CN107629998A - A kind of stem cell in vitro reparation with menstrual blood source is damaged the model of endometrium - Google Patents
A kind of stem cell in vitro reparation with menstrual blood source is damaged the model of endometrium Download PDFInfo
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Abstract
The present invention relates to stem cell regenerating medical domain.Purpose is the endometrium model of the stem cell in vitro reparation damage of foundation menstrual blood extraction, is laid the first stone to repair Endometrial stem cell preclinical study.Technical scheme is：The endometrium model that a kind of stem cell in vitro reparation extracted with menstrual blood damages, comprises the following steps：1) it is primary separation people Endometrial stromal cells (ESCs) and identify, after identification ESCs cultivate to P3 generation and freeze standby；2) the endometrium mescenchymal stem cell (MenSCs) in menstrual blood source, subculture to P3, freezes standby；3) cellular damage is caused for ESCs 2d with finite concentration mifepristone processing P3, then co-cultures 4d with the MenSCs and its that are seeded in transwell nestings；4) complete after co-culturing, detect ESCs change in different aspects respectively；Repairing effects of the MenSCs for ESCs is assessed with this.
Description
Technical field
The present invention relates to stem cell regenerating medical domain, specifically a kind of stem cell body extracted with menstrual blood is dispatched workers to do on-site repairs
The endometrium model damaged again.
Background technology
In regenerative medicine field, the reparation for injury tissue at present is more by the way of stem-cell therapy, wherein bone
The mescenchymal stem cell application in marrow source is most；And for female reproductive system, endometrial impairment is infertile main
Inducement, to repair its function must use stem-cell therapy, the problems such as will considering the source of stem cell.Use is had been reported that before
The mescenchymal stem cell of Cord Blood-Derived repairs endometrium, also there is certain effect；It is generally understood that with identical tissue-derived
Stem cell to be best, be thus born the present invention.
The content of the invention
The purpose of the present invention is to overcome the shortcomings of background technology, and the stem cell in vitro reparation that foundation is extracted with menstrual blood damages
Endometrium model, for repair endometrium stem cell preclinical study lay the first stone.
Technical scheme provided by the invention is：
The endometrium model that a kind of stem cell in vitro reparation extracted with menstrual blood damages, comprises the following steps：
1) it is primary separation people Endometrial stromal cells (ESCs) and identify, after identification ESCs cultivate to P3 generation and freeze
Deposit standby；
2) the endometrium mescenchymal stem cell (MenSCs) in menstrual blood source, subculture to P3, freezes standby；
3) cellular damage is caused for ESCs 2d with certain density mifepristone processing P3, then with being seeded in
MenSCs in transwell nestings co-cultures 4d with it, is damaged to repair cell；
4) complete after co-culturing, detect ESCs change in different aspects respectively；MenSCs is assessed for ESCs with this
Repairing effect.
The different aspects include cellular morphology, cell propagation, apoptosis and protein expression level state.
Certain density mifepristone processing in step 3), is that P3 is replaced by into rice containing 25mg/L for ESCs culture medium
The culture medium of mifepristone, continue to cultivate 2d.
Co-cultivation in step 3), it is by 1 × 10 by MenSCs P3 generations5/ well cell density is inoculated into aperture
In 0.4- μm of transwell nestings, being added to kind every other day has in impaired ESCs orifice plate, and changes culture medium.
The beneficial effects of the invention are as follows：The present invention establishes what a kind of stem cell in vitro reparation extracted with menstrual blood damaged
Endometrium biology external model, it is applied to clinical treatment for later stem cell, improves endometrium quality, open new
Approach.
Brief description of the drawings
Figure 1A is Endometrial stromal cells (ESCs) electron microscope of primary separation.
Figure 1B is the electron microscope of Endometrial stromal cells (ESCs) SABC identification；Dark parts are ESCs.
Fig. 1 C are the electron microscope of the menstrual blood endometrium mescenchymal stem cell (MenSCs) of in vitro culture.
Fig. 2 is that mifepristone processing is illustrated to the inhibition of Endometrial stromal cells (ESCs) growth and cell propagation
Figure.Shown in figure：25mg/L mifepristone processing Endometrial stromal cells 48 hours are optimum experimental condition.
Fig. 3 A are to co-culture to promote endometrial cell to repair by menstrual blood endometrium mescenchymal stem cell (MenSCs)
The control electron microscope of multiple effect.A in figure, d, g are control group ESC (the not adding mifepristone) ESCs of 24,48,72 hours electricity
Mirror figure；B in figure, e, h are dosing damage group ESC+Mfp (after 25mg/L mifepristones are handled 48 hours) 24,48,72 hours
ESCs electron microscopes；C in figure, f, i be co-cultivation group ESC+Mfp+MenSC (mifepristone handle 48 hours after withdraw, then with
MenSCs is co-cultured) the ESCs electron microscopes of 24,48,72 hours.As illustrated, the ESCs cell fusions handled through mifepristone
Degree is obvious to be reduced, and after menstrual blood endometrium mescenchymal stem cell (MenSCs) co-cultures with impaired ESCs, the life of its cell
It is long to recover, degrees of fusion increase.
Fig. 3 B are the cell propagation detection table of comparisons.Shown in figure：Endometrium can be significantly improved by being co-cultured through hemocytoblast
The cell proliferation rate of interstitial cell.
Fig. 4 A are that flow cytometry (Annexin V-FITC/PI methods) detection Endometrial stromal cells (ESCs) wither
Die the two-dimentional point diagram of situation.Left-to-right is followed successively by the two-dimentional point diagram of control group, dosing damage group and co-cultivation group in figure.Show in figure
Show：Mifepristone induces ESCs apoptosis, and stem cell medicine can reverse this process.
Fig. 4 B are the statistical form of Apoptosis flow cytometry.
Fig. 5 A are the cell migration ability compares figure that cell scratch experiment detects Endometrial stromal cells (ESCs).From a left side
Control group, dosing damage group and co-cultivation group are followed successively by the right side, cultivating different time respectively, (0,4,8,12,24,48,72 is small
When) after cell cut change width.Shown in figure：Mifepristone induction ESCs cell migrations ability reduces, menstrual blood
Endometrium mescenchymal stem cell (MenSCs) can promote impaired ESCs transfer abilities to recover.
Fig. 5 B are the statistical result table (being corresponded with Fig. 5 A Each point in time) to the change of cell scratch width.
Fig. 6 A, Fig. 6 B are blood in immunoblotting (Western blot) detection Endometrial stromal cells (ESCs)
The expression variation diagram of endothelial tube growth factor (VEGF) and beta chain albumen (β-catenin).
After Fig. 6 A is cultures different time (24,48,72,96 hours), VEGF and β in Western blot detections ESCs-
The protein band figure of catenin expression, in each time group protein band be followed successively by from left to right control group, dosing damage group and
Co-cultivation group, using GAPDH as internal reference albumen.
Fig. 6 B are the statistical result table to vegf protein band in Fig. 6 A.
Embodiment
The uterus damaged below by way of specific embodiment to the stem cell in vitro reparation extracted involved by the present invention with menstrual blood
Interior membrane modle is described further.
The endometrium mesenchyma in present invention application more preferable feminine menstrual blood source of compatibility compared with other stem cells
Stem cell co-cultures as instrument, and by the Endometrial stromal cells of mifepristone damage, establishes external reparation damage
Endometrium model, reach the purpose for repairing its function damage.
First, material and reagent
Tissue collecting
Womb internal film tissue is derived from Medical College of Zhejiang Univ. Shao Yi husband obstetrics and gynecology hospital hysteroscope operation patients.Through suffering from
Person's informed consent, preoperative signature letter of consent, voluntarily hospital is transferred to handle or for scientific research institution endometrial tissue.Operation moves ahead
The detections such as HBV antigens, Anti-HBV activity antibody, HCV antigen/antibody combination, ANTI-HIV DRUGS, microspironema pallidum and mycoplasma are negative.Sterile bar
It is placed in after gathering endometrial tissue under part in culture medium, 4 DEG C of preservation digested processing of 4h.
Experiment reagent：
DMEM/F12 (Hangzhou Ji Nuo biological medicine technologies Co., Ltd)；
Hyclone FBS (Thermo Fischer Scient Inc.)；
Trypsase (Thermo Fischer Scient Inc.)；
Mifepristone【Sigma-Aldrich (Shanghai) trade Co., Ltd】；
Dimethyl sulfoxide (DMSO)【Sigma-Aldrich (Shanghai) trade Co., Ltd】；
Collagenase I (Thermo Fischer Scient Inc.)；
200 mesh filter screens (BD biotech companies)；
4% paraformaldehyde (Beijing Ding Guo biotechnologys Co., Ltd)；
Immunohistochemical kit (Fuzhou Maixin biotechnology Development Co., Ltd)；
MenSC culture mediums (Hangzhou S-Evans Biosciences Co., Ltd.)；
CCK-8 (the green skies Bioisystech Co., Ltd in Shanghai)；
V/FITC-PI of Annexin apoptosis kits (hero's life technology Co., Ltd of the U.S.)；
Cytokeratin and vimentin Vimentin antibody (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；
VEGF VEGF antibody【Ai Bokang (Shanghai) trade Co., Ltd】；
Beta chain albumen (β-catenin) antibody【Menaphtame leads to (Shanghai) biological reagent Co., Ltd】；
Transwell nestings (Corning Incorporated).
Cell line：Menstrual blood endometrium mescenchymal stem cell (MenSCs) is purchased from the limited public affairs of Hangzhou Yi Wensai biotechnologys
Department.
2nd, experimental procedure
1st, ESCs separation
Washed with the D-Hanks cushioning liquid containing mycillin and remove endometrial tissue remaining blood, tissue shear is broken to
1mm fritter, 0.1% Collagenase I and DNase (DNA enzymatic) are added, 37 DEG C digest 45 minutes.10%FBS DMEM/ is used afterwards
Digestion is terminated in F12 culture mediums, 200 mesh (aperture be 74- μm) strainer filtering, is collected into cell suspension, with 500g rotating speed from
The heart 2 times, 5 minutes every time, supernatant is removed, add 10%FBS DMEM/F12 culture mediums that cell is resuspended, be placed in 37 DEG C, volume fraction
5%CO2Cultivated in incubator.Cell culture removes a small amount of residual tissue block after 3 days, full dose changes culture medium.It is primary thin
Born of the same parents were cultivated to 10-14 days, were passed on after Trypsin Induced, respectively for cell up to being passed on during more than 90% fusion after P1.
2nd, the identification of Endometrial stromal cells (ESCs)
Endometrial cell after P1 passages is inoculated on cell climbing sheet, with 4% poly when degrees of fusion reaches more than 90%
Formaldehyde is fixed, and the expression of SABC detection mesenchymal cell markers vimentin (Vimentin), it is then ESCs that the positive, which is presented,
(P2)。
3rd, the foundation of Endometrial stromal cells (ESCs) damage model
Endometrial stromal cells are passaged to P3, are inoculated into 6 orifice plates, when its cell fusion degree reaches 70% or so,
The 2%FBS-DMEM/F12 of the mifepristone containing 25mg/L is replaced medium to, continues culture 48 hours (that is, using meter Fei
Ketone is taken charge of to handle 48 hours), 2%FBS-DMEM/F12 is now replaced medium to, cellular morphology is observed afterwards and changes and detect thin
Born of the same parents' function.
4th, the culture of menstrual blood endometrium mescenchymal stem cell (MenSCs)
The alternative complete mediums of menstrual blood endometrium mescenchymal stem cell P3 are resuspended, by 1 × 105/ well cell
Density is inoculated into the transwell nestings that aperture is 0.4- μm, and being added to kind every other day has in impaired ESCs orifice plate, changes culture
Base is 2%FBS-DMEM/F12.
5th, menstruation hemocytoblast, which co-cultures, repairs endometrium
Repair process is co-cultured by Endometrial stromal cells/menstruation hemocytoblast (ESC/MenSC) to realize, is used
ESCs P3 generations are resuspended in 10%FBS-DMEM/F12 culture mediums, by 3 × 105The cell density in/hole is inoculated into 6 orifice plates and cultivated
Night.Example of spatial compartmentalizationis is 2%FBS-DMEM/F12.6 hole versions are divided into three groups, are respectively：(1) control group (does not add rice
Mifepristone)；(2) dosing damage group (addition 25mg/L mifepristones)；(3) (processing 48 of 25mg/L mifepristones is small for co-cultivation group
When after addition be inoculated with MenSC transwell).Three groups uniformly replace medium to 2%FBS-DMEM/ after cultivating 48 hours
F12 (is free of mifepristone), wherein the 3rd group of addition is inoculated with MenSC transwell cells.Continue culture 24,48,72
With 96 hours, detect ESCs cellular morphology and changes of function.
6th, the expression of the cellular morphology of detection Endometrial stromal cells (ESCs), cell propagation, apoptosis and albumen.
(1) changed with microscope to observe cellular morphology.
(2) bred with CCK-8 methods to detect cell.After each group ESCs is digested and counted first, by 8000/hole
Cell density be inoculated into 96 orifice plates, three multiple holes of every group of setting.CCK-8 is added separately in every hole, 37 DEG C of incubation 2h, enzyme
Instrument measure Reinhoit Zahl OD450nm/OD630nm is marked, judges the change of cell proliferation rate.
(3) immunoblotting (Western blot) detection each group VEGF (VEGF) and beta chain
Albumen (β-catenin；Bata-catenin i.e. in accompanying drawing) protein expression.
Endometrial stromal cells total protein is extracted first and carries out protein quantification with BCA methods to determine its concentration；Then
Albumen is separated with SDS- polyacrylamide gel electrophoresises (SDS-PAGE), then albumen is transferred on pvdf membrane, 5%
Skimmed milk power-TBST is closed 1 hour and gently shaken.The working concentration recommended with antibody specification, is diluted with 1%BSA-TBST
Every kind of primary antibody (VEGF, β-catenin and GAPDH), wherein GAPDH are albumen internal reference.Pvdf membrane 4 DEG C of overnight incubations of primary antibody
And gently shake.Take the film out every other day, be put into TBST quick concussion to elute unnecessary primary antibody, wash three times, every time 10 minutes.With
The secondary antibody that horseradish peroxidase (HRP) is crosslinked is diluted to recommended density by 1%BSA-TBST, by film immersion wherein, room temperature
It is incubated 1 hour.Secondary antibody is discarded, film is put into a large amount of TBST into acutely concussion, to elute unnecessary secondary antibody, washes three times, every time 10 points
Clock.Enhanced chemical luminescence method determines protein concentration, according to 1:The A liquid and B liquid of 1 ratio mixing ECL kits, are configured to
ECL reagents, film immersion is stood into 3-5 minutes wherein.Film is clipped to rapidly in clean transparent plastic clip prevents from drying up, and is placed in
Exposure is with capture images in ECL imaging devices (LAS4000, Fuji).Analysis band gray value then is compared with ImageJ softwares,
The final relative expression levels for calculating VEGF (VEGF) and beta chain albumen (β-catenin).
As a result prompt, mifepristone processing cause Endometrial stromal cells VEGF (VEGF) and
The expression of beta chain albumen (β-catenin) significantly reduces, and the co-cultivation of menstrual blood Endometrial stem cell can significantly reverse this
One process, there is repair to impaired endometrium.
7th, statistical method
Statistical analysis is carried out using the softwares of SPSS 17.0.Experimental data is represented with MEAN ± SD.Compare between two groups using t
Examine.
8th, conclusion
It is optimal that the mifepristone that concentration is 25mg/L handles Endometrial stromal cells (ESCs) 48 hours damage effects；Month
Menses endometrium mescenchymal stem cell (MenSCs) can effectively recover impaired ESCs cellular morphologies, improve ESCs cells propagation
Rate, reduce ESCs apoptosis rates so that impaired ESCs VEGFs (VEGF) expression raises, at the same time beta chain egg
(β-catenin) expression also has similar trend in vain.This prompting MenSCs, which is co-cultured, can reverse intrauterine caused by mifepristone
Theca cell damages, and the stem cell in menstrual blood source is probably effective sharp weapon that regenerative medicine field is used for repair tissue damage, with
After be possibly used for clinical treatment, improve endometrium quality.
Claims (4)
1. the endometrium model that a kind of stem cell in vitro reparation extracted with menstrual blood damages, comprises the following steps：
1) Endometrial stromal cells of primary separation people and identification, ESCs is cultivated to P3 generations and frozen standby after identification；
2) the endometrium mescenchymal stem cell in menstrual blood source, subculture to P3, freezes standby；
3) cellular damage is caused for Endometrial stromal cells 2d with certain density mifepristone processing P3, then with being seeded in
Endometrium mescenchymal stem cell in transwell nestings co-cultures 4d with it, is damaged to repair cell；
4) complete after co-culturing, detect ESCs change in different aspects respectively；MenSCs repairing for ESCs is assessed with this
Multiple effect.
2. the endometrium model that the stem cell in vitro reparation according to claim 1 extracted with menstrual blood damages, it is special
Sign is：Certain density mifepristone processing in step 3), it is to change P3 for the culture medium of Endometrial stromal cells
For the culture medium of the mifepristone containing 25mg/L, continue to cultivate 2d.
3. the endometrium model that the stem cell in vitro reparation according to claim 2 extracted with menstrual blood damages, it is special
Sign is：Co-cultivation in step 3), it is by 1 × 10 by endometrium mescenchymal stem cell P3 generations5/ well cell density connects
In the transwell nestings that kind is 0.4- μm to aperture, being added to kind every other day has in the orifice plate of impaired Endometrial stromal cells, and
Change culture medium.
4. the endometrium model that the stem cell in vitro reparation according to claim 3 extracted with menstrual blood damages, it is special
Sign is：Different aspects in step 3), including cellular morphology, cell propagation, apoptosis and protein expression level state.
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Cited By (8)
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| CN108998411A (en) * | 2018-08-15 | 2018-12-14 | 山西医科大学 | The isolated culture method of people's endometrial tissue derived mesenchymal stem cell |
| CN109251890A (en) * | 2018-10-22 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
| CN110408673A (en) * | 2019-07-01 | 2019-11-05 | 南通大学附属医院 | Study the method that Hsa_circ_0111659 repairs the mechanism of action in ESC in WJ-MSCs |
| CN113134115A (en) * | 2021-05-08 | 2021-07-20 | 安徽医科大学 | Preparation method of collagen scaffold composite human menstrual blood stem cells for treating endometrial injury |
| CN113249301A (en) * | 2021-05-10 | 2021-08-13 | 博品(上海)生物医药科技有限公司 | Kit based on stem cell exosome freeze-dried powder and application |
| CN115300612A (en) * | 2022-07-29 | 2022-11-08 | 海南博鳌未来医院有限公司 | Stem cell preparation for repairing endometrium and application thereof |
| CN117801091A (en) * | 2023-12-28 | 2024-04-02 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
| CN117925533A (en) * | 2024-01-25 | 2024-04-26 | 广东圆康再生医学科技开发有限公司 | Application of endometrium stem cells in vaginal connective tissue elastic mucosa repair |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998411A (en) * | 2018-08-15 | 2018-12-14 | 山西医科大学 | The isolated culture method of people's endometrial tissue derived mesenchymal stem cell |
| CN109251890A (en) * | 2018-10-22 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
| CN110408673A (en) * | 2019-07-01 | 2019-11-05 | 南通大学附属医院 | Study the method that Hsa_circ_0111659 repairs the mechanism of action in ESC in WJ-MSCs |
| CN113134115A (en) * | 2021-05-08 | 2021-07-20 | 安徽医科大学 | Preparation method of collagen scaffold composite human menstrual blood stem cells for treating endometrial injury |
| CN113249301A (en) * | 2021-05-10 | 2021-08-13 | 博品(上海)生物医药科技有限公司 | Kit based on stem cell exosome freeze-dried powder and application |
| CN115300612A (en) * | 2022-07-29 | 2022-11-08 | 海南博鳌未来医院有限公司 | Stem cell preparation for repairing endometrium and application thereof |
| CN117801091A (en) * | 2023-12-28 | 2024-04-02 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
| CN117801091B (en) * | 2023-12-28 | 2024-05-31 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
| CN117925533A (en) * | 2024-01-25 | 2024-04-26 | 广东圆康再生医学科技开发有限公司 | Application of endometrium stem cells in vaginal connective tissue elastic mucosa repair |
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