CN109666629A - The excretion body in human pluripotent stem cells source, preparation and purposes based on the excretion body - Google Patents
The excretion body in human pluripotent stem cells source, preparation and purposes based on the excretion body Download PDFInfo
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- CN109666629A CN109666629A CN201710953106.7A CN201710953106A CN109666629A CN 109666629 A CN109666629 A CN 109666629A CN 201710953106 A CN201710953106 A CN 201710953106A CN 109666629 A CN109666629 A CN 109666629A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The present invention relates to the excretion bodies in human pluripotent stem cells source, preparation and purposes based on the excretion body, belong to cell biology, molecular biology and medicament research and development technical field.The purposes of human pluripotent stem cells excretion body or the preparation based on human pluripotent stem cells excretion body includes: A, as the maintenance adult stem cell of in vitro culture stemness and inhibit the additive application of its aging, B, the additive application of the proliferation activity of the various tissue in primary culture cells of maintenance as in vitro culture, C, application in the drug of preparation treatment bone and cartilage degradation, D, application in the drug of preparation prevention bone and cartilage degradation, E, osteonecrosis is treated in preparation, bone nonunion, fracture, cartilage damage, tendon injury, application in the drug of the orthopaedic diseases such as frozen shoulder or adhesion of tendon.Compared with prior art, the excretion body of multipotential stem cell secretion of the present invention also has powerful promotion histocyte regeneration, prevents and treats aging, treats the function of bone class disease.
Description
Technical field
The invention belongs to cell biology, molecular biology and medicament research and development technical fields, dry more particularly, to people's multipotency
The excretion body of cell origin, preparation based on the excretion body and application thereof.
Background technique
With advancing age, each organ function of human body will appear gradually aging, and cause the generation of related disease.With
Chinese society aging aggravation, cardiovascular and cerebrovascular disease, bone and joint disease, the nervous system disease and metabolic disease etc.
Disease incidence significantly rises, and the harm of geriatric disease becomes increasingly conspicuous.The whole world has more than 200,000,000 sufferers of osteoporosis face at present, and
Age is more than to have 1/3 generation osteoporotic fracture in 50 years old women, have it is very high disable, lethality, seriously threaten public strong
Health brings great social economical burden.In addition, osteoarthritis is a kind of clinically very common chronic degenerative disease
Disease is listed in the primary cause of disease of the elderly's ability to act decline, seriously affects people's lives quality.Research and development can effectively prevent bone
Degenerative disease occurs to be particularly important with the technology of development.
Stem cell regenerating medical technology is currently most possibly to prevent and treat the technology of these degenerative diseases, studies have shown that moving
The tissue precursor cell treatment related disease planted adult stem cell or human pluripotent stem cells differentiation is induced has obvious curative effects.But
It is that directly transplanting stem cell is faced with cytometaplasia always, tumorigenesis risk, transport preservation difficulty, is difficult to the problems such as industrialization.Closely
Year it has been found that the excretion body that stem cell generates, has the function similar with stem cell height, can also promote regeneration,
Therefore the very big concern of numerous scholars is caused, brings new hope for the Transformation Application of stem cell regenerating medical technology.
Excretion body is the extracellular vesica of a kind of diameter about 30-150nm secreted by cell, carries donor cell sources
Various bioactivators include the entrance effects cell such as DNA, RNA, albumen, Effective Regulation body cell signal transduction.It grinds
The excretion body for showing stem cell secretion is studied carefully by the bioactive substance to effector cell's transmitting source of human stem cell, and physiological is repaired
Cell that is multiple or removing body injury, lesion and aging, play it is anti-inflammatory, adjust it is immune, promote effector cell's proliferation, migration and
Differentiation promotes the functions such as angiogenesis.
Since induction human pluripotent stem cells have totipotency similar with embryonic stem cell, the excretion body of secretion may be wrapped
The factor relevant to versatility, transcription factor, mRNA, microRNA etc. have been wrapped up in, has been shown than adult tissue's stem cell excretion body
More powerful function.The excretion body that human pluripotent stem cells generate can inhibit cellular senescence process, change target cell epigenetic
Feature reverses effector cell to low differentiation state, so that repair tissue be promoted to damage.Human pluripotent stem cells source is outer simultaneously
Body is secreted, the effect similar with tissue stem cell can also be played, blood vessel is such as repaired, inhibits inflammation, promotes tissue stem cell proliferation
The functions such as differentiation.
Embryonic stem cell (embryonic stem cell, ESCs) and induction human pluripotent stem cells (induced
Pluripotent stem cells, iPSCs) characteristic in vitro culture infinite multiplication, self-renewing and Multidirectional Differentiation.
ESCs and iPSCs has versatility (Pluripotency), i.e. ESCs and iPSCs can be divided into tridermic arbitrary cell, has
There is the ability for developing into Various Tissues, participates in the formation of portion of tissue.And adult stem cell only has a variety of differentiation potentials
(multipotency), i.e., can only break up as a limited number of kind of cell.Thus ESCs and iPSCs and tissue adult stem cell phase
It is more with better function than in terms of promoting tissue and organ regeneration update.But since there are oncogenicity by ESCs or iPSCs, so cannot be straight
Connect the prevention and treatment that transplanting is used for disease.And the excretion body of ESCs or iPSCs secretion may wrap up the factor relevant to versatility, turn
The factor, mRNA, microRNA etc. are recorded, the function more powerful than adult tissue's stem cell excretion body may be shown.
Although having application of the excretion body of many documents and patent report source of human stem cell in repair tissue damage,
The excretion body that mesenchymal stem cell secretion such as has been reported has the function of promoting osteanagenesis repairing bone defect, by iPSCs
Excretion body secreted by the mescenchymal stem cell (iPSCs-MSCs) that directional induction obtains, which also has, promotes osteanagenesis, blood vessel new
Raw, regenerating bone or cartilage, for repairing the reparation of lower limb ischemia, skin wound, fracture, skull defeci, Osteoarthritis, osteonecrosis etc.
Treatment.Chinese 101890050 A of granted patent CN discloses human umbilical cordmesenchymal stem cell-derived exosome (excretion body) and is treating
Function in terms of liver, kidney or skin injury disease.Reported above belongs to adult stem cell, since ESCs or iPSCs belongs to people
Multipotential stem cell, has promotion regeneration more stronger than adult stem cell and anti-senescence function, and the excretion body of secretion is also answered
With similar power.But have not yet to see the excretion body prevention and treatment degenerative bone disease directly secreted using ESCs or iPSCs
The research report of disease.
Summary of the invention
The object of the invention is to next in order to provide a kind of human pluripotent stem cells (i.e. ESCs or iPSCs) with better function
Purposes of excretion body, preparation method, the preparation based on the excretion body and its preparation in source in terms of preventing and treating orthopaedic disease.
The purpose of the present invention can be achieved through the following technical solutions:
First aspect present invention:
A kind of preparation method of human pluripotent stem cells excretion body is provided, comprising the following steps:
Culture medium is collected in serum free culture system culture people ESCs or iPSCs using no feeder layer cultural method, is received
Collect the excretion body in pure medium.
In one embodiment, serum free culture system selects commercially available model TeSRTM-E8TMOr the training of mTeSR1
Support base.
In one embodiment, the method for the excretion body in pure medium is collected are as follows: low by rotation combining ultrafiltration
Warm Ultracentrifugation Method collects the excretion body in pure medium.
In one embodiment, after collecting excretion body, by the methods of Electronic Speculum, granularmetric analysis, immunoblotting to excretion
The characteristic of body is identified, excretion body characteristics: diameter range 50-150nm are as a result met, and is in duplicature under transmission electron microscope
CD9, the markers such as CD63 are contained in cystic structures, membrane structure surface.
Second aspect of the present invention:
A kind of human pluripotent stem cells excretion body is provided, the preparation method of first aspect present invention is used to obtain.
The human pluripotent stem cells excretion body includes people's ESCs excretion body and people iPSCs excretion body.
In one embodiment, the human pluripotent stem cells excretion body uses deep-bed drying, the deep-bed drying
Refer to that -80 DEG C or less save.
Third aspect present invention:
Preparation based on the human pluripotent stem cells excretion body is provided.
A, suspending agent: the human pluripotent stem cells excretion body is dissolved in solvent, is existed in the form of suspending agent, and solvent is
Physiological saline or phosphate buffer or basal cell culture medium.
Suspending agent based on the human pluripotent stem cells excretion body is using human pluripotent stem cells excretion body as functional component.
Suspending agent based on the human pluripotent stem cells excretion body can be damaged by oral, intravenous injection, or directly in tissue
Traumatic part position injection (such as intraarticular injection, fracture inject) or the forms such as spraying carry out using.
B, it is sustained the compound of excretion body: forming answering for sustained release excretion body in conjunction with carrier by human pluripotent stem cells excretion body
Close object.
The carrier includes various hydrogels, biomembrane, bioceramic material, biomaterial of nanostructure etc..
The compound of the sustained release excretion body is used in the form of implanting tissue damage location.
C, contain the additive of human pluripotent stem cells excretion body: adding using human pluripotent stem cells excretion body as functional component
Add agent.
Additive containing human pluripotent stem cells excretion body can be added to the treatment that disease is used in various drugs, or add
Enter to various cultivating systems for various cell culture.
Fourth aspect present invention:
The purposes of human pluripotent stem cells excretion body is provided.
A, inhibit the additive application of its aging as the stemness of the maintenance adult stem cell of in vitro culture:
The adult stem cell includes mescenchymal stem cell and tissue-specific stem cells, and the mescenchymal stem cell includes
Various tissue-derived mescenchymal stem cells, as fat mesenchymal stem cell, mesenchymal stem cell, synovial membrane mesenchyma are dry thin
Born of the same parents, the mescenchymal stem cell in urine source, dental pulp mescenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell,
Amnion mesenchymal stem cell etc.；The tissue-specific stem cells include liver stem cells, endothelial progenitor cells, neural stem cell, pancreas islet
Stem cell, epithelial stem cell, kidney stem cell, lung epithelial stem cell, epidermal stem cells, hair follicle stem cells, limbal stem cell,
Melanocyte stem cells, sweat gland stem cell, tendon stem cell, cartilage precursor cells etc..
In one embodiment, human pluripotent stem cells excretion body or preparation based on human pluripotent stem cells excretion body are pressed
Ratio is added in the culture medium of people's adult stem cell of in vitro culture, is observed it and is tieed up to the stemness for the people's adult stem cell cultivated
Hold and inhibit the effect of aging.
B, the additive application as the proliferation activity of the various tissue in primary culture cells of maintenance of in vitro culture:
The tissue in primary culture cell includes that vascular endothelial cell, dermal melanin cell, epidermal cell, liver epithelial are thin
Born of the same parents, nerve cell, corneal epithelial cell, endothelial cell, fibroblast, beta Cell of islet, osteoblast, cartilage cell
With osteoclast etc..
C, the application in the drug of preparation treatment bone and cartilage degradation, or as treatment bone and cartilage degeneration
The medicinal application of lesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body observe reverse of the excretion body to osteoporosis, osteoarthritis
Effect.
In experimentation, using aged animal as experimental model, intravenous injection or the oral or direct locally injecting in affected part or spray
Human pluripotent stem cells excretion body or the preparation based on human pluripotent stem cells excretion body are applied, observation excretion body dredges aged animal sclerotin
Loose, osteoarthritis reversing effect.
D, the application in the drug of preparation prevention bone and cartilage degradation, or as prevention bone and cartilage degeneration
The medicinal application of lesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body, observation excretion body occur its osteoporosis, osteoarthritis pre-
Anti- effect.
In experimentation, using young animals as experimental model, intravenous injection or the oral or direct locally injecting in affected part or spray
Human pluripotent stem cells excretion body or the preparation based on human pluripotent stem cells excretion body are applied, as the age of experimental animal increases, is seen
Examine the preventive effect that its osteoporosis, osteoarthritis occur for excretion body.
E, in bones such as preparation treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder or adhesions of tendon
Application in the drug of section's disease, as treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder, tendon
The medicinal application of the orthopaedic diseases such as adhesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body.
In experimentation, osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury animal model, intravenous injection are constructed
Or the oral or direct locally injecting in affected part or spraying human pluripotent stem cells excretion body or the system based on human pluripotent stem cells excretion body
Agent.
Fifth aspect present invention:
The purposes of preparation based on human pluripotent stem cells excretion body is provided.
A, inhibit the additive application of its aging as the stemness of the maintenance adult stem cell of in vitro culture:
The adult stem cell includes mescenchymal stem cell and tissue-specific stem cells, and the mescenchymal stem cell includes
Various tissue-derived mescenchymal stem cells, as fat mesenchymal stem cell, mesenchymal stem cell, synovial membrane mesenchyma are dry thin
Born of the same parents, the mescenchymal stem cell in urine source, dental pulp mescenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell,
Amnion mesenchymal stem cell etc.；The tissue-specific stem cells include liver stem cells, endothelial progenitor cells, neural stem cell, pancreas islet
Stem cell, epithelial stem cell, kidney stem cell, lung epithelial stem cell, epidermal stem cells, hair follicle stem cells, limbal stem cell,
Melanocyte stem cells, sweat gland stem cell, tendon stem cell, cartilage precursor cells etc..
In one embodiment, human pluripotent stem cells excretion body or preparation based on human pluripotent stem cells excretion body are pressed
Ratio is added in the culture medium of people's adult stem cell of in vitro culture, is observed it and is tieed up to the stemness for the people's adult stem cell cultivated
Hold and inhibit the effect of aging.
B, the additive application as the proliferation activity of the various tissue in primary culture cells of maintenance of in vitro culture:
The tissue in primary culture cell includes that vascular endothelial cell, dermal melanin cell, epidermal cell, liver epithelial are thin
Born of the same parents, nerve cell, corneal epithelial cell, endothelial cell, fibroblast, beta Cell of islet, osteoblast, cartilage cell
With osteoclast etc..
C, the application in the drug of preparation treatment bone and cartilage degradation, or as treatment bone and cartilage degeneration
The medicinal application of lesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body observe reverse of the excretion body to osteoporosis, osteoarthritis
Effect.
In experimentation, using aged animal as experimental model, intravenous injection or the oral or direct locally injecting in affected part or spray
Human pluripotent stem cells excretion body or the preparation based on human pluripotent stem cells excretion body are applied, observation excretion body dredges aged animal sclerotin
Loose, osteoarthritis reversing effect.
D, the application in the drug of preparation prevention bone and cartilage degradation, or as prevention bone and cartilage degeneration
The medicinal application of lesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body, observation excretion body occur its osteoporosis, osteoarthritis pre-
Anti- effect.
In experimentation, using young animals as experimental model, intravenous injection or the oral or direct locally injecting in affected part or spray
Human pluripotent stem cells excretion body or the preparation based on human pluripotent stem cells excretion body are applied, as the age of experimental animal increases, is seen
Examine the preventive effect that its osteoporosis, osteoarthritis occur for excretion body.
E, in bones such as preparation treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder or adhesions of tendon
Application in the drug of section's disease, as treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder, tendon
The medicinal application of the orthopaedic diseases such as adhesion:
In one embodiment, be injected intravenously or take orally the direct locally injecting in affected part or spraying human pluripotent stem cells
Excretion body or preparation based on human pluripotent stem cells excretion body.
In experimentation, osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury animal model, intravenous injection are constructed
Or the oral or direct locally injecting in affected part or spraying human pluripotent stem cells excretion body or the system based on human pluripotent stem cells excretion body
Agent.
Compared with prior art, the present invention has the following advantages and beneficial effects:
The excretion body in the present inventor's multipotential stem cell source is from ESCs or iPSCs multipotential stem cell, prior art master
If studying the excretion body in adult stem cell source；ESCs and iPSCs have versatility (Pluripotency), i.e. ESCs and
IPSCs can be divided into tridermic arbitrary cell, have the ability for developing into Various Tissues, participate in the formation of portion of tissue.And
Adult stem cell only has a variety of differentiation potentials (multipotency), i.e., can only break up as a limited number of kind of cell.Multipotency
Stem cell promotes the function of histocyte regeneration prevention and treatment aging obviously stronger than adult stem cell.The excretion body of multipotential stem cell secretion
Also it is regenerated with powerful promotion histocyte, prevents and treats aging, treat the function of bone class disease, thus show bigger application
Value.
Detailed description of the invention
Excretion body (ES-exo) grain size distribution of Fig. 1 human pluripotent stem cells secretion；
The identification of Fig. 2 excretion body marker；
Fig. 3 Micro CT scan result；A group: control group；B group: Experiment on therapy group；C group: prevention experimental group.
A group is compared with B group and C group, and cortical bone is thinning, and bone trabecula is sparse, and osteoporosis is more apparent；C group bone compared with B group
The loose phenomenon of matter mitigates.
Fig. 4 alkaline phosphatase and Tartrate resistant acid phosphatase ELISA testing result.
* significant difference (P < 0.05) compared with the control group is indicated
Fig. 5 Runx2, Osterix gene PCR testing result.
* significant difference (P < 0.05) compared with the control group is indicated
Fig. 6 Runx2, Osterix albumen Western blot testing result.
Fig. 7 HE coloration result.
Model group (A) bone trabecula obviously tails off, and form is thinning, and vacuole and karyopycnosis occurs in osteocyte；ES-exo intervention group
(B) bone trabecula has mild necrosis, the vacuolar degeneration of rare osteocyte；Control group (C) normal bone tissues.
Fig. 8 microCT scanning result.
The bone trabecula of model group (A) substantially reduces, and the region that structure completely disappears occurs；ES-exo intervention group (B)
Trabecular bone structure keeps relatively complete；Control group (C) normal bone tissues.
Fig. 9 angiographic results.
The blood vessel number of model group (A) significantly reduces；The number of blood vessel of ES-exo intervention group (B) is significantly more than model group；It is right
According to group (C) normal bone tissues.
The photo of Figure 10 mirco structure microscopically observation articular surface.
The visible normal cartilage performance of Normal group (A) and ES-exo treatment group (B), and arthritis model group (C) is visible
Cartilage wear, subchondral bone exposure, surface fiber hyperblastosis.
Figure 11 Micro-CT scanning result.
Model group (C) only has a small amount of area of new bone；β-TCP transplantation group (B) has newborn ostosis, but the both ends of bone defect are simultaneously
Do not connect；There is a large amount of area of new bone in ES-exo/ β-TCP group (A), newborn poroma is fully connected defect.
Form under mirror when Figure 12 BMSCs is cultivated to P10.
A shows the BMSCs of conventional medium culture；B shows the BMSCs cultivated in the culture medium containing ES-exo.
The growth curve that Figure 13 P10 is cultivated in the culture medium containing ES-exo and conventional medium for BMSCs.
Figure 14 senile cell coloration result.Coloured portions are half glycosides lactose enzyme positive of β in B, show senile cell.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1:
The culture of human embryo stem cell (ESCs) and the extraction and identification of excretion body
In one layer of embryonic stem cell matrigel of culture dish middle berth (ESC-Qualified BD Matrigel, BDSparks, MD, USA), ESCs is moved into the culture dish, mTeSR1 serum free medium is added
(StemCell Vancouver, BC, Canada), and in incubator (37 DEG C, 5%CO2, saturated humidity) and training
It supports, collects the culture medium replaced daily.Culture medium is centrifuged 30 points by 0.22 micron pore size membrane filtration and 4 DEG C of 10000g
Clock removes cell fragment；Using the super filter tube of 100KD molecular weight, the excretion being centrifuged in (3500g, 15min) retention concentration supernatant
Body obtains excretion body concentrate；Concentrate is transferred to 30% sucrose/heavy water density pad (1.210g/cm3), 4 DEG C of 100000g
Bottom 5ml sucrose/heavy water density pad is collected in centrifugation 210 minutes, PBS dilution is added, 100KD molecular weight can be retained by being transferred to
In ultra-filtration centrifuge tube, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, is finally settled to certain volume by subsequent experimental requirement, is obtained
To excretion body suspension ES-exo, packing is saved to -80 DEG C.
The form of ES-exo is observed by transmission electron microscope (TEM).Load sample copper mesh (aperture 2nm) is fixed on bracket, it will
20 μ L samples are added drop-wise on copper mesh, after being stored at room temperature 3 minutes, are sucked liquid with filter paper in copper mesh side, and 3% phosphotungstic acid is added dropwise
30 μ L of solution carries out negative staining (room temperature, 5 minutes) to sample.Negative staining liquid is sucked with filter paper, copper mesh is transferred to transmission electron microscopy
Under mirror, excretion volume morphing is observed.
The partial size and concentration of ES-exo is examined by nanoparticle analysis system (iZon qNano, New Zealand)
It surveys, according to operational manual conditioning instrumentation parameters: Nanopore (NP100) being installed to below well, is adjusted
40 μ L PBS are added into well by Stretch to 43cm, stablize electric current within the scope of 100-120nA.PBS is sucked, is added
The 40 diluted CPC100 standard items (partial size 70nm) of μ L 1:100 measure population and concentration, obtain standard curve by software.
Standard items are sucked, PBS is washed 3 times, and the 40 diluted samples to be tested of μ L 1:1000 are added, and measures population and concentration, duplicate measurements 3
It is secondary.Data analysis is carried out by software, obtains analysis report.Particle size range is 50-150nm as the result is shown, sees Fig. 1.
The expression of ES-exo specific surfaces marker CD9 and CD63 are detected by western blot.Specific steps: outer
Body total protein extraction is secreted, sample protein concentration is detected by BCA protein assay kit, glue prepares 10% separation gel, electricity
Swimming, transferring film, closing and antibody incubation, chemiluminescence imaging analyzer observe band development situation.As a result extracted excretion body
Equal expression specificity surface marker CD9 and CD63.See Fig. 2.
People induces the culture of human pluripotent stem cells (iPSCs) and the extraction and identification of excretion body
In one layer of embryonic stem cell matrigel of culture dish middle berth (ESC-Qualified BD Matrigel, BDSparks, MD, USA), iPSC is moved into the culture dish, mTeSR1 serum free medium is added
(StemCell Vancouver, BC, Canada), and in incubator (37 DEG C, 5%CO2, saturated humidity) and training
It supports, collects the culture medium replaced daily.Culture medium is centrifuged 30 points by 0.22 micron pore size membrane filtration and 4 DEG C of 10000g
Clock removes cell fragment；Using the super filter tube of 100KD molecular weight, the excretion being centrifuged in (3500g, 15min) retention concentration supernatant
Body obtains excretion body concentrate；Concentrate is transferred to 30% sucrose/heavy water density pad (1.210g/cm3), 4 DEG C of 100000g
Bottom 5ml sucrose/heavy water density pad is collected in centrifugation 210 minutes, PBS dilution is added, 100KD molecular weight can be retained by being transferred to
In ultra-filtration centrifuge tube, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, is finally settled to certain body by subsequent experimental requirement with PBS
Product, obtains excretion body suspension iPS-exo, and packing is saved to -80 DEG C.
The form of iPS-exo is observed by transmission electron microscope (TEM).Load sample copper mesh (aperture 2nm) is fixed on bracket, it will
20 μ L samples are added drop-wise on copper mesh, after being stored at room temperature 3 minutes, are sucked liquid with filter paper in copper mesh side, and 3% phosphotungstic acid is added dropwise
30 μ L of solution carries out negative staining (room temperature, 5 minutes) to sample.Negative staining liquid is sucked with filter paper, copper mesh is transferred to transmission electron microscopy
Under mirror, excretion volume morphing is observed.
The partial size and concentration of iPS-exo is examined by nanoparticle analysis system (iZon qNano, New Zealand)
It surveys, according to operational manual conditioning instrumentation parameters: Nanopore (NP100) being installed to below well, is adjusted
40 μ L PBS are added into well by Stretch to 43cm, stablize electric current within the scope of 100-120nA.PBS is sucked, is added
The 40 diluted CPC100 standard items (partial size 70nm) of μ L 1:100 measure population and concentration, obtain standard curve by software.
Standard items are sucked, PBS is washed 3 times, and the 40 diluted samples to be tested of μ L 1:1000 are added, and measures population and concentration, duplicate measurements 3
It is secondary.Data analysis is carried out by software, obtains analysis report.
The expression of iPS-exo specific surfaces marker CD9 and CD63 are detected by western blot.Specific steps:
Excretion body total protein extraction detects sample protein concentration by BCA protein assay kit, and glue prepares 10% separation gel,
Electrophoresis, transferring film, closing and antibody incubation, chemiluminescence imaging analyzer observe band development situation.As a result extracted excretion
The equal expression specificity surface marker CD9 and CD63 of body.
Embodiment 2
Preventive and therapeutic effect of the excretion body in human pluripotent stem cells source to osteoporosis
Prevention experiment: 6 monthly age SAMP6 senescence-acceleratad mice models of selection, two groups of experiment point, every group of 10 mouse, experiment
People 2~5 × 10 is given in the daily stomach-filling of group8A ES-exo or iPS-exo is intervened, the physiology of control group stomach-filling same volume
Salt water.3 months execution mouse after intervention, materials detection.
Experiment on therapy: the SAMP6 senescence-acceleratad mice model in selection August age, two groups of experiment point, every group of 10 mouse are real
It tests the daily stomach-filling of group and gives people 2~9 × 108A ES-exo is intervened, the physiological saline of control group stomach-filling same volume.In
3 months execution mouse after intervention, materials detection.
Mouse leaves and takes blood before putting to death, ELISA method measures serum Tartrate resistant acid phosphatase and bone alkaline phosphatase water
It is flat.The femur for leaving and taking every mouse is saved respectively by the requirement of Testing index.Toy tomographic system (microCT) is right
Bone tissue is scanned, and detects bone tissue microstructure, and the parameter of assessment bone tissue micro-structure includes opposite bone volume (BV/
TV), Connection Density (Conn.Dn), bone trabecula quantity (Tb.N), bone mineral density (BMD), bone trabecula thickness (Tb.Th) and
Structure model index (SMI).Gene Runx2, Osterix base of regulation osteanagenesis is detected by real-time quantitative fluorescence PCR technology
The expression of cause.Western blot detects Runx2, Osterix protein expression level.Histopathology and immunohistochemistry inspection
Survey: interception femur, normal saline flushing are placed on formalin and fix (4 DEG C, 24-48h), pass through ethylenediamine tetra-acetic acid (EDTA)
Decalcification processing, using ethanol solution serial dehydration, dimethylbenzene is transparent, and paraffin longitudinally embeds bone tissue, continuously cuts with a thickness of 5um
Piece, for one Yihong of haematoxylin (HE) dyeing, TRAP dyeing, masson dyeing and immunohistochemical staining.
By hematoxylin eosin staining the results show that the bone structure destruction of control group and prevention experimental group and Experiment on therapy
Group is serious compared to obvious, and the femur for preventing experimental group and Experiment on therapy group has newborn bone trabecula to be formed, and trabecular bone structure is complete
It is whole, number is more, it is seen that osteoblast be more than control group.MicroCT testing result shows that control group cortical bone is thinning, and bone is small
Beam is sparse, and osteoporosis is obvious；Compared with the control group, the cortical bone for preventing experimental group and Experiment on therapy group is thicker, bone trabecula compared with
It is close.See Fig. 3.
Experimental result shows that compared with the control group, the alkaline phosphatase levels of Experiment on therapy group are significantly raised, anti-tartaric acid
Acid phosphatase enzyme level is substantially reduced, and difference has significant (P < 0.05), sees Fig. 4.The regulation bone shape of Experiment on therapy group
At Runx2, Osterix gene level be all remarkably higher than control group (P < 0.05), see Fig. 5；The Runx2 of Experiment on therapy group,
Osterix protein expression level is all remarkably higher than control group (P < 0.05), sees Fig. 6.
Result above prompt, the excretion body in human pluripotent stem cells source have the generation and development of osteoporosis apparent
Preventive and therapeutic effect.
Embodiment 3
Preventive and therapeutic effect of the excretion body in human embryo stem cell source to femoral head ischemic necrosis
The culture of human embryo stem cell and the extraction of excretion body are the same as embodiment 1.Experiment is big using the male SD of 300-320g
Mouse makes hormone-inducible femoral head necrosis model: SD rat intramuscular injection methylprednisolone (40mg/kg), 3 times a week, for 3 weeks.It is real
Test grouping: model group (hormone induction caput femoris necrosis, n=10)；ES-exo intervention group (give while hormone induction by vein
ES-exo is intervened, n=10)；Normal group (gives the same dose of PBS, n=10).It, will be big after the completion of all injections
Mouse, which is placed in cage, normally to be raised 3 weeks.Then materials assessment femoral head angiogenesis situation and osteonecrosis situation.
Femoral head bony structure is detected by microCT (Skyscan, 1076scanner, Kontich, Belgium)
Situation of change: being placed in detection platform for femoral head sample, tomoscan carried out after resolution adjustment to 18mm, soft by Nrecon
Part (Version 1.5.1.4, Skyscan) rebuilds femoral head 3D rendering, and calculates bone volume/total volume (BV/TV), bone surface
Product/bone volume, bone trabecula thickness (Tb.Th) and bone trabecula quantity (Tb.N).Femoral head position is observed by microangiography
Vascular distribution and density change: after each group rat anesthesia, cutting off thoracic cavity and abdominal cavity, ligature aorta ascendens, use intravenous infusion needle
Left ventricle is punctured, rinses the chambers of the heart with heparin-saline, with 4% paraformaldehyde fixing organization and blood vessel, is then transfused to ventricle
Microfil contrast agent (Microfil MV-122, Flow Tech, Carver, MA, USA), is placed in 4 DEG C for the rat being perfused
Then refrigerator overnight is removed femoral head, fixed with 4% paraformaldehyde, carries out decalcification processing with 10%EDTA, sample is most laggard
Row microCT detection.Histological stain: the sample after decalcification is subjected to paraffin embedding, slice, every 5 μ m-thicks of slice, HE dye
Color, optical microphotograph is under the microscope.
Experimental result: HE coloration result shows that model group bone trabecula obviously tails off, and form is thinning, and vacuole occurs in osteocyte
And karyopycnosis；ES-exo intervention group bone trabecula has mild necrosis, the vacuolar degeneration (Fig. 7) of rare osteocyte；ES-exo intervention group
The ratio of vacuole be 15.5 ± 2.1%, substantially less than 38 ± 4.8% (P < 0.05) of model group.MicroCT testing result is aobvious
Show, the bone trabecula of model group substantially reduces, is thinning, and the region that trabecular bone structure completely disappears occurs；ES-exo intervention group
Trabecular bone structure keeps relatively complete (Fig. 8), and BV/TV, Tb.Th and Tb.N value are all remarkably higher than model group (P < 0.05).Blood vessel
Visualisation shows that the number of blood vessel of ES-exo intervention group is significantly more than model group (Fig. 9), and blood vessel volume and percentage, blood
Pipe surface is long-pending and blood vessel thickness is obviously higher than model group (P < 0.05).
The above experimental result prompt, ES-exo can prevent the generation and development of caput femoris necrosis.
Embodiment 4
The excretion body in human embryo stem cell source prevents and treats Osteoarthritis
The culture of human embryo stem cell and the extraction of excretion body are same as above.6-8 week old C57 mouse is selected to make Osteoarthritis
Model (model group): mouse anesthesia is placed on mouse plate, exposure knee joint, shaving, iodophor disinfection, and knee joint midsection is shown
Knee joint is isolated under micro mirror, micro syringe injects 5-10uL Collagenase V II.ES-exo treatment group: the 7th after modeling, 14,
21 days, difference joint cavity injection ES-exo 5-10 μ l (1~9 × 1010/ml).Arthritis model group and Normal group are in joint
The PBS of chamber injection same volume.The 28th day execution each group mouse after modeling, in the horizontal detachment shin of knee joint after mouse execution
Bone.Gross examination of skeletal muscle: separating tibial plateau surrounding tissue and meniscus under microscope, sufficiently shows tibial plateau articular surface, high
Times histology microscopically observation articular surface is simultaneously taken pictures.Histology: EDTA solution decalcification 1 week, paraffin embedding, tibial plateau
Coronal slice, 5um thickness, row HE dyeing, the fast green dyeing of sarranine-, immunohistochemical staining (COL-1, COL-2).
Experimental result shows that the visible cartilage wear of arthritis model group, subchondral bone exposes, surface fiber hyperblastosis；
And the visible normal cartilage performance of Normal group and ES-exo treatment group, see Figure 10.Histology is the results show that normal control
Group typical hyaline cartilage appearance visible with ES-exo treatment group, cartilage surface is smooth, and cartilage cell's marshalling, collagen content is just
Often；Arthritis model group is in cartilage wear, and bone falls to be formed, and collagen content is reduced, surface fiber hyperblastosis.ICRS scoring knot
Fruit: Normal group=ES-exo treatment group > arthritis model group；Immunohistochemical staining the results show that Normal group and
ES-exo treatment group COL-2 is negative in strong positive (dark-brown), COL-1 dyeing；And arthritis model group COL-2 coloring is very
Shallowly；COL-1 dyeing is in strong positive (dark-brown).
The above experimental result prompt, ES-exo can prevent the generation and development of Osteoarthritis
Embodiment 5
The excretion body in human embryo stem cell source is carried on β-TCP and repairs large segmental bone defect
The culture of human embryo stem cell and the extraction of excretion body are same as above.Select the tricresyl phosphate with three-dimensional porous nano structure
Calcium (β-TCP) adsorbs certain density ES-exo.Large segmental bone defect model is constructed using SD male rat (300 ± 50g).Experiment
Points three groups: model group (not doing any intervention after modeling), β-TCP transplantation group (transplanting β-TCP after modeling at bone defect) and
ES-exo/ β-TCP transplantation group (transplants ES-exo/ β-TCP) at bone defect after modeling.After rat anesthesia is fixed, exposure femur
Middle section, after electric drill drilling, near end of thighbone and distal end one piece of screw of each implantation on femur, then adopt stainless steel plate fixation
Femur middle section 6mm long bone tissue is ground off with dentistry abrasive drilling, β-TCP and USCs/ β-TCP are transplanted to defect respectively and is consolidated
Fixed, model control group is not implanted into any material.The healing of material degradation and bone defect is observed in progress x-ray inspection in postoperative 4 and 8 weeks
Repair situation.12 weeks after operation puts to death animal, takes out operation femur specimen, 4% paraformaldehyde is fixed for 24 hours, using toy
Micro-CT is scanned reconstruction to femur specimen, and specific sweep parameter is provided that voltage 600kV, 300 μ A of electric current, scanning
Time 900ms, 18 μm of spatial resolution.ROI, length 15mm are set by femoral surgery position, the longitudinal axis and femoral shaft are vertical
Axis is parallel.Initial data is analyzed using CT analysis analysis software, analysis indexes include opposite bone volume (bone
Volume/tissue volume, BV/TV) and bone density (bone mineral density, BMD).Bone tissue carries out conventional
Decalcification passes through New born formation at conventional H E dyeing and Masson dyeing display bone defect.
X-ray inspection result shows that model group 4 weeks and 8 weeks all without obvious New born formation；β-TCP transplantation group is at 4 weeks without obvious
New born formation, material is gradually degraded after 8 weeks, it is seen that newborn ostosis；ES-exo/ β-TCP group is in 4 weeks materials in conjunction with the broken ends of fractured bone
There is newborn ostosis at position, and material is almost degraded after 8 weeks, and bone defect position covers with area of new bone.Micro-CT result
It has been shown that, model group only have a small amount of area of new bone；β-TCP transplantation group has newborn ostosis, but the both ends of bone defect do not connect,
Still there is a large amount of β-TCP to be filled in bone defect position；There is a large amount of area of new bone in ES-exo/ β-TCP group, newborn poroma is scarce
Damage position is fully connected, and only a small amount of β-TCP is shown in Figure 11 at bone defect.Further bone density (bone mineral
Density, BMD) analysis shows that, model group, β-TCP transplantation group and ES-exo/ β-TCP transplantation group are respectively 683.1 ± 38.6,
844.5 ± 65.7 and 854.5 ± 68.7mg/cm3.Diaphysis fraction (bone volume fraction, BV/TV) analysis shows that
Model group, β-TCP and ES-exo/ β-TCP transplantation group diaphysis fraction be respectively 6.8 ± 2.9%, 9.2 ± 3.8% and 18.3 ±
3.2%.Being shown in ES-exo/ β-TCP transplantation group has more newborn ostosis.HE coloration result show three groups all without apparent
Inflammatory reaction.HE dyeing and Masson trichrome stain all show that ES-exo/ β-TCP transplantation group has a large amount of area of new bone；β-TCP group
Equally there is area of new bone but quantity is much smaller than ES-exo/ β-TCP transplantation group；In model group without apparent area of new bone, there is a large amount of fibre
Tie up connective tissue filling.
The above result shows that ES-exo load biomaterial can remarkably promote osteanagenesis, clinical intractable big section bone is repaired
Defect.
Embodiment 6
The excretion body in human embryo stem cell source inhibits the mesenchymal stem cell aging of in vitro culture
The culture of human embryo stem cell and the extraction of excretion body are same as above.The culture of human marrow mesenchymal stem cell (BMSCs):
The bone tissue discarded in clinical operation, normal saline flushing cancellous bone, the cell suspension of acquisition, cell warp are obtained through informed consent
Supernatant (300g, 5 minutes) is abandoned in centrifugation after being sieved through filter, is inoculated into Tissue Culture Flask after being resuspended with DMEM complete medium,
Be placed in cell incubator (37 DEG C, 5%CO2) in culture.Full dose changes liquid after 24 hours, changes the liquid once later every 3 days.To cell
Cell clone in culture bottle merges to form monolayer adherence cell after, with 0.25% trypsin digestion and cell, with containing serum
DMEM culture medium stop to digest and be centrifuged to abandon supernatant (300g, 5 minutes), be resuspended and cell and be inoculated into DMEM complete medium
It is expanded in new Tissue Culture Flask.Well-grown 5th generation cell is taken to be tested.2 groups of experiment point, one group routinely square
Method continues secondary culture (control group), and another group is added 1X10 in cell culture medium7/ ml ES-exo, secondary culture to control
When slow cell Proliferation and form aging occurs in group, cell-proliferation activity is detected with CCK-8 kit, passes through half glycosides of cell ageing β
Lactose enzyme staining reagent kit detects the aging aspects of two groups of cells.
Experimental result shows, the BMSCs of the conventional culture methods culture cell when secondary culture is to ten generations (P10) is given birth to
The agings forms such as length is slowly, particle increases, cell space becomes larger, are shown in Figure 12；CCK-8 testing result is shown with the training containing ES-exo
The ability of cell proliferation for supporting base culture is significantly stronger than control group, sees Figure 13；The discovery of half glycosides lactase coloration result of β is with containing ES-
The cell that the cell cytosol of the culture medium culture of exo is blue is seldom, and a large amount of endochylemas of the cell of routine culture appearance are blue
Senile cell, see Figure 14.The result shows that ES-exo can effectively prevent the BMSCs aging of in vitro culture.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of preparation method of human pluripotent stem cells excretion body, comprising the following steps: using no feeder layer cultural method, in nothing
Serum free culture system system culture people ESCs or iPSCs collect culture medium, collect the excretion body in pure medium.
2. the preparation method of human pluripotent stem cells excretion body according to claim 1, which is characterized in that collect purifying culture
The method of excretion body in base are as follows: the excretion in pure medium is collected by rotation combining ultrafiltration low temperature Ultracentrifugation Method
Body.
3. the preparation method of human pluripotent stem cells excretion body according to claim 1, which is characterized in that free serum culture body
System selects commercially available model TeSRTM-E8TMOr the culture medium of mTeSR1.
4. using the human pluripotent stem cells excretion body of the preparation of preparation method described in claim 1.
5. human pluripotent stem cells excretion body according to claim 4, which is characterized in that the human pluripotent stem cells excretion
Body includes the excretion body that human embryo stem cell and people induce multi-potent stem cell source.
6. the preparation based on human pluripotent stem cells excretion body, which is characterized in that the preparation selects any one of following form:
A, suspending agent: the human pluripotent stem cells excretion body is dissolved in solvent, is existed in the form of suspending agent；
B, it is sustained the compound of excretion body: forming the compound of sustained release excretion body in conjunction with carrier by human pluripotent stem cells excretion body
Object；
C, contain the additive of human pluripotent stem cells excretion body: using human pluripotent stem cells excretion body as the addition of functional component
Agent.
7. the preparation according to claim 6 based on human pluripotent stem cells excretion body, which is characterized in that the carrier includes each
Kind of hydrogel, biomembrane, bioceramic material, nanostructure biomaterial.
8. the application of human pluripotent stem cells excretion body as claimed in claim 5, which is characterized in that including following purposes:
A, inhibit the additive application of its aging as the stemness of the maintenance adult stem cell of in vitro culture；
B, the additive application as the proliferation activity of the various tissue in primary culture cells of maintenance of in vitro culture；
C, the application in the drug of preparation treatment bone and cartilage degradation；
D, the application in the drug of preparation prevention bone and cartilage degradation；
E, the bone including preparation treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder or adhesion of tendon
Application in the drug of section's disease.
9. the application of the preparation as claimed in claim 6 based on human pluripotent stem cells excretion body, which is characterized in that including using below
On the way:
A, inhibit the additive application of its aging as the stemness of the maintenance adult stem cell of in vitro culture；
B, the additive application as the proliferation activity of the various tissue in primary culture cells of maintenance of in vitro culture；
C, the application in the drug of preparation treatment bone and cartilage degradation；
D, the application in the drug of preparation prevention bone and cartilage degradation；
E, the bone including preparation treatment osteonecrosis, bone nonunion, fracture, cartilage damage, tendon injury, frozen shoulder or adhesion of tendon
Application in the drug of section's disease.
10. application as claimed in claim 8 or 9, which is characterized in that the adult stem cell includes mescenchymal stem cell and group
Knit specific stem cells,
The mescenchymal stem cell include fat mesenchymal stem cell, mesenchymal stem cell, Synovial Mesenchymal Stem Cells,
Mescenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, the amnion mesenchymal stem cell in urine source；
The tissue-specific stem cells include that liver stem cells, endothelial progenitor cells, neural stem cell, pancreatic stem cell, epithelium are dry thin
Born of the same parents, kidney stem cell, epidermal stem cells, hair follicle stem cells, limbal stem cell, melanocyte stem cells, sweat gland stem cell, tendon
Stem cell, cartilage precursor cells；
The tissue in primary culture cell include vascular endothelial cell, dermal melanin cell, epidermal cell, liver epithelial cell,
Nerve cell, corneal epithelial cell, endothelial cell, fibroblast, beta Cell of islet, osteoblast, cartilage cell and broken
Osteocyte.
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