CN108904534A - A kind of preparation method of stem cell secretion factor flexible lipidosome - Google Patents
A kind of preparation method of stem cell secretion factor flexible lipidosome Download PDFInfo
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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
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/55—Phosphorus compounds
- A61K8/553—Phospholipids, e.g. lecithin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Abstract
The present invention relates to a kind of preparation methods of stem cell secretion factor flexible lipidosome, the vitamin E of the phosphatide of 0.100-0.150g, the cholesterol of 0.020-0.030g, 0.2-0.3mg are dissolved with 10ml organic solvent, vacuum rotary steam removes organic solvent and forms adipose membrane, stem cell secretion factor solution and lipid-film hydration are formed into stem cell secretion factor flexible lipidosome stoste, gained stem cell secretion factor flexible lipidosome stoste is subjected to homogenization to get stem cell secretion factor flexible lipidosome is arrived.The present invention effectively improves the holding time of mescenchymal stem cell secretion factor, and it is conducive to drug and skin adherence and absorbs, it reduces stimulation and reduces adverse reaction, enhance penetrating power and reaches tissue deep layer, the effect curative effect for enhancing stem cell secretion factor, improves the availability of mescenchymal stem cell and has no toxic side effect.
Description
Technical field
The present invention relates to medicine effect liposome field more particularly to a kind of stem cell secretion factor flexibility lipid
The preparation method of body.
Background technique
Wound repair is an extremely complex biological process, and it comprises a variety of repair cells and cell factor etc. are all
The multifactor cytology participated in jointly, immunology, molecular biology processes.Wound repair process includes granulation tissue generation, glue
Original filling, wound are shunk, epithelium covers and the stages such as collagen remodeling.Scar is the inevitable outcome of wound healing process.But mistake
Pathologic scar caused by degree is repaired is formed, then can cause the dysfunction of disfigurement and varying degree.Pathologic scar
It mainly include hyperplastic scar and keloid, histological characteristic is a large amount of fibroblast proliferations, collagen, egg in extracellular matrix
The excess depositions such as white polysaccharide, glycoprotein, arrangement of collagen fibers disorder.Its clinical manifestation be cacesthesia, Tumor like hyperplasia and with
Different degrees of dysfunction.Patient needs in face of a series of physiology, psychology, beauty and social concern.Up to now, pathology
The treatment of property scar is still extremely complex and difficult.
Umbilical cord mesenchymal stem cells, which can be secreted, generates a large amount of cell bio-activity factor, including transforming growth factor
(transforming growth factor- β) TGF-β, epidermal growth factor (Epidermal Growth Factor) EGF,
Fibroblast growth factor (fibroblast growth factor) FGF, vascular endothelial growth factor (vascular
Endothelial growth factor) VEGF etc., these cell bio-activity factors can be with Effective Regulation body cell signal
Conduction, activating human body stem cell, and then physiological reparation or substitution body injury, lesion and the cell of aging etc..They pass through
The mode of autocrine or paracrine improves body local microenvironment, promotes endogenous retinal stem cells to the migration of wound location and divides
Change, the cell for being on the verge of apoptosis is saved, to achieve the purpose that repair tissue.Especially can effectively it make with Skin Cell
With participation inflammation occurs and wound healing.
Currently, clinically only having the clinical report that the single cell factor is applied to scar repairing, curative effect is very good.Compared to list
One cell factor, stem cell supernatant are more suitable for the network adjustment of the body surface of a wound and cicatricial tissue microenvironment.However, stem cell point
Secreting the factor is bioactive macromolecule substance, cannot be directed through skin and reach basal layer of epidermis and skin corium, or infiltration
Drug effect is played to tissue deep layer.Secondly, the store method of stem cell secretion factor directly affect its bioactivity height and
The length of holding time.The application of stem cell secretion factor of these factors affects.
Summary of the invention
It is an object of the present invention in place of solving the above deficiencies in the existing technologies.
To achieve the above object, the present invention provides a kind of preparation method of stem cell secretion factor flexible lipidosome, including
Following steps：Step 1, the primary of stem cell isolate and purify and culture identification：It is logical that umbilical cord Fahrenheit is separated from Freshman umbilical cord
Glue obtains umbilical cord mesenchymal stem cells by tissue block adherent cultivation；Step 2, the acquisition of stem cell secretion factor：To navel
Band mescenchymal stem cell degrees of fusion is when reaching 80% or more, replaces fresh not antibiotic dry without phenol red serum-free mesenchyma
Cell culture medium is cultivated, and collects the cell culture supernatant in the 4th~10 generation of umbilical cord mesenchymal stem cells, supernatant is used
0.22 μm of sterilizing filter filtering, takes filtrate to get stem cell secretion factor；Step 3, flexible mescenchymal stem cell secretion
It is prepared by the micro-capsule of the factor：The phosphatide of 0.100-0.150g, the cholesterol of 0.020-0.030g, 0.2- are dissolved with 10ml organic solvent
The vitamin E of 0.3mg, vacuum rotary steam remove organic solvent and form adipose membrane, and stem cell secretion factor solution and adipose membrane water are hydrated
Stem cell secretion factor flexible lipidosome stoste is formed, gained stem cell secretion factor flexible lipidosome stoste is carried out at homogeneous
It manages to get stem cell secretion factor flexible lipidosome is arrived.
Preferably, mescenchymal stem cell secretion factor solution and adipose membrane water are hydrated to form the rouge containing bioactie agent
Plastid stoste, specifically includes：It measures 10mL stem cell secretion factor solution to be added in dry film, while 0.2- is added
The vitamin C of 0.3mg shakes aquation and forms emulsion to get stem cell secretion factor flexible lipidosome stoste is arrived.
Preferably, stem cell secretion factor is that the bioactie agent of human umbilical cord mesenchymal stem cells secretion or source of people are done
The bioactie agent of cell secretion.
Preferably, organic solvent is chloroform, dehydrated alcohol or methanol.
Preferably, organic solvent is at least two mixture of following organic solvent：Chloroform, dehydrated alcohol, first
Alcohol.
Preferably, the liposome preparation of stem cell secretion factor can also be added protective agent and be made into freeze-dried powder.
Preferably, the particle size range of stem cell secretion factor flexible lipidosome is 50-200nm.
Preferably, phosphatide is phospholipid of natural soybean, hydrogenated phospholipid, dimyristoylphosphatidylcholine, two palmityl phosphatidyls
Choline, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine, distearyl acid phosphorus
Acyl ethanol amine, dipalmitophosphatidic acid or Polyethylene Glycol 2000-Distearoyl Phosphatidylethanolamine.
Human umbilical cord mesenchymal stem cells secretion factor is prepared as flexible lipidosome by the present invention, can be applied to tissue damage
In pathologic scar reparation.Human umbilical cord mesenchymal stem cells secretion factor is directly used in wound location, absorptivity and transdermal
Rate is lower, and is unfavorable for the maintenance of cytokine bioactivity.Mescenchymal stem cell secretion factor is prepared into flexible lipidosome,
The holding time of mescenchymal stem cell secretion factor is effectively improved, and is conducive to drug and skin adherence and absorbs, reduces thorn
Swash and reduce adverse reaction, enhancing penetrating power reaches tissue deep layer, enhances the effect curative effect of stem cell secretion factor, improve
It the availability of mescenchymal stem cell and has no toxic side effect.In addition, mescenchymal stem cell secretion factor flexible lipidosome is directly used
In wound, the deep structure of tissue can be penetrated into, improves the hair that local microenvironment promotes wound hair follicle and body of gland
Raw and generation and epithelialization, to reach the healing of wound.
Detailed description of the invention
It is the schematic diagram of umbilical cord tissue extraction mescenchymal stem cell that Fig. 1, which is provided in an embodiment of the present invention,；
Fig. 2 is the morphology schematic diagram of umbilical cord mesenchymal stem cells provided in an embodiment of the present invention；
Fig. 3 is a kind of umbilical cord mesenchymal stem cells streaming qualification result figure provided in an embodiment of the present invention；
Fig. 4 is the alizarin red dye that a kind of umbilical cord mesenchymal stem cells provided in an embodiment of the present invention induce differentiation into osteocyte
Color result figure；
Fig. 5 is the oil red O that a kind of umbilical cord mesenchymal stem cells provided in an embodiment of the present invention induce differentiation into fat cell
Coloration result figure；
Fig. 6 is a kind of umbilical cord mesenchymal stem cells secretion factor liposome figure provided in an embodiment of the present invention；
Fig. 7 is a kind of umbilical cord mesenchymal stem cells secretion factor flexible lipidosome droplet measurement provided in an embodiment of the present invention
Figure；
Fig. 8 is that rat wound model provided in an embodiment of the present invention repairs situation wound tissue pathological section；
Fig. 9 is wound tissue's pathological section histological score statistical chart provided in an embodiment of the present invention.
Specific embodiment
Combined with specific embodiments below, technical scheme of the present invention will be described in further detail.
The embodiment of the present invention provides a kind of preparation method of stem cell secretion factor flexible lipidosome, includes the following steps：
Step 1, the primary of stem cell isolate and purify and culture identification：Umbilical cord Fahrenheit is separated from Freshman umbilical cord and leads to glue, passes through tissue
Block stationary culture obtains umbilical cord mesenchymal stem cells；Step 2, the acquisition of stem cell secretion factor：It is dry thin to umbilical cord mesenchyma
When born of the same parents' degrees of fusion reaches 80% or more, replace it is fresh it is not antibiotic without phenol red serum-free mescenchymal stem cell culture medium into
Row culture, collects the cell culture supernatant in the 4th~10 generation of umbilical cord mesenchymal stem cells, by 0.22 μm of sterile mistake of supernatant
Filter filtering, takes filtrate to get stem cell secretion factor；Step 3, the micro-capsule preparation of flexible mescenchymal stem cell secretion factor：
The vitamin E of the phosphatide of 0.100-0.150g, the cholesterol of 0.020-0.030g, 0.2-0.3mg are dissolved with 10ml organic solvent,
Vacuum rotary steam removes organic solvent and forms adipose membrane, by stem cell secretion factor solution and adipose membrane water be hydrated to form stem cell secretion because
Gained stem cell secretion factor flexible lipidosome stoste is carried out homogenization to get stem cell is arrived by sub- flexible lipidosome stoste
Secretion factor flexible lipidosome.
Embodiment 1
Step 1, the primary of stem cell isolate and purify and culture identification：Fresh umbilical cord is cut into the trifle of 1-2cm long, is cutd open
Fetal membrane is opened, vein and two arteries is rejected, then extracts colloid substance, be cut into the tissue block of 1~2mm3 size, be inoculated in cell
In culture dish, complete medium is added after half an hour, is placed in 37 DEG C, is cultivated in the cell incubator of 5%CO2, changes liquid after 3 days.
After 10 days, under inverted microscope, it can be seen that there is mescenchymal stem cell to climb out of around tissue.When cell culture to 3 generation,
Umbilical cord mesenchymal stem cells are purified, and carry out stemness identification to cell：1. Morphological Identification：Mescenchymal stem cell is pasted in shuttle shape
Parietal cell；2. surface marker is identified：Mescenchymal stem cell height expresses CD29, CD44, CD105, the table of low expression CD45, CD34
It reaches；3. differentiation and identification：Under specific circumstances, mescenchymal stem cell can break up osteoblast, fat cell.
Step 2, the acquisition of stem cell secretion factor：When cell grows to 80% or so, former culture medium is discarded, with buffering
After liquid rinses cell, use instead not antibiotic without phenol red serum free medium culture, 3~10 generation cell supernatants of collection, general
Supernatant is filtered with 0.22 μm of sterilizing filter, takes filtrate to get umbilical cord mesenchymal stem cells secretion factor, freezen protective.
Step 3, the micro-capsule preparation of flexible mescenchymal stem cell secretion factor：With 10ml Liposomal formulation, compositing formula
For：Phosphatidase 0 .100g, cholesterol 0.020g, mescenchymal stem cell secretion factor collection liquid 6mL, vitamin E 0.2mg, vitamin
C 0.2mg。
(1) phosphatide, cholesterol, vitamin E are weighed by above-mentioned formula and is dissolved in chloroform, set in a round bottom flask；
(2) under bath temperature 30 DEG C of constant temperature, vacuum conditions, rotary evaporation removes organic solvent, makes lipid solution in bottle
Wall forms one layer of dry film；
(3) above-mentioned formula precise volume mescenchymal stem cell secretion factor and vitamin C are pressed, is added in dry film, water
Change forms emulsion to get stem cell secretion factor flexible lipidosome stoste is arrived；
(4) stem cell secretion factor flexible lipidosome stoste ultrasound homogeneous is obtained into stem cell secretion factor flexibility lipid
Body is placed 4 DEG C of refrigerators and is saved.
White suspension is presented in the resulting stem cell secretion factor flexible lipidosome of the present embodiment, and standing bottom has cotton-shaped
Object, shake can be changed uniform milky, and particle size is that (117.3 ± 2.89) nm, Zate current potential size is (- 43.56 ± 1.03)
Mv, it is (72.06 ± 0.36) % that ultracentrifugation combination EILSA method, which measures encapsulation rate,.
In one example, the ultrasonic homogeneous in (4) can also change extruder homogeneous into.
In one example, stem cell secretion factor is the bioactie agent or its that human umbilical cord mesenchymal stem cells are secreted
The bioactie agent of the bioactie agent of other people stem-cell-secreteds or the source of people stem cell secretion after concentration or it is known at
The composite bio-active factor divided.
In one example, phosphatide is the natural phospholipid and synthetic phospholipid of high-purity.
In one example, stem cell secretion factor liposome also includes other anti-oxidation protection preparations, wherein anti-oxidant
The protective agent mild antioxidant non-stimulated to human body, such as ascorbic acid, vitamin E, dextran, sucrose, lactose or sea
At least one of algae sugar.
In one example, the liposome preparation of stem cell secretion factor can also be added protective agent and be made into freeze-drying
Powder.The freeze-dried powder can be applied in function cosmetics, and externally applied formulation or ejection preparation can also be used as in wound repair.
In one example, phosphatide be native soy lecithin, hydrogenated phospholipid, dimyristoylphosphatidylcholine (DMPC),
Dipalmitoylphosphatidylcholine (DPPC), GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG), dipalmitoylphosphatidylglycerol (DPPG),
Dipalmitoylphosphatidylethanolamine (DPPE), distearyl acid phosphatidyl-ethanolamine (DSPE), dipalmitophosphatidic acid (DPPA) or
Polyethylene Glycol 2000-Distearoyl Phosphatidylethanolamine (DSPE-PEG2000).
In one example, stem cell secretion factor flexible lipidosome preferable particle size range is 50-200nm.
In one example, stem cell secretion factor also includes suspending agent, and suspending agent is glycerol or high molecular material, such as
PVP etc..
Embodiment 2
In step 3, with 10ml Liposomal formulation, compositing formula ratio is：Phosphatidase 0 .150g, cholesterol 0.030g, stem cell
Secretion factor concentrate 6mL, vitamin E 0.3mg, vitamin C 0.3mg.Other steps are same as Example 1.
Embodiment 3
In step 3, with 10ml Liposomal formulation, compositing formula ratio is：Phosphatidase 0 .120g, cholesterol 0.025g, stem cell
Secretion factor concentrate 6mL, vitamin E 0.25mg, vitamin C 0.25mg.Other steps are same as Example 1.
Liposome stability is measured
Umbilical cord mesenchymal stem cells secretion factor flexible lipidosome obtained by Example 1 saves under the conditions of 4 DEG C, point
Its partial size, Zeta potential, encapsulation rate were not measured in 0 day, 15 days and 30 days, and observes appearance, to determine its stability, result
As shown in table 1：
1 umbilical cord mesenchymal stem cells secretion factor flexible lipidosome stability result of table
As it can be seen from table 1 umbilical cord mesenchymal stem cells secretion factor flexible lipidosome is good in 4 DEG C of condition stability inferiors
Good, every result does not have significant difference (p within 0 day, 15 days and 30 days>0.05) stabilization of stem cell flexible lipidosome, can be predicted
Property is good.
Wound observation repair is used for 3 gained umbilical cord mesenchymal stem cells secretion factor flexible lipidosome of embodiment：
The SD rat of healthy male 220g or so is anaesthetized through intraperitoneal injection 10% chloraldurate (0.35mL/100g), to
Its response cannot after be fixed and carry out back depilation, washing back skin cuts off two in rat back vertebra two sides respectively
Diameter is the full thickness dermal wound of 1cm, sub-cage rearing.The different wounds of every rat are given at different drug wrappings
It manages and is grouped, daily dressing is once observed one week.Postoperative 2,3,5,7d timing observation wound repair situation photograph to record and collect
Tissue samples, wherein sample includes surface of a wound neoplastic skin tissue and a little normal skin of surrounding, is grouped as follows：Gauze is cut into
Appropriately sized strip is soaked in each medical fluid, and the sliver that medical fluid is soaked is covered on wound, and oily yarn, yarn are covered on sliver
Cloth finally fixes auxiliary material with bandage winding rat abdomen, and the specification for the strip that gauze is cut into can be 1cm × 2cm.
The tissue samples of collection fix 1d, serial dehydration, paraffin embedding, 4um serial section, routine with 4% neutral formalin
Hematoxylin eosin staining, neutral gum mounting, optical microphotograph microscopic observation neoplastic skin pathological change.To wound tissue's disease
Reason slice carries out histological score statistical analysis.
Fig. 1 is that the umbilical cord tissue block stationary culture separation and Extraction umbilical cord mesenchyma of one kind provided in an embodiment of the present invention is dry
Cell results figure has umbilical cord mesenchymal stem cells to climb out of around umbilical cord tissue block.
Fig. 2 is the morphology figure of the umbilical cord mesenchymal stem cells of one kind provided in an embodiment of the present invention after purification, and cell is in
Spindle shape swirl shape illustrates that the form of separated umbilical cord mesenchymal stem cells meets mescenchymal stem cell characteristic.
Fig. 3 is a kind of umbilical cord mesenchymal stem cells surface marker streaming qualification result figure provided in an embodiment of the present invention,
A, B, C are the expression of hUC-MSCs surface marker PROTEIN C D105, CD44, CD29 respectively, positive rate is respectively 99.7%,
95.7%, 99.4%；D, E is the expression of hUC-MSCs surface marker PROTEIN C D45, CD34 respectively, positive rate is respectively 1.1%,
0.1%.Illustrate that the surface marker of separated umbilical cord mesenchymal stem cells meets mescenchymal stem cell characteristic.
Fig. 4 is the alizarin red dye that a kind of umbilical cord mesenchymal stem cells provided in an embodiment of the present invention induce differentiation into osteocyte
Color calcium tubercle takes on a red color result figure.Illustrate that umbilical cord mesenchymal stem cells can be induced to differentiate into osteocyte.
Fig. 5 is the oil red O that a kind of umbilical cord mesenchymal stem cells provided in an embodiment of the present invention induce differentiation into fat cell
Dyeing fat drips take on a red color result figure.Illustrate that umbilical cord mesenchymal stem cells can be induced to differentiate into fat cell.Fig. 4 and Fig. 5 explanation
The differentiation capability of separated umbilical cord mesenchymal stem cells meets mescenchymal stem cell characteristic.
Fig. 6 is a kind of umbilical cord mesenchymal stem cells secretion factor liposome figure provided in an embodiment of the present invention, is said
Bright umbilical cord mesenchymal stem cells secretion factor flexible lipidosome is in irregular spherical shape, and size is inhomogenous, but is dispersed more equal
It is even.
Fig. 7 is a kind of umbilical cord mesenchymal stem cells secretion factor flexible lipidosome droplet measurement provided in an embodiment of the present invention
Figure illustrates that umbilical cord mesenchymal stem cells secretion factor flexible lipidosome partial size is in quasi- normal distribution, particle size range 37.84nm~
3580.00nm average grain diameter 262.3nm.
Fig. 8 is that rat wound model provided in an embodiment of the present invention repairs situation wound tissue pathological section, wherein An is
Wound healing situation, Bn are that blank cultures group HE dyes histopathologic slide, and Cn is blank cultures flexible lipidosome group HE
Histopathologic slide is dyed, Dn is that umbilical cord mesenchymal stem cells secretion factor group HE dyes histopathologic slide, and En is between umbilical cord
Mesenchymal stem cells secretion factor flexible lipidosome group HE dye histopathologic slide, n=1,2,3,4 be respectively wound the 2nd, 3,5,
7 days reparation situations.
Fig. 9 is wound tissue's pathological section histological score statistical chart provided in an embodiment of the present invention, between the 2nd day umbilical cord
Mesenchymal stem cells secretion factor group and umbilical cord mesenchymal stem cells secretion factor flexible lipidosome group do not have statistical difference (P=
0.730), P < 0.05 between other each groups, there is statistical difference.
Skin trauma basal layer and shallow is lured containing the cell factor for largely facilitating wound reparation in stem cell secretion factor
The generation of hair follicle and body of gland and epithelialization are at layer connective tissue to promote skin wound reparation, stem cell secretion factor liposome
Cell-secretion factor can be delivered to wound deep layer by the phosphatide in vesica, induced skin deep layer connective tissue hair follicle and body of gland
Generation and epithelialization.
Above specific embodiment has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
Illustrate, it should be understood that the above is only a specific embodiment of the invention, the protection model that is not intended to limit the present invention
It encloses, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the present invention
Protection scope within.
Claims (7)
1. a kind of preparation method of stem cell secretion factor flexible lipidosome, which is characterized in that comprise the steps of：
Step 1, the primary of stem cell isolate and purify and culture identification：Umbilical cord Fahrenheit is separated from Freshman umbilical cord and leads to glue, is passed through
Tissue block adherent cultivation obtains umbilical cord mesenchymal stem cells；
Step 2, the acquisition of stem cell secretion factor：When umbilical cord mesenchymal stem cells degrees of fusion reaches 80% or more, more renew
It is fresh it is not antibiotic cultivated without phenol red serum-free mescenchymal stem cell culture medium, collect umbilical cord mesenchymal stem cells the
0.22 μm of sterilizing filter of supernatant is filtered, takes filtrate to get stem cell point by the cell culture supernatant in 4~10 generations
Secrete the factor；
Step 3, the micro-capsule preparation of flexible mescenchymal stem cell secretion factor：0.100-0.150g is dissolved with 10ml organic solvent
Phosphatide, the cholesterol of 0.020-0.030g, 0.2-0.3mg vitamin E, vacuum rotary steam removes organic solvent and forms adipose membrane,
Stem cell secretion factor solution and adipose membrane water are hydrated to form stem cell secretion factor flexible lipidosome stoste, by gained stem cell
Secretion factor flexible lipidosome stoste carries out homogenization to get stem cell secretion factor flexible lipidosome is arrived.
2. preparation method according to claim 1, which is characterized in that stem cell secretion factor solution and the adipose membrane by between
Water is hydrated to form the liposome stoste containing bioactie agent, specifically includes：
It measures 10mL stem cell secretion factor solution to be added in the dry film, while the vitamin of 0.2-0.3mg is added
C shakes aquation and forms emulsion to get stem cell secretion factor flexible lipidosome stoste is arrived.
3. preparation method according to claim 1, which is characterized in that the stem cell secretion factor is human umbilical cord mesenchymal
The bioactie agent of stem cell secretion or the bioactie agent of source of people stem cell secretion.
4. preparation method according to claim 1, which is characterized in that the organic solvent is chloroform, dehydrated alcohol
Or methanol.
5. preparation method according to claim 1, which is characterized in that the organic solvent be following organic solvent at least
Two kinds of mixture：Chloroform, dehydrated alcohol, methanol.
6. preparation method according to claim 1, which is characterized in that the grain of the stem cell secretion factor flexible lipidosome
Diameter range is 50-200nm.
7. preparation method according to claim 1, which is characterized in that the phosphatide be phospholipid of natural soybean, hydrogenated phospholipid,
Dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two palmityl phosphatidyls are sweet
Oil, dipalmitoylphosphatidylethanolamine, distearyl acid phosphatidyl-ethanolamine, dipalmitophosphatidic acid or polyethylene glycol 2000-two
Stearyl phosphatidyl ethanol amine.
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| CN109394563A (en) * | 2018-11-06 | 2019-03-01 | 福建省海西细胞生物工程有限公司 | A kind of preparation method of stem cell media liposome and the application in skin care item |
| CN109568270A (en) * | 2018-12-27 | 2019-04-05 | 佛山科学技术学院 | A kind of liposome and preparation method thereof of fatty stem cell secretion object |
| CN110917063A (en) * | 2019-11-15 | 2020-03-27 | 上海瑞思德生物科技有限公司 | Application of epithelial cell active peptide compound nano liposome and preparation thereof |
| CN110917333A (en) * | 2019-11-15 | 2020-03-27 | 上海瑞思德生物科技有限公司 | Application of nerve stem cell active factor compound nano liposome and preparation thereof |
| CN111374934A (en) * | 2020-03-19 | 2020-07-07 | 厚朴生物科技（苏州）有限公司 | Preparation of liposome-encapsulated human stem cell factor and skin injury repair detection method |
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| CN111617040A (en) * | 2020-07-16 | 2020-09-04 | 卡杜兰(广州)生命基因工程科技有限公司 | Preparation method of lipid microcapsule wrapping stem cell secretion factors |
| CN113730361A (en) * | 2021-10-19 | 2021-12-03 | 陕西科美致尚生物科技有限公司 | Exosome preparation with needle-free injection effect suitable for mucous membrane and preparation method thereof |
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| CN109394563A (en) * | 2018-11-06 | 2019-03-01 | 福建省海西细胞生物工程有限公司 | A kind of preparation method of stem cell media liposome and the application in skin care item |
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| CN110917063A (en) * | 2019-11-15 | 2020-03-27 | 上海瑞思德生物科技有限公司 | Application of epithelial cell active peptide compound nano liposome and preparation thereof |
| CN110917333A (en) * | 2019-11-15 | 2020-03-27 | 上海瑞思德生物科技有限公司 | Application of nerve stem cell active factor compound nano liposome and preparation thereof |
| CN111374934A (en) * | 2020-03-19 | 2020-07-07 | 厚朴生物科技（苏州）有限公司 | Preparation of liposome-encapsulated human stem cell factor and skin injury repair detection method |
| CN111481510A (en) * | 2020-05-26 | 2020-08-04 | 南开大学 | Nanoparticle for wrapping cell secretory component and preparation method and application thereof |
| CN111617040A (en) * | 2020-07-16 | 2020-09-04 | 卡杜兰(广州)生命基因工程科技有限公司 | Preparation method of lipid microcapsule wrapping stem cell secretion factors |
| CN113730361A (en) * | 2021-10-19 | 2021-12-03 | 陕西科美致尚生物科技有限公司 | Exosome preparation with needle-free injection effect suitable for mucous membrane and preparation method thereof |
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