CN110478369A - A kind of composite dry cell bioagent - Google Patents
A kind of composite dry cell bioagent Download PDFInfo
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- CN110478369A CN110478369A CN201910880985.4A CN201910880985A CN110478369A CN 110478369 A CN110478369 A CN 110478369A CN 201910880985 A CN201910880985 A CN 201910880985A CN 110478369 A CN110478369 A CN 110478369A
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- stem cell
- fat stem
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- 239000002131 composite material Substances 0.000 title claims abstract description 33
- 210000000130 stem cell Anatomy 0.000 claims abstract description 53
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- 239000002904 solvent Substances 0.000 claims abstract description 19
- RXUWDKBZZLIASQ-UHFFFAOYSA-N Puerarin Natural products OCC1OC(Oc2c(O)cc(O)c3C(=O)C(=COc23)c4ccc(O)cc4)C(O)C(O)C1O RXUWDKBZZLIASQ-UHFFFAOYSA-N 0.000 claims abstract description 13
- HKEAFJYKMMKDOR-VPRICQMDSA-N puerarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 HKEAFJYKMMKDOR-VPRICQMDSA-N 0.000 claims abstract description 13
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 claims abstract description 12
- QXMNTPFFZFYQAI-IMDKZJJXSA-N beta-sitosterol 3-O-beta-D-glucopyranoside Natural products CC[C@H](CC[C@@H](C)[C@H]1CC[C@H]2[C@@H]3CC=C4C[C@H](CC[C@]4(C)[C@H]3CC[C@]12C)O[C@@H]5C[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)C(C)C QXMNTPFFZFYQAI-IMDKZJJXSA-N 0.000 claims abstract description 9
- NPJICTMALKLTFW-OFUAXYCQSA-N daucosterol Chemical compound O([C@@H]1CC2=CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CC[C@@H](CC)C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O NPJICTMALKLTFW-OFUAXYCQSA-N 0.000 claims abstract description 9
- QDFKFNAHVGPRBL-UHFFFAOYSA-N daucosterol Natural products CCC(CCC(C)C1CCC2C1CCC3C2(C)CC=C4CC(CCC34C)OC5OC(CO)C(O)C(O)C5O)C(C)C QDFKFNAHVGPRBL-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims description 22
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- 238000000034 method Methods 0.000 claims description 9
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- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000012797 qualification Methods 0.000 claims description 8
- 239000003124 biologic agent Substances 0.000 claims description 7
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- 102100022464 5'-nucleotidase Human genes 0.000 claims description 6
- 102000029816 Collagenase Human genes 0.000 claims description 6
- 108060005980 Collagenase Proteins 0.000 claims description 6
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 6
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 claims description 6
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 6
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 claims description 6
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 6
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 6
- 102100025304 Integrin beta-1 Human genes 0.000 claims description 6
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 6
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 230000007910 cell fusion Effects 0.000 claims description 6
- 229960002424 collagenase Drugs 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 5
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- 150000003431 steroids Chemical class 0.000 claims 1
- 230000035876 healing Effects 0.000 abstract description 7
- 206010072170 Skin wound Diseases 0.000 abstract description 4
- 230000008929 regeneration Effects 0.000 abstract description 4
- 238000011069 regeneration method Methods 0.000 abstract description 4
- 208000027418 Wounds and injury Diseases 0.000 description 18
- 206010052428 Wound Diseases 0.000 description 17
- 230000029663 wound healing Effects 0.000 description 9
- 208000018875 hypoxemia Diseases 0.000 description 7
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
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- 229920001436 collagen Polymers 0.000 description 2
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- 230000001225 therapeutic effect Effects 0.000 description 2
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- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 101150014221 Son gene Proteins 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
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- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940076810 beta sitosterol Drugs 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 210000000630 fibrocyte Anatomy 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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Abstract
The invention discloses a kind of composite dry cell bioagent, including fat stem cell, daucosterol, Puerarin, cupreol, solvents.The fat stem cell is 1 × 107‑5×107A/mL, daucosterol are 5-10 μ g/mL, and Puerarin is 5-10 μ g/mL, and cupreol is 4-6 μ g/mL.Composite dry cell bioagent raw material sources of the invention extensively, using convenient, and can effectively accelerate the healing rate of skin wound, promote the reparative regeneration of skin.
Description
Technical field
The present invention relates to biologic product technology field more particularly to a kind of composite dry cell bioagents.
Background technique
Wound healing is the complex process that body carries out repairing recovery to formed defect, including blood coagulation, inflammation occur, blood
Pipe new life, fibr tissue formation, extracellular matrix deposition, cicatricial tissue is formed and the stages such as epithelial tissue regeneration.Refractory wounds
The forfeiture that will lead to skin function brings great pain to daily life, and the problem treated at present.Stem cell
It can promote skin by being divided into tissue relevant cell, participating in cambium building and changing the approach such as surface of a wound local microenvironment
The healing of the surface of a wound, researching value with higher.Wherein, fat stem cell has from a wealth of sources, materials convenience, to body injury
Small advantage, application prospect are preferable.But single fat stem cell is not ideal enough to the therapeutic effect of wound healing, limitation
It is effective to apply.
Summary of the invention
Technical problems based on background technology, the invention proposes a kind of composite dry cell bioagent, raw materials
It is from a wealth of sources, using convenient, and can effectively accelerate the healing rate of skin wound, promote the reparative regeneration of skin.
A kind of composite dry cell bioagent proposed by the present invention, including fat stem cell, daucosterol, Puerarin, β-
Sitosterol, solvent.
Preferably, the fat stem cell is 1 × 107-5×107A/mL, daucosterol are 5-10 μ g/mL, and Puerarin is
5-10 μ g/mL, cupreol are 4-6 μ g/mL.
Preferably, the composite dry cell bioagent further includes cell-protecting, and the cell-protecting is seaweed
The mixture of sugar, human serum albumin or both；Preferably, the cell-protecting is 30-50 μ g/mL.
Preferably, the solvent is PBS solution or physiological saline.
Preferably, the preparation method of the composite dry cell bioagent includes the following steps:
S1, it separated from adipose tissue, cultivate fat stem cell；
S2, obtained fat stem cell is subjected to Phenotypic examination；
S3, the fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, preparation obtains compound
Stem cell biological preparation.
Preferably, the preparation method of the composite dry cell bioagent further include will cultivate obtained fat stem cell into
Row hypoxic preconditioning.
Preferably, the condition of culture of the hypoxic preconditioning specifically: 1%O2, 5%CO2, 94%N2, 37 DEG C.
Preferably, the hypoxic preconditioning method specifically: the fat stem cell trypsin solution for obtaining secondary culture
Digestion, is then seeded into serum free medium, first in 5%CO2, then 37 DEG C of normal oxygen culture 2-3d replace fresh culture,
In 1%O2, 5%CO2, 94%N2, hypoxemia culture 1-2d under the conditions of 37 DEG C.
Preferably, in the step S1, the separation of fat stem cell, cultural method specifically: take adipose tissue, use 0.2-
After 0.3% collagenase solution digestion, it is inoculated into serum free medium and cultivates, fresh culture is replaced after 2-3d, it is later every
1-2d replacement is primary, passes on after cell fusion reaches 80-90%.
Preferably, in the step S3, Phenotypic examination qualification is referred specifically to: CD31, CD34 and CD45 < 1%, CD29,
CD73, CD90 > 95%.
Beneficial effects of the present invention are as follows:
The present invention compounds fat stem cell, daucosterol, Puerarin, cupreol to obtain compound stem cell biological system
Agent, the reparative regeneration for skin.Wherein, fat stem cell can secrete various active cell factor, such as vascular endothelial growth factor
Son, platelet derived growth factor, transforming growth factor etc., and the activity for being adjustable fibroblast, horn cell etc., thus plus
Fast collagen deposition promotes re-epithelialization, is conducive to the healing of wound and the reparation of skin；Suitable daucosterol, Puerarin,
Cupreol cooperation, energy efficient activation ERK signal path can not only improve the proliferation activity of fat stem cell, and then improve wound
The secretion level of active cytokine at mouthful, moreover it is possible to promote the proliferation of wound endothelial cell, accelerate wound healing and skin
Repair speed；Daucosterol, Puerarin, cupreol can also play anti-oxidation, can reduce oxidation caused by
Fibrocyte apoptosis promotes wound healing to accelerate collagen deposition.In composite dry cell bioagent of the invention, may be used also
Appropriate cell-protecting, such as trehalose, human serum albumin is added, plays the role of protecting fat stem cell, improve fat
Stem cell helps preferably to play wound healing promoting effect in the survival rate and activity of wound.Composite dry of the invention
Fat stem cell used in cell bioagent can also carry out hypoxic preconditioning, improve its wound proliferative capacity with
And cell activity, and then the secretion level of wound active cytokine is improved, preferably performance wound healing promoting effect.This hair
Bright composite dry cell bioagent using fat stem cell as main component, not only have raw material sources extensively, using convenient
Advantage, and can effectively accelerate the healing rate of skin wound, promote the reparative regeneration of skin, therapeutic effect is good.
Specific embodiment
In the following, technical solution of the present invention is described in detail by specific embodiment.
Embodiment 1
A kind of composite dry cell bioagent, including 1 × 107The fat stem cell of a/mL, 5 μ g/mL daucosterols, 5 μ
The Puerarin of g/mL, the cupreol of 4 μ g/mL, the trehalose and solvent of 30 μ g/mL, wherein solvent is PBS solution.
Composite dry cell bioagent the preparation method comprises the following steps:
(1) mouse groin adipose tissue is taken, after 0.2% collagenase solution digestion, is inoculated into serum free medium
Middle culture replaces fresh culture after 2d, and every 1d replacement is primary later, passes on after cell fusion reaches 80%；
(2) P2 fat subsitutes stem cell is digested with trypsin solution, is then seeded into serum free medium, first 5%
CO2, then 37 DEG C of normal oxygen culture 2d replace fresh culture, in 1%O2, 5%CO2, 94%N2, hypoxemia culture under the conditions of 37 DEG C
1d；
(3) fat stem cell for obtaining hypoxemia culture carries out Phenotypic examination, Phenotypic examination criterion of acceptability are as follows: CD31,
CD34 and CD45 < 1%, CD29, CD73, CD90 > 95%；
(4) fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, preparation obtains compound
Stem cell biological preparation.
Embodiment 2
A kind of composite dry cell bioagent, including 3 × 107The fat stem cell of a/mL, 8 μ g/mL daucosterols, 6 μ
The Puerarin of g/mL, the cupreol of 5 μ g/mL, the cell-protecting and solvent of 45 μ g/mL.
Wherein, cell-protecting is the mixture of trehalose and human serum albumin, the weight of trehalose and human serum albumin
Than for 1:1；Solvent is PBS solution.
Composite dry cell bioagent the preparation method comprises the following steps:
(1) mouse groin adipose tissue is taken, after 0.25% collagenase solution digestion, is inoculated into serum free medium
Middle culture replaces fresh culture after 2d, and every 1d replacement is primary later, passes on after cell fusion reaches 80%；
(2) P2 fat subsitutes stem cell is digested with trypsin solution, is then seeded into serum free medium, first 5%
CO2, then 37 DEG C of normal oxygen culture 2d replace fresh culture, in 1%O2, 5%CO2, 94%N2, hypoxemia culture under the conditions of 37 DEG C
1d；
(3) fat stem cell for obtaining hypoxemia culture carries out Phenotypic examination, Phenotypic examination criterion of acceptability are as follows: CD31,
CD34 and CD45 < 1%, CD29, CD73, CD90 > 95%；
(4) fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, preparation obtains compound
Stem cell biological preparation.
Embodiment 3
A kind of composite dry cell bioagent, including 5 × 107The fat stem cell of a/mL, 10 μ g/mL daucosterols, 10
The Puerarin of μ g/mL, the cupreol of 6 μ g/mL, the trehalose and solvent of 50 μ g/mL, wherein solvent is physiological saline.
Composite dry cell bioagent the preparation method comprises the following steps:
(1) mouse groin adipose tissue is taken, after 0.3% collagenase solution digestion, is inoculated into serum free medium
Middle culture replaces fresh culture after 3d, and every 2d replacement is primary later, passes on after cell fusion reaches 80%；
(2) P2 fat subsitutes stem cell is digested with trypsin solution, is then seeded into serum free medium, first 5%
CO2, then 37 DEG C of normal oxygen culture 2d replace fresh culture, in 1%O2, 5%CO2, 94%N2, hypoxemia culture under the conditions of 37 DEG C
1d；
(3) fat stem cell for obtaining hypoxemia culture carries out Phenotypic examination, Phenotypic examination criterion of acceptability are as follows: CD31,
CD34 and CD45 < 1%, CD29, CD73, CD90 > 95%；
(4) fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, preparation obtains compound
Stem cell biological preparation.
Embodiment 4
A kind of composite dry cell bioagent, including 1 × 107The fat stem cell of a/mL, 5 μ g/mL daucosterols, 5 μ
The Puerarin of g/mL, the cupreol and solvent of 4 μ g/mL, wherein solvent is PBS solution.
Composite dry cell bioagent the preparation method comprises the following steps:
(1) mouse groin adipose tissue is taken, after 0.2% collagenase solution digestion, is inoculated into serum free medium
Middle culture replaces fresh culture after 2d, and every 1d replacement is primary later, passes on after cell fusion reaches 80%；
(2) P2 fat subsitutes stem cell is subjected to Phenotypic examination, Phenotypic examination criterion of acceptability are as follows: CD31, CD34 and CD45 <
1%, CD29, CD73, CD90 > 95%；
(3) fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, preparation obtains compound
Stem cell biological preparation.
Test example
Mouse skin wound repairing test
Back wool will be removed after 60 mouse anesthesias, forms the skin injury wound that diameter is 1.5cm at back with sterile cut
Face.It is divided into 5 processing groups, each processing group 12 after modeling success.Wherein, 1 is handled: the biological agent of injection embodiment 1；Place
Reason 2: the biological agent of injection embodiment 2；Processing 3: the biological agent of injection embodiment 3；Processing 4: the biology of injection embodiment 4
Preparation；Processing 5: injection single fat stem cell biological preparation, ingredient are as follows: contain 1 × 107The PBS of a/mL fat stem cell is molten
Liquid.Injecting method is as follows: injecting biological agent, every 100 μ l around wound 5 injection methods of Zhou Caiyong.By mouse after end of operation
It puts back in cage and raises, free diet, and be observed continuously.Respectively at the 3rd, 7,10 day, each processing group is acquired using digital camera
Surface of a wound image, Image-Pro Plus 6.0 analyze software and measure surface of a wound area.Wound healing rate is calculated using following formula: wound
Face healing rate={ (the current surface of a wound area of original surface of a wound area -)/original surface of a wound area } × 100%.Test result such as 1 institute of table
Show:
1 each group Wound healing rate of table
As can be seen from the above table, biological agent of the invention, can be significantly compared with single fat stem cell biological preparation
Healing speed is promoted, there is excellent wound healing effect.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of composite dry cell bioagent, which is characterized in that including fat stem cell, daucosterol, Puerarin, β-paddy steroid
Alcohol, solvent.
2. composite dry cell bioagent according to claim 1, which is characterized in that the fat stem cell is 1 × 107-
5×107A/mL, daucosterol are 5-10 μ g/mL, and Puerarin is 5-10 μ g/mL, and cupreol is 4-6 μ g/mL.
3. composite dry cell bioagent according to claim 1 or 2, which is characterized in that it further include cell-protecting, institute
State the mixture that cell-protecting is trehalose, human serum albumin or both；Preferably, the cell-protecting is 30-50 μ g/
mL。
4. composite dry cell bioagent according to claim 1-3, which is characterized in that the solvent is PBS
Solution or physiological saline.
5. composite dry cell bioagent according to claim 1-4, which is characterized in that the compound stem cell
The preparation method of biological agent includes the following steps:
S1, it separated from adipose tissue, cultivate fat stem cell；
S2, obtained fat stem cell is subjected to Phenotypic examination；
S3, the fat stem cell of Phenotypic examination qualification is resuspended with solvent, remaining raw material is then added, it is thin that preparation obtains composite dry
Born of the same parents' biological agent.
6. composite dry cell bioagent according to claim 5, which is characterized in that the composite dry cell bioagent
Preparation method further include that will cultivate obtained fat stem cell to carry out hypoxic preconditioning.
7. composite dry cell bioagent according to claim 6, which is characterized in that the culture item of the hypoxic preconditioning
Part specifically: 1%O2, 5%CO2, 94%N2, 37 DEG C.
8. composite dry cell bioagent according to claim 6 or 7, which is characterized in that the hypoxic preconditioning method
Specifically: the fat stem cell for obtaining secondary culture is digested with trypsin solution, is then seeded into serum free medium, is first existed
5%CO2, then 37 DEG C of normal oxygen culture 2-3d replace fresh culture, in 1%O2, 5%CO2, 94%N2, low under the conditions of 37 DEG C
Oxygen culture 1-2d.
9. according to the described in any item composite dry cell bioagents of claim 5-8, which is characterized in that in the step S1,
The separation of fat stem cell, cultural method specifically: adipose tissue is taken, after the collagenase solution digestion of 0.2-0.3%, inoculation
It is cultivated into serum free medium, fresh culture is replaced after 2-3d, every 1-2d replacement is primary later, reaches to cell fusion
It is passed on after 80-90%.
10. according to the described in any item composite dry cell bioagents of claim 5-9, which is characterized in that in the step S3,
Phenotypic examination qualification refers specifically to: CD31, CD34 and CD45 < 1%, CD29, CD73, CD90 > 95%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210131231A (en) * | 2020-04-23 | 2021-11-02 | 동국대학교 경주캠퍼스 산학협력단 | Composition Comprising Puerarin for Wound Healing |
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| CN106727706A (en) * | 2016-12-26 | 2017-05-31 | 湖南新起源医疗技术有限公司 | A kind of preparation method of the cell preparation repaired for skin injury |
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| CN106727706A (en) * | 2016-12-26 | 2017-05-31 | 湖南新起源医疗技术有限公司 | A kind of preparation method of the cell preparation repaired for skin injury |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210131231A (en) * | 2020-04-23 | 2021-11-02 | 동국대학교 경주캠퍼스 산학협력단 | Composition Comprising Puerarin for Wound Healing |
| KR102669861B1 (en) | 2020-04-23 | 2024-05-28 | 동국대학교 와이즈캠퍼스 산학협력단 | Composition Comprising Puerarin for Wound Healing |
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