CN107080754A - A kind of Endometrial stem cell preparation and its application - Google Patents
A kind of Endometrial stem cell preparation and its application Download PDFInfo
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- CN107080754A CN107080754A CN201710289230.8A CN201710289230A CN107080754A CN 107080754 A CN107080754 A CN 107080754A CN 201710289230 A CN201710289230 A CN 201710289230A CN 107080754 A CN107080754 A CN 107080754A
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- stem cell
- endometrial stem
- ginsenoside
- endometrial
- cell preparation
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Classifications
-
- 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
- A61K35/48—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Reproductive Health (AREA)
- Zoology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Gynecology & Obstetrics (AREA)
- Molecular Biology (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application belongs to regenerative medicine and biology techniques field, and in particular to a kind of Endometrial stem cell preparation and its application.Endometrial stem cell preparation provided by the present invention is included：Endometrial stem cell, ginsenoside Rb1 and ginsenoside Re, source of human stem cell, convenient material drawing, non-invasive, no ethics limitation are used as from Endometrial stem cell；Endometrial stem cell and ginsenoside play synergy, effectively inhibit premature ovarian failure, greatly shorten the tissue repair time.Present invention also offers application of the above-mentioned Endometrial stem cell preparation in treatment premature ovarian failure medicine is prepared.
Description
Technical field
The invention belongs to regenerative medicine and biology techniques field, and in particular to a kind of Endometrial stem cell preparation and its
Using.
Background technology
Premature ovarian failure (premature ovarian failure, POF) refers to the female of oneself formation rule of menstruation before 40 years old
Property, occur the symptoms such as irregular menstruation, amenorrhoea, sexual organ atrophy due to ovarian failure, and it is often sharp along with sexual gland is promoted
The rising of plain level and the decline of estrogen level.Premature ovarian failure is the common disease and frequently-occurring disease in gynemetrics's clinical position,
Through the main cause of disease as current woman's acyesis.At present, ovum is mainly recovered by methods such as hormone therapy and immunization therapies
Nest function, but the above method can only temporarily relief of symptoms, it is impossible to the Regeneration and Repair of self-inflicted injury ovary tissue is effectively facilitated,
Can not radical curing of disease.It is clinical practice in recent years by the treatment of injection of bone marrow mescenchymal stem cell alternative medicine and operative treatment
In be used for treating the new tool of premature ovarian failure.But mesenchymal stem cells MSCs must could be obtained by bone marrow aspiration, not only
Sampling is difficult, and certain somatic damage is easily caused to patient, hinders the wish that healthy population contributes marrow.In addition,
The content of mesenchymal stem cells MSCs is very rare in marrow, and multiplication capacity is relatively low.
The content of the invention
In view of this, it is an object of the invention to provide a kind of Endometrial stem cell preparation, ovary is treated applied to preparing
The medicine of early ageing, source of human stem cell is convenient, non-invasive.
The concrete technical scheme of the present invention is as follows：
The invention provides a kind of Endometrial stem cell preparation, comprising：Endometrial stem cell, ginsenoside Rb1 and
Ginsenoside Re.
Endometrial stem cell (endometrial stem cells, EnSCs) is a kind of adult stem cell, with self
The potential of renewal, Multidirectional Differentiation and hyperproliferation, carcinogenic risk is low, is disputed on by ethics few.Endometrial stem cell have with
Biological characteristics similar BMMSCs, expresses the special surface mark of the mescenchymal stem cells such as CD29, CD44, CD73 and CD90
Will, the endothelial cells such as CD14, CD34, CD45, CD133 and Stro-1, hemopoietic stem cell surface mark are not expressed, MHC- is not expressed
I, expresses MHC-II on a small quantity, does not express or few expression of HLA-DR, illustrates that EnSCs has compared with low immunogenicity.EnSCs can be from warp
Noninvasive in blood to obtain, growth rate is fast, and passage is stable, and immunogenicity is low, with stronger paracrine ability.
It is preferred that, the concentration of the Endometrial stem cell is 1.0 × 107~5.0 × 107Individual/mL.
General ginsenoside Central nervous system, cardiovascular system, digestive system, immune system, internal system, uropoiesis
Reproductive system has extensive effect, so as to improve people's muscle power, the mobility of intelligence, non-spy of the enhancing body to destructive stimulus
Different in nature resistance.
It is preferred that, the concentration of the ginsenoside Rb1 is 0.1~2mg/L, more preferably 0.5~1.5mg/L, most preferably
For 1mg/mL.
It is preferred that, the concentration of the ginsenoside Re is 0.1~2mg/L, more preferably 0.5~1.5mg/L, is most preferably
1mg/mL。
It is preferred that, the Endometrial stem cell preparation is also included：Physiological saline.
Present invention also offers a kind of preparation method of above-mentioned Endometrial stem cell preparation, by Endometrial stem cell,
Ginsenoside Rb1, ginsenoside Re and physiological saline mixing, obtain the Endometrial stem cell preparation.
The endometrium obtained present invention also offers above-mentioned Endometrial stem cell preparation or above-mentioned preparation method is dry thin
Application of born of the same parents' preparation in treatment premature ovarian failure medicine is prepared.
It is preferred that, the Endometrial stem cell preparation is ejection preparation.
Compared with prior art, Endometrial stem cell preparation of the invention has advantages below：
1) present invention is used as seed cell, convenient material drawing, non-invasive, no ethics limitation from Endometrial stem cell；
And the Endometrial stem cell preparation immunogenicity is low, immunological rejection can be avoided, it is safe and reliable；
2) Endometrial stem cell preparation of the present invention also includes ginsenoside Rb1 and ginsenoside Re, and ginsenoside can be straight
The anti-adversity ability of enhancing senile cell is connect, Endometrial stem cell repairs premature ovarian failure tissue, ginseng soap by paracrine action
Glycosides and Endometrial stem cell synergy, effectively inhibit premature ovarian failure, greatly shorten the tissue repair time；
3) invention formulation is from physiological saline as solvent, and its osmotic pressure is consistent with the osmotic pressure of human body, can avoid damaging
Hinder its bioactivity under ovary tissue, Cord blood stable；
4) Endometrial stem cell preparation of the present invention is ejection preparation, can avoid blood flow and cause the loss of preparation；
5) preparation process of invention formulation is simple, takes short, achievable large-scale production.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is the ovarian follicle quantity result that each group ovary tissue is cut into slices.
Embodiment
By substituting stem cell from Endometrial stem cell, while optimize the constituent in preparation, the present invention
There is provided a kind of Endometrial stem cell preparation, applied to the medicine for preparing treatment premature ovarian failure, stem cell survival is stable, come
Source is convenient, non-invasive, and the preparation time of preparation is short, efficiency high.
The present invention is not particularly limited to Endometrial stem cell, ginsenoside Rb1 and the source of ginsenoside Re, is used
Conventional Endometrial stem cell well-known to those skilled in the art, ginsenoside Rb1 and ginsenoside Re, or use
Endometrial stem cell, ginsenoside Rb1 and ginseng that routine techniques well-known to those skilled in the art is prepared
Saponin(e Re.
Technical scheme is clearly and completely described below in conjunction with the specific embodiment of the invention, it is clear that
Described embodiment is a part of embodiment of the invention, rather than whole embodiments.Those skilled in the art should manage
Solution, modifies to the specific embodiment of the present invention or some technical characteristics is replaced on an equal basis, without departing from the present invention
The spirit of technical scheme, all should cover in the scope of protection of the invention.
In following examples, from Shaanxi, intelligent section plant exploitation has the ginsenoside Rb1 and ginsenoside Re used
Limit company.
The preparation of the Endometrial stem cell of embodiment 1
1st, after agreeing to through consulting, according to menses collection suit (Hangzhou Yi Wensai, S-Evans biosciences) specification
Collect healthy women through blood sample；Then these samples are transferred to the phosphorus containing dual anti-(penicillin/streptomycin) and heparin
In phthalate buffer, preserved under the conditions of 4 DEG C, sample is sent to laboratory centrifugal within 24h~48h；Then take supernatant and
Quick detection whether there is germ contamination under aseptic condition, if feminine gender then continues to be separately cultured and identify, table 1 is endometrium
The streaming qualification result of stem cell.Wherein, Endometrial stem cell be separately cultured and qualification process refer to following documents：
Zhou Hua, Song Lina, quick people's Endometrial stem cell in-vitro separation, culture and the Chinese traditional Chinese medicine academic periodicals of identification
[J].2014.32(8):1907-1908。
Table 1
| CD73 | CD90 | CD105 | CD11b | CD19 | CD34 | CD45 | HLA-DR | |
| Expression quantity | 98.4% | 97.5% | 99.3% | 1.8% | 1.2% | 1.0% | 0.4% | 1.4% |
Embodiment 2
(1) it is formulated：
Endometrial stem cell 5 × 107Individual/mL；
Ginsenoside Rb1 1mg/mL；
Ginsenoside Re 1mg/mL；
Physiological saline is supplied.
(2) prepare：
1) ginsenoside Rb1 and ginsenoside Re are dissolved using physiological saline, obtains ginsenoside solution；Its
In, the concentration of ginsenoside Rb1 and ginsenoside Re are 1mg/mL；
2) using step 1) Endometrial stem cell obtained in embodiment 1 is resuspended in obtained ginsenoside solution, obtain
The Endometrial stem cell preparation, the wherein concentration of Endometrial stem cell are 5 × 107Individual/mL；
3) step 2) obtained Endometrial stem cell preparation dispensed into syringe, is then put into sterile sylphon
In, the sylphon for turning to have preparation is transferred in the storage box for being placed with ice bag, preserved at 0~4 DEG C.
Embodiment 3
Endometrial stem cell 1 × 107Individual/mL；
Ginsenoside Rb1 0.1mg/mL；
Ginsenoside Re 0.1mg/mL；
Physiological saline is supplied.
Embodiment 4
Endometrial stem cell 2.5 × 107Individual/mL；
Ginsenoside Rb1 0.5mg/mL；
Ginsenoside Re 0.5mg/mL；
Physiological saline is supplied.
Embodiment 5
1st, 2-3 monthly ages for being provided from experimental animal portion of Traditional Chinese Medicine University Of Guangzhou, 200 ± 20g of body weight, with normally emotionally
The wistar female rats in cycle 120, adaptability raise 1 week after, be randomly divided into 4 groups, be followed successively by control group 1, control group 2,
Control group 3 and experimental group, every group each 30.
Build the early failure model of rat ovary：Disposable celiac injection cyclophosphamide solution (120mg/kg) and busulfan solution
(12mg/kg) each 0.1mL, daily 8：00 beats easily vaginal fluid smear, then pap staining with cotton swab, observes vaginal exfoliated
Change, the phase between continuous 10d observes Constant Estrus, is considered as premature ovarian failure and models successfully.Wherein build rat ovary early ageing mould
The detailed process of type refers to following document：
Yingying Zhang, Yin Huiqun, Ni Feng Bone Marrow Mesenchymal Stem Cells Transplantations should in premature ovarian failure mouse ovarian reconstruction
With Agricultural University Of Anhui journal, 2017,44 (1):1-6.
2nd, after modeling success, by rat back after every experimental rat intraperitoneal injection sodium phenobarbital 60mg/kg, 15min
It is fixed on upwards on animal operating table, sterilizes skin, hold ophthalmic tweezers and clutch lower abdomen median line skin, make small horizontal stroke with clipper for surgical use
Otch, scissors blunt separation fully exposes surgical field of view, cuts manadesma, and fat pad, ovary are hauled out with tweezers.Fixed again with tweezers
Firmly preparation described in table 2, is then injected separately into rat ovary tissue by ovary with micro syringe, and injection dosage is 20 μ L/.
Table 2
3rd, participating in the important gene of apoptosis regulation has many kinds, wherein Bcl-2 and Bax gene pairs muroid ovarian granulosa
Apoptosis plays a decisive role.Wherein, Bax increases promotion Apoptosis, and Bcl-2 increases suppression Apoptosis.
(1) expression of method test experience rat ovary Bax, Bcl-2 albumen of immunohistochemical staining is used,
Its detailed process is：The ovary tissue serial section of 6 μ m-thicks is taken, using SABC method row immunohistochemical stainings, ovary tissue section
Air drying；20mL 30%H2O2With the mixing of 200mL distilled water, then add in ovary tissue section, room temperature 10min is to go out
Endogenous peroxydase living, then with distilling water washing 3 times, each 5min；Carried out with micro-wave oven after the multiple antigen of hot repair, with 5%
BSA confining liquids room temperature close 30min, then respectively be added dropwise Bcl-2 and Bax primary antibodies mixed liquor (volume ratio is 1：200) at 4 DEG C
Overnight；On next day, biotinylated goat anti-rabbit IgG effect 10min are added dropwise at room temperature, then SABC effect 30min are added dropwise, then DAB
Developed the color 3~10min, and haematoxylin is redyed, and running water returns blue 15min；Conventional gradients dehydration of alcohol, dimethylbenzene are transparent, neutral gum
Mounting.
Table 3 is the expression of results of each group experimental rat ovary Bax, Bcl-2 albumen, as shown in table 3, the He of control group 2
Bax, Bcl-2 protein expression of control group 3 are compared with control group 1, P<0.05, with statistical significance, show medulla mesenchyma
Stem cell and Endometrial stem cell can suppress premature ovarian failure；Control group 2 is close with the result of control group 3, shows between marrow
The effect of the reparation premature ovarian failure of mesenchymal stem cells and Endometrial stem cell is close, Endometrial stem cell can effectively replace bone
Bone marrow-drived mesenchymal stem is used as the seed cell for treating premature ovarian failure.Experimental group is compared with control group 1, P<0.01, have
Notable statistical significance, shows that the effect of experimental group suppression premature ovarian failure is obvious；Compared with control group 2 and control group 3, experimental group
The effect for suppressing premature ovarian failure is more excellent.
The expression of each group ovary Bax, Bcl-2 albumen of table 3
| Group | Experiment mice number | Bax | Bcl-2 |
| Control group 1 | 10 | 0.3469±0.0068 | 0.2684±0.0215 |
| Control group 2 | 10 | 0.2845±0.0042* | 0.2978±0.0160* |
| Control group 3 | 10 | 0.2797±0.0063* | 0.2961±0.0152* |
| Experimental group | 10 | 0.1964±0.0059# | 0.3428±0.0124# |
* represent, compared with control group, P<0.05, with statistical significance；
# is represented, compared with control group, P<0.01, tool is statistically significant.
(2) using the expression of Western blot test experience rat ovary Bax, Bcl-2 albumen, its detailed process
For：Every group takes 10 rat cervical dislocation execution, peels off and takes out bilateral ovaries, is shredded；0.5mL lysates are added,
12000g/min at 30min, 4 DEG C is homogenized on ice and centrifuges 30min, takes supernatant, protein quantification is carried out using BCA methods, calculate sample
Protein concentration；According to volume ratio 5:1 ratio adds sample-loading buffer, and 5min, cooled on ice, after being denatured are boiled in water-bath
Albumen；By the albumen equivalent loading after denaturation, SDS- polyacrylamide gel electrophoresises are then carried out；Using wet method transferring film by gel
In protein delivery to pvdf membrane on, transferring film condition is：60v transferring films 2h；Pvdf membrane (is prepared) with 5% skimmed milk power with TBST
Room temperature closes 1h, is separately added into Bax (1：200)、Bcl-2(1：200) with β-actin (1：200), 4 DEG C of overnight incubations, TBST is washed
Film 10min × 3 time；The secondary antibody (1 that pvdf membrane and horseradish peroxidase (HRP) are marked：5000) 1h is incubated at room temperature,
TBST washes film 10min*3 times；Pvdf membrane is placed on preservative film, A, B luminescent solution press 1：1 ratio is mixed, and A, B mixed liquor is added dropwise,
Imaging system is automatically analyzed through gel to be imaged, IMAQ and analysis.
Table 4 is each group rat ovary Bax and Bcl-2 protein expression result, as shown in the result of table 4, control group 2 and control group
3 Bax, Bcl-2 protein expression is compared with control group 1, P<0.05, with statistical significance, show mesenchymal stem cells MSCs
It can suppress premature ovarian failure with Endometrial stem cell；Control group 2 is close with the result of control group 3, shows that medulla mesenchyma is done
The effect of the reparation premature ovarian failure of cell and Endometrial stem cell is close, is filled between Endometrial stem cell can effectively replace marrow
Matter stem cell is used as the seed cell for treating premature ovarian failure.Experimental group is compared with control group 1, P<0.01, with notable system
Meter learns meaning, shows that the effect of experimental group suppression premature ovarian failure is obvious；Compared with control group 2 and control group 3, experimental group suppresses ovum
The effect of nest early ageing is more excellent.
The expression of results of each group rat ovary Bax and the Bcl-2 albumen of table 4
| Group | Experiment mice number | Bax | Bcl-2 |
| Control group 1 | 10 | 1.12±0.082 | 0.49±0.046 |
| Control group 2 | 10 | 0.84±0.051* | 0.67±0.063* |
| Control group 3 | 10 | 0.93±0.047* | 0.72±0.059* |
| Experimental group | 10 | 0.52±0.026# | 0.95±0.038# |
* represent, compared with control group, P<0.05, with statistical significance；
# is represented, compared with control group, P<0.01, tool is statistically significant.
4th, HE dyeing observation ovary tissue section
Every group takes remaining 10 rat cervical dislocations to put to death, break end, peel off and take out bilateral ovaries, with 4% poly first
Aldehyde is fixed and FFPE is used after 24h, then serial section, and one, 5 μm of slice thick are taken every 20 μm；Take 20 sections, conventional H E dyes
After color under 400 times of light microscopics, the ovarian follicle situation in each stage in the section of four groups of samples is observed, while observing the follicle number in ovary
Amount.Fig. 1 is the ovarian follicle quantity result that each group ovary tissue is cut into slices.
Claims (8)
1. a kind of Endometrial stem cell preparation, comprising：Endometrial stem cell, ginsenoside Rb1 and ginsenoside Re.
2. Endometrial stem cell preparation according to claim 1, it is characterised in that the Endometrial stem cell it is dense
Spend for 1.0 × 107~5.0 × 107Individual/mL.
3. Endometrial stem cell preparation according to claim 1, it is characterised in that the concentration of the ginsenoside Rb1
For 0.1~2mg/mL.
4. Endometrial stem cell preparation according to claim 1, it is characterised in that the concentration of the ginsenoside Re is
0.1~2mg/mL.
5. Endometrial stem cell preparation according to claim 1, it is characterised in that also include：Physiological saline.
6. the preparation method of the Endometrial stem cell preparation described in a kind of claim 5, it is characterised in that do endometrium
Cell, ginsenoside Rb1, ginsenoside Re and physiological saline mixing, obtain the Endometrial stem cell preparation.
7. preparation method as described in the Endometrial stem cell preparation or claim 6 as described in claim 1 to 5 any one is obtained
To Endometrial stem cell preparation prepare treatment premature ovarian failure medicine in application.
8. application according to claim 7, it is characterised in that the Endometrial stem cell preparation is ejection preparation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108815190A (en) * | 2018-09-27 | 2018-11-16 | 天津欣普赛尔生物医药科技有限公司 | It is a kind of for treating the preparation and preparation method of premature ovarian failure |
| CN111686119A (en) * | 2020-08-10 | 2020-09-22 | 上海中医药大学附属岳阳中西医结合医院 | Traditional Chinese medicine monomer for treating early endometriosis and application thereof |
| CN119285704A (en) * | 2024-10-17 | 2025-01-10 | 西安晶彩生物科技有限公司 | Use of a stem cell composition in endometrial repair |
| WO2025030262A1 (en) * | 2023-08-04 | 2025-02-13 | Peking University Third Hospital | Compositions of bioactive agents |
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| CN105055450A (en) * | 2015-02-13 | 2015-11-18 | 中国福利会国际和平妇幼保健院 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
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| CN105055450A (en) * | 2015-02-13 | 2015-11-18 | 中国福利会国际和平妇幼保健院 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108815190A (en) * | 2018-09-27 | 2018-11-16 | 天津欣普赛尔生物医药科技有限公司 | It is a kind of for treating the preparation and preparation method of premature ovarian failure |
| CN111686119A (en) * | 2020-08-10 | 2020-09-22 | 上海中医药大学附属岳阳中西医结合医院 | Traditional Chinese medicine monomer for treating early endometriosis and application thereof |
| WO2025030262A1 (en) * | 2023-08-04 | 2025-02-13 | Peking University Third Hospital | Compositions of bioactive agents |
| CN119285704A (en) * | 2024-10-17 | 2025-01-10 | 西安晶彩生物科技有限公司 | Use of a stem cell composition in endometrial repair |
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