pancreatin reaction, collects cell, is then centrifuged 6min in 1300rpm, regathers cell, then renewed vaccination is into another culture bottle
It is expanded, finally obtains umbilical cord mesenchymal stem cells.
The preparation method of the Amniotic Fluid-derived Mesenchymal Stem Cells: in aseptic working platform, amniotic fluid 40mL, 1300rpm centrifugation are taken
6min abandons supernatant and takes precipitating, cell is resuspended with the dedicated cell culture medium of AmnioMAX II complete amniotic fluid, according to cell number
It is 5 × 104It is a to be inoculated in T25 bottles, with AmnioMAX II complete cell culture medium polishing to 5ml, it is placed in 37 DEG C, CO2It is dense
The CO that degree is 5%2It is cultivated in incubator.Liquid is changed after 4~6d of cell culture, when cell growth fusion degree reaches 80%~90%
When, it collects cell renewed vaccination and is expanded into another culture bottle, finally obtain Amniotic Fluid-derived Mesenchymal Stem Cells.
The preparation method of the fat mesenchymal stem cell: in aseptic working platform, the adipose tissue that liposuction is obtained divides
In centrifuge tube, 1300rpm is centrifuged 6min, and the bulk fat tissue on the upper layer after centrifugation is poured into another centrifuge tube, is added
Enter physiological saline or PBS cleaning, be centrifuged 6min in 1300rpm, abandons supernatant and take precipitating, clean 3-5 times repeatedly in the same way.
Then the Type I collagen enzyme that the mass percentage of 3 times of volumes is 0.075%, which is added, (takes 7.5g clostridiopetidase A to be dissolved in 100ml sterilizing
In deionized water, 0.22 zut filter is sub-packed in new 50ml centrifuge tube, 100 times of dilutions when use).In 37 DEG C of water-baths
In pot, 15-30min is digested, every 5min, which is shaked gently, to be shaken up once.After digestion, the DMEM with liquid aliquot in pipe is added
Digestion is terminated, the filtration of 200 mesh screens, the fragment tissue generated after removal digestion, then 1300rpm is centrifuged 6min, abandons supernatant, adds
Enter 20ml physiological saline and cell is resuspended, counts.It is centrifuged 6min in 1300rpm, abandon supernatant and the DMEM culture containing 10%FBS is added
Base weight hangs cell, by 2~3 × 106In a cell/T75 culture bottle, and with the DMEM containing 10%FBS by liquid in culture bottle
11ml is complemented to, the CO of 37 DEG C, 5% is placed in2It is cultivated in incubator.It changes the liquid once within originally culture every 3~5 days, when cell is grown
When degrees of fusion reaches 80%, collects cell renewed vaccination and expanded into another culture bottle, it is dry to finally obtain fat mesenchymal
Cell.
The preparation method of platelet rich plasma (PRP) lysate: in aseptic working platform, no infectiousness, tumour are taken
Property, disease in the blood system medical history peripheric venous blood 200ml, first survey Whole blood platelet number > 105 × 109/ L, whole blood with 200~
7001300r/min is centrifuged 10min, and whole blood is divided into three layers, i.e. upper layer supernatant in heparin tube at this time, and milky middle layer is under
Layer red blood cell is collected supernatant, milky middle layer and 2mm red blood cell layer and is placed in new centrifuge tube, again 700r/min
Lower centrifugation 15min, collecting 1/4 yellow supernatant is PRP, after Automatic Blood Cell Analyzer detection is qualified.PRP is sub-packed in
It in EP pipe, is stored in -80 DEG C of refrigerators, when use takes out PRP from -80 DEG C of refrigerators, puts into 37 DEG C of water-baths rapidly, makes it quickly
It thaws.It is then centrifuged for, takes supernatant through 0.22 μm of sterile filters filtering as platelet rich plasma lysate；Wherein PRP is qualified
Standard 4 times or more of platelet count when be PRP platelet count being whole blood.
Specific embodiment 6: present embodiment is unlike specific embodiment five: step 2 middle finger target is qualified
Refer to: virus and Bacteria Detection are feminine gender, and immunophenotype positive expression rate is 95% or more, and negative expression rate is lower than 2%, carefully
Born of the same parents' motility rate is at least 95%.It is other identical as specific embodiment five.
Specific embodiment 7: present embodiment is unlike specific embodiment five or six: step 3 stem cell system
Final concentration of 10~40 μm of ol/L of Fasudil solution in agent.It is other identical as specific embodiment five or six.
To verify beneficial effects of the present invention, the following experiment of progress:
Beneficial effects of the present invention are verified by following tests:
The preparation method for testing the stem cell medicine of 1, this test of cure chronic ulcer of skin sequentially includes the following steps:
One, to fat mesenchymal stem cell secondary culture, P3 fat subsitutes mescenchymal stem cell is taken；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function inspection is carried out to P3 fat subsitutes mescenchymal stem cell
It surveys, the preparation of stem cell medicine is used for after indices are qualified；
Three, platelet rich plasma lysate is prepared；
Four, fat mesenchymal stem cell is resuspended in platelet rich plasma lysate, and the method that concentration is 20mM is then added and relaxes
You obtain stem cell medicine by solution on ground, that is, complete；The concentration of mescenchymal stem cell is (0.5~5) × 10 in stem cell medicine7It is a
Cell/mL, the concentration of Fasudil solution is 15 μm of ol/L in stem cell medicine, in the platelet rich plasma lysate after resuspension
Containing mescenchymal stem cell (0.5~5) × 107A/mL.
The preparation method of fat mesenchymal stem cell described in step 1: in aseptic working platform, by the fat of liposuction acquisition
Tissue is divided in centrifuge tube, and 1300rpm is centrifuged 6min, and the bulk fat tissue on the upper layer after centrifugation is poured into another centrifuge tube
In, physiological saline cleaning is added, is centrifuged 6min in 1300rpm, abandons supernatant and takes precipitating, clean 4 times repeatedly in the same way.So
The Type I collagen enzyme that the mass percentage that 3 times of volumes are added afterwards is 0.075% (takes 7.5g clostridiopetidase A to be dissolved in going for 100ml sterilizing
In ionized water, 0.22 zut filter is sub-packed in new 50ml centrifuge tube, 100 times of dilutions when use).In 37 DEG C of water-baths
In, 25min is digested, every 5min, which is shaked gently, to be shaken up once.After digestion, it is added and disappears with the DMEM termination of liquid aliquot in pipe
Change, the filtration of 200 mesh screens, the fragment tissue generated after removal digestion, then 1300rpm is centrifuged 6min, abandons supernatant, and 20ml is added
Cell is resuspended in physiological saline, counts.It is centrifuged 6min in 1300rpm, abandon supernatant and the DMEM culture medium resuspension containing 10%FBS is added
Cell, by 2~3 × 106In a cell/T75 culture bottle, and liquid in culture bottle is complemented to the DMEM containing 10%FBS
11ml is placed in the CO of 37 DEG C, 5%2It is cultivated in incubator.It changes the liquid once within originally culture every 5 days, when cell growth fusion degree reaches
When 80%, collects cell renewed vaccination and expanded into another culture bottle, finally obtain fat mesenchymal stem cell.
Central bay mesenchymal stem cells preparation can treat the curative effect of chronic ulcer of skin as evidence, fill between containing after preparation respectively
Matter stem cell, PRP lysate and Fasudil stem cell medicine；Contain mescenchymal stem cell and platelet rich plasma lysate
Stem cell medicine；The stem cell medicine of mescenchymal stem cell and Fasudil；The preparation of the mescenchymal stem cell containing equivalent is distinguished
Rat skin affected part is entered using intracutaneous injection.This experiment use SD Immune deficient mice, half male and half female, weight about 200~250g,
It is randomly divided into control group and treatment group.It is burnt by chemical method and establishes skin injury model, be at random A, B by therapeutic component,
C, D group is individually insulated under identical environment and raises.Injection 2 × 106The mescenchymal stem cell mixed liquor of a cell, control group injection
Same amount of normal saline, the next day additional 1 medication, dose is the same as the first time.The above each group treatment starts rear 3d, 7d, 14d observation wound
Healing state calculates wound healing index (WCI):
WCI (%)=(1- shows surface of a wound area/original face area) × 100%
Specific test is grouped as follows:
Control group: physiological saline；
Treat A group: the fat mesenchymal stem cell of this test preparation；
Treat B group: the fat mesenchymal stem cell of this test preparation+this test preparation PRP lysate；
Treat C group: the fat mesenchymal stem cell of this test preparation+this test preparation HA1077；
Treat D group: the stem cell medicine of this test preparation.
The comparison (average ± standard deviation x ± s, %) of 1 each group Rat Wound Healing WCI of table
This test result is as shown in table 1 and Fig. 1, by figure 1 above and table 1 it is found that the stem cell medicine pair of this test preparation
The therapeutic effect of the rat surface of a wound is better than other groups.
The preparation method for testing the stem cell medicine of 2, this test of cure chronic ulcer of skin sequentially includes the following steps:
One, to Amniotic Fluid-derived Mesenchymal Stem Cells secondary culture, take P3 for Amniotic Fluid-derived Mesenchymal Stem Cells；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function inspection is carried out for Amniotic Fluid-derived Mesenchymal Stem Cells to P3
It surveys, the preparation of stem cell medicine is used for after indices are qualified；
Three, platelet rich plasma lysate is prepared；
Four, Amniotic Fluid-derived Mesenchymal Stem Cells are resuspended in platelet rich plasma lysate, and the method that concentration is 20mM is then added and relaxes
Ground that solution, then it is settled to 1mL with platelet rich plasma lysate, stem cell medicine is obtained, that is, is completed；Among stem cell medicine
The concentration of mesenchymal stem cells is (0.5~5) × 107A cell/mL, the concentration of Fasudil solution is 20 μ in stem cell medicine
Mol/L, (0.5~5) × 10 containing mescenchymal stem cell in the platelet rich plasma lysate after resuspension7A/mL.
The preparation method of the Amniotic Fluid-derived Mesenchymal Stem Cells: in aseptic working platform, amniotic fluid 40mL, 1300rpm centrifugation are taken
6min abandons supernatant and takes precipitating, cell is resuspended with the dedicated cell culture medium of AmnioMAX II complete amniotic fluid, according to cell number
It is 5 × 104It is a to be inoculated in T25 bottles, with AmnioMAX II complete cell culture medium polishing to 5ml, it is placed in 37 DEG C, CO2It is dense
The CO that degree is 5%2It is cultivated in incubator.Liquid is changed after 4~6d of cell culture, when cell growth fusion degree reaches 90%, is collected
Cell renewed vaccination is expanded into another culture bottle, finally obtains Amniotic Fluid-derived Mesenchymal Stem Cells.
Specific test is grouped as follows:
Control group: physiological saline；
Treat A group: the Amniotic Fluid-derived Mesenchymal Stem Cells of this experiment preparation；
Treat B group: the Amniotic Fluid-derived Mesenchymal Stem Cells of this test preparation+this test preparation PRP lysate；
Treat C group: the Amniotic Fluid-derived Mesenchymal Stem Cells of this test preparation+this test preparation HA1077；
Treat D group: the stem cell medicine of this test preparation.
The comparison (average ± standard deviation x ± s, %) of 2 each group Rat Wound Healing WCI of table
This test result is as shown in table 2 and Fig. 2, by figure 2 above and table 2 it is found that the stem cell medicine pair of this test preparation
The therapeutic effect of the rat surface of a wound is better than other groups.
The preparation method for testing the stem cell medicine of 3, this test of cure chronic ulcer of skin sequentially includes the following steps:
One, to umbilical cord mesenchymal stem cells secondary culture, take P5 for umbilical cord mesenchymal stem cells；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function inspection is carried out for umbilical cord mesenchymal stem cells to P5
It surveys, the preparation of stem cell medicine is used for after indices are qualified；
Three, platelet rich plasma lysate is prepared；
Four, umbilical cord mesenchymal stem cells are resuspended in platelet rich plasma lysate, and the method that concentration is 20mM is then added and relaxes
Ground that solution, then it is settled to 1mL with platelet rich plasma lysate, stem cell medicine is obtained, that is, is completed；Among stem cell medicine
The concentration of mesenchymal stem cells is (0.5~5) × 107A cell/mL, the concentration of Fasudil solution is 15 μ in stem cell medicine
Mol/L, (0.5~5) × 10 containing mescenchymal stem cell in the platelet rich plasma lysate after resuspension7A/mL.
The preparation method of the umbilical cord mesenchymal stem cells: in aseptic working platform, umbilical cord is disappeared by 75% alcohol
Poison processing, is then cleaned 2 times with physiological saline, then the section for being 3cm at length by the umbilical cord scissors after cleaning is placed in plate,
Magnificent general formula glue is removed, then is dissected to 0.2~2mm3Bulk, then at 37 DEG C by mass concentration be 0.1% Collagenase I
Digestion 1 hour obtains single cell suspension, counts and adjusts cell concentration, is then inoculated in sterile culture flask, is placed in 37 DEG C, CO2
Volumetric concentration be 5% incubator in cultivate, carry out changing liquid after the every 3~5d of originally culture, to cell fusion degree reach 80% with
After upper, cell culture fluid is collected, then 1300rpm is centrifuged 6min, takes supernatant；By solid formation normal saline flushing two
It is secondary, 3min is then digested at 37 DEG C with the pancreatin that percent by volume is 0.25%, and supernatant is added and terminates pancreatin reaction, collects
Then cell is centrifuged 6min in 1300rpm, regathers cell, then renewed vaccination is expanded into another culture bottle, finally
Obtain umbilical cord mesenchymal stem cells.
Specific test is grouped as follows:
Control group: physiological saline；
Treat A group: the umbilical cord mesenchymal stem cells of this experiment preparation；
Treat B group: the umbilical cord mesenchymal stem cells of this test preparation+this test preparation PRP lysate；
Treat C group: the umbilical cord mesenchymal stem cells of this test preparation+this test preparation HA1077；
Treat D group: the stem cell medicine of this test preparation.
The comparison (average ± standard deviation x ± s, %) of 3 each group Rat Wound Healing WCI of table
This test result is as shown in table 3 and Fig. 3, by table 3 and Fig. 3 it is found that the stem cell medicine of this test preparation is to rat
The therapeutic effect of the surface of a wound is better than other groups.
By test 1,2 and 3 it is found that the stem cell medicine of this test preparation can obviously improve wound healing and shorten wound and is cured
The time is closed, new approach is opened up for skin ulcer or wound healing, and then promote skin wound healing, clinically has wide
Application prospect.
Claims (3)
1. a kind of stem cell medicine for treating chronic ulcer of skin, it is characterised in that said preparation includes mescenchymal stem cell, rich blood
Platelet-poor plasma lysate and Fasudil solution, the mescenchymal stem cell is umbilical cord mesenchymal stem cells, amniotic fluid mesenchyma is dry
Cell or fat mesenchymal stem cell；Wherein mescenchymal stem cell is resuspended in platelet rich plasma lysate, in stem cell medicine
Final concentration of (0.5~5) × 10 of mescenchymal stem cell7A cell/mL；Final concentration of 10~40 μ of Fasudil solution
mol/L。
2. a kind of stem cell medicine for treating chronic ulcer of skin according to claim 1, it is characterised in that the method is relaxed
The concentration of your solution of ground is 20mM.
3. the preparation method of the stem cell medicine for the treatment of chronic ulcer of skin as described in claim 1, it is characterised in that this method
It sequentially includes the following steps:
One, to mescenchymal stem cell secondary culture, take P3 for mescenchymal stem cell；
Two, viral, sterile, immunophenotype, Cell viability, differentiation function detection is carried out for mescenchymal stem cell to P3, it is every
The preparation of stem cell medicine is used for after qualified；
Three, platelet rich plasma lysate is prepared；
Four, mescenchymal stem cell is resuspended in platelet rich plasma lysate, and the Fasudil solution that concentration is 20mM is then added,
Stem cell medicine is obtained, that is, is completed；The mescenchymal stem cell is umbilical cord mesenchymal stem cells, Amniotic Fluid-derived Mesenchymal Stem Cells or rouge
Fat mescenchymal stem cell；(0.5~5) × 10 containing mescenchymal stem cell in platelet rich plasma lysate after resuspension7A/mL,
Wherein indices qualification described in step 2 refers to: virus and Sterility testing are feminine gender, and the positive expression rate of immunophenotype is big
In 95%, negative example reaches less than 2%, and Cell viability reaches 95% or more, has differentiation function.
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| CN105477018A (en) * | 2015-12-07 | 2016-04-13 | 深圳爱生再生医学科技有限公司 | Stem cell-based medicinal product for repairing skin ulcer and preparation method of stem cell-based medicinal product for repairing skin ulcer |
| CN105708860A (en) * | 2016-01-26 | 2016-06-29 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for repairing skin ulcer |
| CN106039293A (en) * | 2016-07-28 | 2016-10-26 | 广州赛莱拉干细胞科技股份有限公司 | Composition for repairing skin ulcers and preparation method thereof |
| CN106176813A (en) * | 2016-07-28 | 2016-12-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN106074605A (en) * | 2016-07-28 | 2016-11-09 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN106668838A (en) * | 2017-01-22 | 2017-05-17 | 广州赛莱拉干细胞科技股份有限公司 | Aminiotic fluid-derived mesenchymal stem cells combined preparation and preparation method and application thereof |
| CN107754002A (en) * | 2017-12-04 | 2018-03-06 | 广州市天河诺亚生物工程有限公司 | A kind of biomaterial preparation method with Stem Cell Activity |
| CN108715834B (en) * | 2018-06-01 | 2021-09-14 | 天晴干细胞股份有限公司 | Preparation method of platelet lysate rich in CD41+ and CD81+ microcapsules |
| CN109700998B (en) * | 2019-02-25 | 2022-02-08 | 曾湘红 | Compound skin injury regeneration repairing agent and preparation method thereof |
| CN112315978A (en) * | 2019-07-19 | 2021-02-05 | 丰泽康生物医药（深圳）有限公司 | Peripheral blood multipotential cell active matter and platelet-rich plasma compound and preparation method and application thereof |
| CN111494423B (en) * | 2020-04-22 | 2022-01-25 | 上海市东方医院（同济大学附属东方医院） | Stem cell composition, application thereof and stem cell dropping liquid |
| CN111467374A (en) * | 2020-04-28 | 2020-07-31 | 杨喻丹 | Novel leukocyte extract mixed factor and preparation method thereof |
| CN113995772A (en) * | 2020-07-27 | 2022-02-01 | 是光隽恒(北京)生物科技有限公司 | Composition for wound repair and preparation method and application thereof |
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