CN108715830B - Method for extracting exosome from urinary stem cell and application of exosome - Google Patents
Method for extracting exosome from urinary stem cell and application of exosome Download PDFInfo
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Abstract
The invention discloses an extraction method of a urinary stem cell exosome and application thereof, wherein the extraction method comprises the following specific processes: 1) centrifuging for the first time after urine collection, adding PBS into the first residual liquid obtained after supernatant liquid is removed, centrifuging for the second time, and resuspending the second residual liquid obtained after the supernatant liquid is removed by using a primary culture medium to obtain a urine-derived stem cell suspension; 2) performing amplification culture on the urine-derived stem cell suspension, and carrying out passage to 2-6 generations for later use; 3) and (3) replacing the culture medium of the cells obtained in the step 2), continuing culturing, collecting culture supernatant, centrifuging, filtering, centrifuging, concentrating, adding an exosome extraction reagent, and extracting to obtain a resuspension of the urine-derived stem cell exosomes. Animal experiments prove that the urine-derived stem cell exosome has a treatment effect on senile osteoporosis, postmenopausal osteoporosis and disuse osteoporosis.
Description
Technical field
The present invention relates to stem cells technology fields, it particularly relates to a kind of extracting method for urinating derived stem cell excretion body
And its application, anti-curing osteoporosis is used for more particularly, to a kind of extracting method for urinating derived stem cell excretion body and its in preparation
Drug, the application in food or health care product.
Background technique
Osteoporosis is a kind of metabolic bone characterized by bone amount reduction, bone micro-structure destruction and bone brittleness increase
Disease often induces fracture, brings heavy burden to personal, family and society.Osteoporosis is divided into primary and secondary two
Major class, wherein primary osteoporosis is divided into post menopausal, senile and idiopathic osteoporosis again, and secondary sclerotin is dredged
Loose disease is mostly caused by disease or other factors, including disuse osteoporosis etc..According to international osteoporosis foundation
(IOF) statistical data, the whole world have more than 200,000,000 patients with osteoporosis.1/3 can undergo sclerotin to dredge in 50 years old or more women
Pine property fracture, and thus bring serious consequence.Current osteosporosis resistant medicament has certain effect to osteanagenesis, but usually controls
It is long, costly and have side effect to treat the period.Therefore, more safe and efficient and cheap treatment or prevention osteoporosis is explored
New strategy seem urgent and necessary.
Stem cell transplantation provides new approaches in the result of study of osteanagenesis medical domain for osteoporosis treatment.Closely
Studies have shown that stem cell has certain application prospect in terms of osteoporosis treatment over year.But stem cell directly transplanting begins
There is certain risk eventually, such as blood vessel embolism, micro- infarct and tumour is caused to be formed, thus can sufficiently be sent out it is necessary to explore one kind
Volatilize the new strategy that cell superior functionality is avoided that stem cell transplantation potential risk again.Recent study discovery, is transplanted in vivo
Stem cell only few a part is directly divided into specific parenchyma, they are more mainly living by some biologies of paracrine
Property molecule mobilize endogenous cell participate in tissue repair regeneration.Excretion body is discharged into after extracellular compartment and cell membrane fusion
The film property vesicles that diameter in extracellular environment is 40~150nm, play the part of particularly important in the paracrine action of cell
Role.Having many research report excretion bodies in recent years has the function of stem-like cell tissue repair, can be independently as a kind of life
Object active constituent is applied to regeneration and repairing and treating.Human urine derived stem cell is the one kind being separately cultured out from the urine of people
Precursor, relative to the stem cell in other sources, human urine derived stem cell is without limitations with source, materials safety is noninvasive, system
Standby numerous advantages such as process is simple, self-renewal capacity is powerful, can be used as good excretion body production " factory ".Research report
Road, human urine derived stem cell have significant rush osteogenesis function.Human urine derived stem cell is strong with it as the great advantage of seed cell
The characteristic and excretion body of big promotion osteanagenesis do thin the mediation of its derived cell function for development and utilization urine source
The extracellular anti-curing osteoporosis of body this new strategy secreted provides theoretical foundation and experiment basis.
CN104212762A discloses a kind of method that property pluripotent stem cell in source is urinated by external small molecule Fiber differentiation, packet
It includes and obtains urine source property cell from human urine sample, external small molecule induction is carried out to obtained primary cell and culture is pasted
P1 generation urine derived stem cell (urine-derived stem cells, the USCs) clone of wall growth, picking growth conditions are good
Clone is inoculated with, and continues secondary culture, obtains cell and spindle shape is presented, in good condition.The hUSCs prepared using this method
Induced that orientable to be divided into osteoblast, fat cell, Skeletal Muscle Cell, nerve cell, fibroblast or smooth muscle thin
Born of the same parents carry out transplanting urine derived stem cell, result of study in vivo to lower extremity ischemia in diabetic patients model and show that it can significantly increase ischemic portion
The restoration of blood flow and angiogenesis of position.Although extraction and the separation method of the open urine derived stem cell of the patent, and for studying sugar
Urine disease, but it is not directed to the prevention and treatment of osteoporosis.
Summary of the invention
For above-mentioned technical problem in the related technology, the present invention proposes a kind of extracting method for urinating derived stem cell excretion body
And its it is dry thin to obtain urine source through centrifugation, filtering, centrifugal concentrating for application, the amplification cultivation after isolating urine derived stem cell in urine
It is extracellular to secrete body.Gained urinates the treatment that derived stem cell excretion body is used for osteoporosis through injecting, and mentions for the prevention and treatment of osteoporosis
For a kind of new approach.
To realize the above-mentioned technical purpose, the technical scheme of the present invention is realized as follows:
On the one hand, the present invention provides a kind of extracting method for urinating derived stem cell excretion body, and detailed process is as follows:
1) it is centrifuged for the first time after collecting urine, PBS is added in the first residual liquid of gained after removal supernatant,
It carries out second to be centrifuged, the second residual liquid of gained is resuspended to obtain with primary culture medium after removing supernatant
Urinate derived stem cell suspension；
2) derived stem cell suspension amplification cultivation will be urinated, after being passaged to for the 2nd~6 generation, for use；
3) continue to cultivate after being replaced the culture medium of cell obtained by step 2), collect culture supernatant,
Excretion body extraction reagent is added after centrifugation, filtering, centrifugal concentrating to extract, obtains urine derived stem cell
The re-suspension liquid of excretion body.
Further, clean the middle urine of the urine from normal adults.
Further, the condition of the first time centrifugation are as follows: 400 × g is centrifuged 10 minutes.
Further, in step 1), the dosage volume ratio of first residual liquid and PBS are 1: 10.
Further, in step 1), the dosage volume ratio of second residual liquid and primary culture medium is 1: 15.
Further, in step 1), the primary culture medium be containing 10% fetal calf serum, 100U/mL penicillin,
100mg/L streptomysin and REGM SingleQuot growth factor additive (Lonza；CC-4127 DMEM/F-12 culture medium).
Further, in step 2), the process of amplification cultivation is as follows: will urine derived stem cell suspension at 37 DEG C, 5%CO2
Under the conditions of cultivate, supplement 1mL amplification culture medium every 48h in 96h, 1mL amplification culture medium replaced every 48h after 96h, to cell
Passage when density reaches 80~90%.
Further, the amplification culture medium is to contain 10% fetal calf serum, 1%GlutaMAX, 1% non-essential amino
Acid, 5ng/mL basic fibroblast growth factor, 5ng/mL platelet-derived growth factor-BB, 5ng/mL epidermal growth factor
The DMEM/F-12 culture medium of son, 100U/mL penicillin and 100mg/L streptomysin adds with the growth factor of SingleQuot containing REGM
Add agent (Lonza；CC-4127 the mixed liquor that the volume ratio of RBM culture medium) is 1: 1.
Further, in step 3), the culture medium is changed to the fresh amplification culture medium without excretion body.Preferably,
The fresh amplification culture medium without excretion body is the fetal calf serum, 1%GlutaMAX, 1% non-for removing excretion body containing 10%
Essential amino acid, 5ng/mL basic fibroblast growth factor, 5ng/mL platelet-derived growth factor-BB, 5ng/mL table
The DMEM/F-12 culture medium and SingleQuot containing REGM of skin growth factor, 100U/mL penicillin and 100mg/L streptomysin are raw
Long factor additive (Lonza；CC-4127 the mixed liquor that the volume ratio of RBM culture medium) is 1: 1.
Further, in step 3), the centrifugation, filtering, detailed process is as follows for centrifugal concentrating: will be in the culture of collection
Clear liquid, centrifugation removal residual cell and cell fragment, 0.22 μm of sterilizing filter filtering, are transferred to 15mL's for filtered fluid
10KD ultra-filtration centrifuge tube, 4000 × g, 4 DEG C are centrifuged about 20 minutes, and ultrafiltrate is made to be concentrated into 1mL；14mL is added into ultrafiltrate
PBS, 4000 × g, 4 DEG C are centrifuged about 15 minutes, so that ultrafiltrate is concentrated into 1mL, obtain concentrate.
Further, the concentrate and excretion body extract reagent and extract according to the ratio of volume ratio 1: 5, extract
Process it is as follows: concentrate and excretion body are extracted after reagent mix, 4 DEG C of incubation 12h are then centrifuged, 1500 × g, and 4 DEG C are centrifuged
30 minutes, supernatant is abandoned, precipitating is resuspended with sterile PBS to get the re-suspension liquid of urine derived stem cell excretion body is arrived.
Further, it is Exoquick-TC that the excretion body, which extracts reagent,TMExcretion body extracts reagent.
On the one hand, the present invention provide a kind of urine derived stem cell excretion body preparation prevention and treatment osteoporosis agents, food or
Application in health care product.
Further, the urine derived stem cell excretion body is for promoting distal femur Grafting Cancellous Bone Bolt volume fraction.
Further, the urine derived stem cell excretion body is for increasing bone trabecula thickness.
Further, the urine derived stem cell excretion body is for promoting bone trabecula quantity.
Further, the urine derived stem cell excretion body is for promoting thickness of cortex of bone.
Further, the osteoporosis includes senile osteoporosis, Postmenopausal Osteoporosis or useless property bone
Matter osteoporosis.
Further, the drug further includes pharmaceutically acceptable carrier.
Further, the drug passes through intravenously administrable.
On the one hand, the present invention provides a kind of for treating the pharmaceutical composition of osteoporosis comprising urine derived stem cell
Excretion body.
It further, further include pharmaceutically acceptable carrier.
Further, the dosage form of described pharmaceutical composition be injection, capsule, tablet, oral preparation, microcapsule formulation,
Ointment or spray etc..
In mouse senile osteoporosis model, the soma prognosis of derived stem cell excretion, mouse femur distal end cancellous bone are urinated
Diaphysis fraction (Tb.BV/TV), bone trabecula quantity (Tb.N), bone trabecula thickness (Tb.Th), epibial circumference (Ps.Pm) and skin
Matter bone thickness (Ct.Th) raising (P < 0.05) more significant than solvent control group, and bone trabecula separating degree (Tb.Sp) is than solvent pair
(P < 0.05) is substantially reduced according to group；In mouse Postmenopausal Osteoporosis model and hind leg disuse osteoporosis model,
Urinate the soma prognosis of derived stem cell excretion, mouse femur distal end Grafting Cancellous Bone Bolt volume fraction (Tb.BV/TV), bone trabecula quantity
(Tb.N), bone trabecula thickness (Tb.Th) and thickness of cortex of bone (Ct.Th) raising (P < 0.05) more significant than solvent control group, and
Bone trabecula separating degree (Tb.Sp) and endoluminal circumference (Es.Pm) are substantially reduced (P < 0.05) than solvent control group.
Beneficial effects of the present invention:
The present invention provide it is a kind of urinate derived stem cell excretion body extracting method and its application, by centrifugation, amplification cultivation, from
Urine derived stem cell excretion body is obtained after the heart, filtering, centrifugal concentrating；Prove urine derived stem cell excretion body for old by zoopery
Year osteoporosis, Postmenopausal Osteoporosis and disuse osteoporosis have therapeutic effect, pass through intravenous injection urine source
Stem cell excretion physical efficiency reaches the bone amount for improving distal femur, plays old, post menopausal and disuse osteoporosis prevention and treatment
Effect.
The urine derived stem cell that the present invention uses is without limitations with source, materials safety is noninvasive, preparation process is simple, self
Numerous advantages such as updating ability is powerful can be used as good excretion body production " factory ", so as to continually obtain urine source
Stem cell excretion body.
Directly transplanting excretion body is advantageously than transplanting stem cell itself.Excretion body is nanoscaled vesicle, opposite micron order
Cell, excretion body activity it is more stable, more easy to maintain, be not easy to cause the complication such as blood vessel embolism, also can avoid chromosome it is abnormal
The problems such as change and abnormal differentiation.
Therefore, urine derived stem cell excretion body can be used for preparing old prevention and treatment, post menopausal and disuse osteoporosis medicine
Object, food or health care product provide new safely and effectively biology and control for prevention and treatment old age, post menopausal and disuse osteoporosis
Treatment means.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only some implementations of the invention
Example, for those of ordinary skill in the art, without creative efforts, can also obtain according to these attached drawings
Obtain other attached drawings.
Fig. 1 a: urinating the transmission electron microscope picture of derived stem cell excretion body, and white scale is 50nm in figure.
The result of the surface marker of Fig. 1 b:Western blot identification urine derived stem cell excretion body.
Fig. 2: each group senile osteoporosis mouse model femur bone micro-structure comparison diagram；That is senile osteoporosis mouse
Model tail vein injection urinated derived stem cell excretion body or isometric solvent (PBS) after 3 months, femur bone micro-structure comparison diagram；
Note: * P < 0.05 shows that difference is statistically significant compared with Control group.
Fig. 3: each group senile osteoporosis mouse model distal femur three-dimensional imaging, white scale is 1mm in figure.
Fig. 4: each group Postmenopausal Osteoporosis mouse model femur bone micro-structure comparison diagram, the i.e. post menopausal of OVX simulation
Osteoporosis mouse model tail vein injection urinated derived stem cell excretion body or isometric solvent (PBS) after 2 months, and femur bone is micro-
Structure Comparison figure.Note: * P < 0.05 shows that difference is statistically significant compared with Sham group；#P < 0.05 shows and OVX group
It compares, difference is statistically significant.
Fig. 5: each group Postmenopausal Osteoporosis mouse model distal femur three-dimensional imaging, white scale is 1mm in figure.
Fig. 6: each group hind leg disuse osteoporosis mouse model femur bone micro-structure comparison diagram, the i.e. hind leg of TS induction
Disuse osteoporosis mouse model tail vein injection urinated derived stem cell excretion body or isometric solvent (PBS) after 3 weeks, femur
Bone micro-structure comparison diagram.Note: * P < 0.05 shows that difference is statistically significant compared with Control group；#P < 0.05 shows
Compared with TS group, difference is statistically significant.
Fig. 7: each group hind leg disuse osteoporosis mouse model distal femur three-dimensional imaging, white scale is in figure
1mm。
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art's every other embodiment obtained belong to what the present invention protected
Range.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.
Except there is a special instruction, the various reagents used in the present invention, raw material be can commodity commercially or
Person can the product as made from well known method.
Embodiment 1
Urine derived stem cell is separately cultured:
(1) collection of derived stem cell is urinated
After obtaining contributor's informed consent, using added with 1% antibacterial-antifungal agent (Antibiotic-
Antimycotic 50mL sterile centrifugation tube) collects the clean the middle urine (30-50mL) of normal adults.
(2) separation of derived stem cell is urinated
The urine of collection is centrifuged 10 minutes through 400 × g, is removed most of supernatant (staying 1mL liquid in tube bottom), is being centrifuged
10mL PBS is added in pipe, and 200 × g is centrifuged after ten minutes, removes most supernatants (staying 0.2mL liquid in tube bottom), uses
Precipitating is resuspended 3mL primary culture medium, then averagely assigns in 3 holes of 12 orifice plates.All operations step carries out at room temperature.
37 DEG C, 5%CO2Under the conditions of cultivate.Primary culture medium is to contain 10% fetal calf serum, 100U/mL penicillin, 100mg/L strepto-
Element and REGM SingleQuot growth factor additive (Lonza；CC-4127 DMEM/F-12 culture medium).
(3) culture of derived stem cell is urinated
After 48h, the fresh amplification culture medium of 1mL is directly added into the hole Xiang Shangshu.After 96h, every hole, which is inhaled, abandons 1mL culture medium, and
Add the fresh amplification culture medium of 1mL.Hereafter, the next day, replaces a subculture.The passage when cell density reaches 80%~90%.
2nd generation is to the 6th alternative in subsequent experimental.Amplification culture medium is to contain 10% fetal calf serum, 1%GlutaMAX, 1% nonessential ammonia
Base acid, 5ng/mL basic fibroblast growth factor, 5ng/mL platelet-derived growth factor-BB, 5ng/mL epidermal growth
The DMEM/F-12 culture medium and the growth factor of SingleQuot containing REGM of the factor, 100U/mL penicillin and 100mg/L streptomysin
Additive (Lonza；CC-4127 the mixed liquor that the volume ratio of RBM culture medium) is 1: 1.
Embodiment 2:
Urinate the extraction and identification of derived stem cell excretion body:
After urine derived stem cell reaches 80% degrees of fusion, continued to cultivate 48h with the fresh amplification culture medium without excretion body.
Cell culture supernatant is collected, low-speed centrifugal removes residual cell and cell fragment in supernatant, 0.22 μm of sterilizing filter mistake
Filtered fluid is transferred to the 10KD ultra-filtration centrifuge tube of 15mL by filter, and 4000 × g, 4 DEG C are centrifuged about 20 minutes, keep ultrafiltrate dense
It is reduced to 1mL.14mL PBS, 4000 × g, 4 DEG C are added into ultrafiltrate to be centrifuged about 15 minutes, ultrafiltrate is made to be concentrated into 1mL.It will surpass
Filtrate is transferred in the sterile eppendorf centrifuge tube of new 1.5mL, and Exoquick-TC is added according to 1: 5 ratioTMExcretion body
Reagent is extracted, after 4 DEG C of incubations 12h, 1500 × g, 4 DEG C are centrifuged 30 minutes, abandon supernatant, and precipitating is resuspended to get to urinating with sterile PBS
Derived stem cell excretion body (urine-derived stem cells-derived exosomes, USC-Exos) suspension.Using saturating
Radio microscopy surveys its diameter and form, using Western blot detect its surface marker CD9, CD63, CD81 and
TSG101, the above results such as Fig. 1 a~1b institute are not.
Embodiment 3:
Urinate the effect of the anti-curing osteoporosis of derived stem cell excretion body:
(1) mouse model:
16 monthly age SPF grade C57BL/6 male mices are selected, as senile osteoporosis model.
2 monthly age SPF grade C57BL/6 female mices are selected, postmenopausal osteoporosis is constructed by bilateral excision ovary (OVX)
Disease model, while sham-operation (Sham) group is established, which only wipes out a little ovary peripheral adipose tissue.
3 month female mouse row tail-suspensions (TS) are selected, mitigates its double hind leg weight bearing, constructs disuse osteoporosis
Disease model, control group are move freely.Experiment mice is bought from Hunan Si Laike scape up to zoopery Co., Ltd.
(2) grouping experiment:
Senile osteoporosis model: it is divided into urine derived stem cell excretion body group i.e. USC-Exos group and isometric solvent
(PBS) control group (Control group).100 μ g USC-Exos of tail vein injection (being dissolved in 100 μ L PBS) or 100 μ L weekly
PBS, continuous 3 months.
Postmenopausal Osteoporosis model: mouse row OVX was divided into two groups after 1 week, gave USC-Exos (OVX+USC-
Exos group) or isometric PBS (OVX group) processing.Sham group also gives isometric PBS in same time point.Tail vein weekly
Inject a 100 μ g USC-Exos (being dissolved in 100 μ L PBS) or 100 μ L PBS, continuous 2 months.
Disuse osteoporosis model: by prepare row TS mouse be divided into two groups, be expert at TS when in corresponding time point
Give USC-Exos (TS+USC-Exos group) or isometric PBS (TS group) processing.It move freely control group (Control group)
Give isometric PBS.Tail vein injection 100 μ g USC-Exos (being dissolved in 100 μ L PBS) or 100 μ L PBS twice weekly, even
It is 3 weeks continuous.
The samples such as mouse bone tissue are collected at corresponding time point after intervening according to the method described above, using μ CT detection mouse stock
Bone distal end Grafting Cancellous Bone Bolt volume fraction (Tb.BV/TV), bone trabecula quantity (Tb.N), bone trabecula thickness (Tb.Th), bone trabecula point
From degree (Tb.Sp), endoluminal circumference (Es.Pm), epibial circumference (Ps.Pm) and thickness of cortex of bone (Ct.Th), it is dry to assess urine source
Treatment or prevention effect of the cell excretion body, that is, USC-Exos to mouse osteoporosis.
Fig. 2 and Fig. 3 is each group mouse femur bone micro-structure after senile osteoporosis mouse model is intervened 3 months respectively
Comparison diagram and distal femur three-dimensional imaging.Fig. 4 and Fig. 5 is after Postmenopausal Osteoporosis mouse model is intervened 2 months, respectively respectively
Group mouse femur bone micro-structure comparison diagram and distal femur three-dimensional imaging.Fig. 6 and Fig. 7 is hind leg disuse osteoporosis respectively
After mouse model is intervened 3 weeks, each group mouse femur bone micro-structure comparison diagram and distal femur three-dimensional imaging.
By Fig. 2~7 it is found that after urine derived stem cell excretion body, that is, USC-Exos intervention, Aged Mice distal femur cancellous bone
Diaphysis fraction (Tb.BV/TV), bone trabecula quantity (Tb.N), bone trabecula thickness (Tb.Th), epibial circumference (Ps.Pm) and skin
Matter bone thickness (Ct.Th) raising (P < 0.05) more significant than solvent control group, and bone trabecula separating degree (Tb.Sp) is than solvent pair
(P < 0.05) is substantially reduced according to group；After USC-Exos intervenes, OVX mouse and TS mouse femur distal end Grafting Cancellous Bone Bolt volume fraction
(Tb.BV/TV), bone trabecula quantity (Tb.N), bone trabecula thickness (Tb.Th) and thickness of cortex of bone (Ct.Th) compare solvent control
Group is significant to increase (P < 0.05), and bone trabecula separating degree (Tb.Sp) and endoluminal circumference (Es.Pm) are more obvious than solvent control group
It reduces (P < 0.05).Therefore, postmenopausal osteoporosis of the urine derived stem cell excretion body for senile osteoporosis, OVX simulation
The hind leg disuse osteoporosis of disease and TS induction is highly effective, can be used for preparing old prevention and treatment, post menopausal and useless use
Property osteoporosis drug or health care product in, provided to treat or prevent old, post menopausal and disuse osteoporosis
New safely and effectively biological therapy means.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (2)
1. a kind of application of urine derived stem cell excretion body in preparation prevention and treatment osteoporosis agents, food or health care product, special
Sign is,
The urine derived stem cell excretion body is obtained by following procedure extraction:
1) be centrifuged for the first time after collecting urine, remove and be added PBS in the first residual liquid of gained after supernatant, carry out second from
The heart, the second residual liquid of gained is resuspended to obtain urine derived stem cell suspension with primary culture medium after removing supernatant；
2) derived stem cell suspension amplification cultivation will be urinated, after being passaged to for the 2nd ~ 6 generation, for use；
3) continue to cultivate after being replaced the culture medium of the urine derived stem cell suspension after step 2 amplification cultivation, collect culture
Supernatant, centrifugation, filtering excretion body are added after centrifugal concentrating extract reagent and extract, and obtain urine derived stem cell excretion body
Re-suspension liquid；
The primary culture medium is to contain 10% fetal calf serum, 100 U/mL penicillin, 100 mg/L streptomysins and REGM
The DMEM/F-12 culture medium of SingleQuot growth factor additive；
In step 3), the culture medium is changed to the fresh amplification culture medium without excretion body；It is described fresh without excretion body
Amplification culture medium are as follows: fetal calf serum, 1% GlutaMAX, 1% nonessential amino acid, the 5 ng/mL alkali for removing excretion body containing 10%
Property fibroblast growth factor, 5 ng/mL platelet-derived growth factor-BB, 5 ng/mL epidermal growth factor, 100 U/
The DMEM/F-12 culture medium of mL penicillin and 100 mg/L streptomysins and the growth factor of SingleQuot containing REGM additive
The volume ratio of RBM culture medium is the mixed liquor of 1:1；
The urine derived stem cell excretion body is for promoting distal femur Grafting Cancellous Bone Bolt volume fraction；The urine derived stem cell excretion body
For increasing bone trabecula thickness；The urine derived stem cell excretion body is for promoting bone trabecula quantity；The urine derived stem cell excretion
Body is for promoting thickness of cortex of bone.
2. application according to claim 1, which is characterized in that the osteoporosis include senile osteoporosis, absolutely
Osteoporosis or disuse osteoporosis after.
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| CN109554335A (en) * | 2018-12-04 | 2019-04-02 | 上海市第六人民医院 | The preparation method and application of normal stem cell are separately cultured in the urine of mitochondria mt3243AG mutation crowd |
| CN110051694B (en) * | 2019-04-22 | 2023-12-01 | 中山大学附属第一医院 | Urine-derived stem cell preparation, preparation thereof and application thereof in preparation of acute immune rejection medicament after organ transplantation |
| CN110305835A (en) * | 2019-06-26 | 2019-10-08 | 中山大学附属第一医院 | Kidney transplant donor specific urinates the preparation method and its application of source cell and its DNA |
| CN111494719A (en) * | 2019-12-31 | 2020-08-07 | 中南大学湘雅医院 | Novel bone tissue engineering scaffold and preparation method thereof |
| CN111388504B (en) * | 2020-03-12 | 2021-02-23 | 成都世联康健生物科技有限公司 | Preparation of tooth epithelial cell exosome, preparation of exosome implant and application of exosome implant |
| CN114317426A (en) * | 2022-01-06 | 2022-04-12 | 上海市同济医院 | Preparation method of mouse adipose-derived stem cell exosome |
| CN114377034A (en) * | 2022-01-19 | 2022-04-22 | 四川大学华西医院 | Biological agent for treating arthritis and preparation method and application thereof |
| CN115054616A (en) * | 2022-06-27 | 2022-09-16 | 中南大学湘雅医院 | Application of urinary stem cell exosome in preparation of anti-aging preparation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003044166A3 (en) * | 2001-11-15 | 2003-12-31 | Univ California | METHODS AND COMPOSITIONS FOR REGULATION OF GENE EXPRESSION THROUGH MODULATION OF mRNA TURNOVER |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
| CN106967673A (en) * | 2017-04-26 | 2017-07-21 | 溯源生命科技股份有限公司 | Kidney stem cell excretion preparation |
-
2018
- 2018-05-04 CN CN201810420845.4A patent/CN108715830B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003044166A3 (en) * | 2001-11-15 | 2003-12-31 | Univ California | METHODS AND COMPOSITIONS FOR REGULATION OF GENE EXPRESSION THROUGH MODULATION OF mRNA TURNOVER |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
| CN106967673A (en) * | 2017-04-26 | 2017-07-21 | 溯源生命科技股份有限公司 | Kidney stem cell excretion preparation |
Non-Patent Citations (4)
| Title |
|---|
| Chun-Yuan Chen et al..Exosomal DMBT1 from human urine-derived stem cells facilitates diabetic wound repair by promoting angiogenesis.《Theranostics》.2018,第8卷(第6期), * |
| Exosomal DMBT1 from human urine-derived stem cells facilitates diabetic wound repair by promoting angiogenesis;Chun-Yuan Chen et al.;《Theranostics》;20180207;第8卷(第6期);第1607-1623页 * |
| Exosomes secreted by human urine-derived stem cells could prevent kidney complications from type I diabetes in rats;Zhen-zhen Jiang et al.;《Stem Cell Research & Therapy》;20161231;第7卷;第1-13页 * |
| 人尿源干细胞来源的外泌体对1型糖尿病大鼠的肾脏保护作用及其机制研究;姜珍珍;《中国学位论文全文数据库》;20151203;第3-22页 * |
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