CN108841787A - The influence for the excretion body that EPO discharges mescenchymal stem cell - Google Patents
The influence for the excretion body that EPO discharges mescenchymal stem cell Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/14—Erythropoietin [EPO]
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Abstract
The present invention relates to the methods of the quantity or performance of a kind of excretion body of raising mescenchymal stem cell secretion, the method is by adding EPO when cultivating mescenchymal stem cell, so that the excretion body quantity of MSC release increases, there is stronger rush endothelial cell proliferation ability and angiogenesis promoting ability.The present invention is applied to clinic for MSC excretion body and provides powerful.
Description
Technical field
The present invention relates to excretion body fields more particularly to erythropoietin(EPO) (EPO) to change mescenchymal stem cell release
Excretion body aspect of performance application.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSC) is that a group has skeletonization, at cartilage and at fat
The adult stem cell of function.MSC can be played by way of paracrine and be adjusted immune, promotion angiogenesis and hematopoiesis support etc.
Function.MSC source is abundant, is present in the Various Tissues such as placenta, umbilical cord, marrow, fat.Therefore, MSC is as a drug,
By multiple state approvals, for controlling for the diseases such as graft versus host disease(GVH disease), ulcerative colitis, myocardial infarction
It treats.However no matter venoclysis, intramuscular injection or intramyocardial injection, the MSC of external source transplanting is dead mostly in 72h.So, extremely
How the MSC died plays above-mentioned effect again？The past studies have shown that the MSC of in vitro culture in hypoxemia and serum deprivation
Under the conditions of, a large amount of excretion body can be discharged.This excretion body can promote endothelial cell proliferation in vitro, promote endothelial cell
The netted structure of capillary is formed, the recovery of ischemia model blood flow is accelerated.In fact, the excretion body of MSC source comes into and faces
Bed experimental stage, the treatment for diabetes and renal insufficiency.Therefore, the excretion body of MSC source is used as " cell-free " cell
The material for the treatment of has potential practical value.To improve therapeutic effect, the way for obtaining and there is more powerful excretion body is explored
Diameter is very necessary to the clinical experimental study for carrying out MSC excretion body.
The data of the past shows that erythropoietin(EPO) (erythropoietin, EPO) can promote MSC secretion liver cell raw
The long factor is the known factor with very strong angiogenesis promoting.It will inquire into whether EPO can stimulate MSC to discharge excretion body herein,
And whether this excretion body has the function of stronger angiogenesis promoting.The result shows that, EPO promotes MSC release function more herein
Strong excretion body is the clinical experimental study of the excretion body of MSC source, provides beneficial data.
Summary of the invention
The present invention provides the method for the quantity or performance of a kind of excretion body of raising mescenchymal stem cell secretion, the sides
Method includes the following steps：(1) MSC is suspended in MSC serum free medium, is inoculated in culture dish, overnight incubation pastes cell
Wall；(2) culture medium is removed, uses α-MEM culture medium instead, and erythropoietin(EPO) (EPO) is added, continues to cultivate 72h；(3) it collects
Supernatant, centrifugation removal cell fragment are collected by centrifugation precipitating, precipitating are suspended in buffer through membrane filtration, i.e. acquisition quantity
The excretion body improved with performance.
Preferably, the performance refers to the rush endothelial cell proliferation ability and angiogenesis promoting ability of excretion body.
Preferably, wherein in step (1) according to 5 × 106Cell/ware is inoculated in culture dish.
Preferably, wherein the concentration of EPO is 1U/ml in step (2).
Preferably, wherein the centrifugal condition of removal cell fragment is 1500g in step (3), 30min, after membrane filtration
Centrifugal condition is 100,000g, is centrifuged 1h.
Preferably, precipitating is wherein suspended in KCl containing 5mmol/L, 1mmol/L MgCl2 and 136mmol/ in step (3)
In the buffer of L NaCl.
Preferably, the mescenchymal stem cell is people's umbilical cord MSC.
The present invention also provides erythropoietin(EPO) (EPO) in the quantity or property for improving the excretion body that mescenchymal stem cell is secreted
The application of energy aspect, wherein the performance refers to the rush endothelial cell proliferation ability and angiogenesis promoting ability of excretion body.
The present invention also provides the quantity for the excretion body that erythropoietin(EPO) (EPO) is secreted in preparation raising mescenchymal stem cell
Or the application in the reagent of performance, wherein the performance refers to the rush endothelial cell proliferation ability and angiogenesis promoting energy of excretion body
Power.
Preferably, the mescenchymal stem cell is people's umbilical cord MSC.
The present invention has the positive effect that：
The study find that the excretion body quantity of MSC release increases, with stronger rush endothelial cell after EPO is stimulated
Proliferative capacity and angiogenesis promoting ability.The research is that MSC excretion body is applied to clinic, provides necessary information and strong
Tool.
Detailed description of the invention
Fig. 1 is the morphologic observation of the particle of human mesenchymal stem cell release：MSC culture supernatant is harvested through ultracentrifugation
Particle, projection Electronic Speculum observation.Bar:200nm.
Fig. 2 is the surface molecular expression analysis of the particle from human mesenchymal stem cell：Collect mescenchymal stem cell supernatant
Particle and CTR, CD9, CD63, CD81 antibody response in liquid.X-axis is relative intensity of fluorescence, and Y-axis is to count.
Fig. 3 is the influence for the excretion body endothelial cell proliferation that the MSC of different condition of culture secretes：People's umbilical-cord endothelial cells
Culture medium in the excretion body that MSC under different condition of culture secretes, its proliferative conditions of MTT experimental observation are added.Y-axis：
OD490nm.As a result from independent experiment twice.Untreated is plus does not stimulate the experimental group of excretion body, and EPO is plus EPO is stimulated
The experimental group of MSC excretion body.
Fig. 4 is the influence that the excretion body Human Umbilical Vein Endothelial Cells reticular structure that the MSC of different condition of culture secretes is formed：
Untreated is plus does not stimulate the experimental group of excretion body, and EPO is plus EPO stimulates the experimental group of MSC excretion body.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
Reagent and instrument
People's MSC serum free medium is purchased from the advantageous Bioisystech Co., Ltd in Beijing three.Acetylation latex bead (3.9 μ of diameter
It m) is U.S. Invitrogen Products.Mouse anti human CD9, CD63 and the CD81 monoclonal antibody of FITC label is the U.S.
Becton Dicbnson Products.H.7650 for Hitachi, Ultracentrifuge is U.S. Beckman to electron microscope
Products.Flow cytometer FACSCalibur is U.S. company BD product.
Embodiment 1：The culture and identification of people's umbilical cord MSC
People's umbilical cord Specimen origin is voluntarily contributed in PLA General Hospital normal labor fetus, by puerpera, and sample application meets
People's sample experimental applications specification that Ethics Committee formulates.By people's MSC serum free medium operational manual provide method into
Row.In short, collector's umbilical cord, removes outer membrane and wherein arteriovenous, final concentration of 0.2% collagenase type I is added after shredding,
37 DEG C of digestion are overnight.Later, final concentration of 0.05% trypsase is added, reacts at room temperature 1h.Vitellophag is collected by centrifugation, is inoculated with
In the culture dish that diameter is 150mm.Non-adherent cell, and had digestive transfer culture are removed after 1 week.Take the 3rd generation cell for following realities
It tests.According to laboratory conventional method, morphological observation, the detection of surface molecular feature, external skeletonization are carried out to cell and divided at rouge
Change identification, it was demonstrated that obtained cell meets people's MSC feature.
Embodiment 2：The preparation and authentication of MSC source excretion body
1, the preparation of excretion body
Trypsinization harvests third generation people umbilical cord MSC, counts cell number, is suspended in people's MSC serum free medium,
According to 5 × 106Cell/ware is inoculated in the culture dish that diameter is 150mm, and 37 DEG C, 5%CO2, overnight incubation makes under 95% humidity
Cell is adherent.Culture medium is removed, uses α-MEM culture medium instead, and EPO (1U/ml) is added, continues to cultivate 72h.Supernatant is collected, from
The heart (1500g, 30min) removes cell fragment.Diameter is 0.22 μm of membrane filtration culture supernatant, to remove apoptosis that may be present
The particles such as corpusculum.It is centrifuged under the conditions of 4 DEG C, 100,000g, is centrifuged 1h, collect precipitating.Precipitating is suspended in KCl containing 5mmol/L,
1mmol/L MgCl2, in 136mmol/L NaCl solution, ibid centrifuge washing 2 times is to remove the protein of remaining.Precipitating is outstanding
Float in above-mentioned buffer, Bradford method measures protein concentration.Packing, be stored in -80 DEG C it is spare.This research is used outer
Secrete the MSC that body derives from three sample cultures.
As a result：By MSC culture supernatant after 0.22 μm of membrane filtration, ultracentrifugation harvests particle.Project Electronic Speculum observation
It was found that particle mean size obtained is 80 μm or so, in hollow capsule structure, meet the Morphological Characteristics of excretion body
(Fig. 1).Show to can get the excretion body of MSC source using ultracentrifugation method.
2, the identification of excretion body
By the method that document provides, excretion body surface face developed by molecule is carried out using flow cytometry and is analyzed.In short, by newborn
Glue bead is washed with MES buffer, in conjunction with excretion body overnight.Glycine closing is added after washed, is added after washing again
Mouse anti human CD9, CD63 and the CD81 monoclonal antibody of FITC label.Room temperature reaction is washed after twenty minutes, flow cytometer
(FACS-Calibur, BD) collects at least 10,000 data point.Result is analyzed using WinMdi2.9 software.According to above-mentioned text
The method for offering offer is taken pictures using the form of the obtained excretion body of transmission electron microscope observing, and under 10,000 times of amplification factors.
WinMdi software analysis shows that, this method obtain particle uniform expression CD9, CD63 and CD81.As a result, it was confirmed that logical
Crossing ultracentrifugation particle obtained is excretion body (Fig. 2).
Embodiment 3：Influence of the growth factor to the MSC excretion body secreted
Growth factor handle MSC by influence its secretion excretion body in a manner of (EPO) with embodiment 2.
1, protein content determination
After tentatively understanding EPO processing, people's umbilical cord MSC discharges the variation of excretion body quantity.It collects supernatant and is used for excretion body
It isolates and purifies, meanwhile, cell number is counted after trypsin digestion.Using protein content in Bradford method measurement excretion body, and push away
Calculate 108The quantity of the discharged excretion body of a cell.The result shows that not stimulating group, the excretion body protein content of EPO group, respectively
256±124μg/108Cell, 1021 ± 392 μ g/108Cell, after EPO is stimulated, the quantity that people's umbilical cord MSC discharges excretion body is aobvious
It writes and increases (P<0.01).As a result it prompts, EPO processing can promote MSC to discharge excretion body.
2, MTT experiment
Utilize the influence of MTT experiment observation separate sources MSC excretion body endothelial cell proliferation.People's umbilical-cord endothelial cells by
Experimental hematology research department of Institute of Radiation Medicine of Military Medical Science Institute provides.It is real conventionally to carry out MTT
It tests.In short, people's umbilical-cord endothelial is inoculated in 96 orifice plates according to 2000 cells/wells.Be separately added into cultivating system without
The excretion body of cell factor processing and the excretion body of EPO processing, the concentration of excretion body is 10 μ g/ml, every group of 3 multiple holes.
After cultivating 72h, MTT is added, continues to cultivate 4h.Later, dimethyl sulfoxide is added.Measure 490nm OD value.
As a result as shown in Figure 3.MSC is after EPO and processing, compared with untreated control group, the excretion body of release
With stronger rush endothelial cell proliferation activity.EPO group OD value is significantly higher than untreated fish group (P<0.05).The above results suggest that
After EPO is handled, the excretion body that MSC is discharged has stronger rush endothelial cell proliferation ability.
3, capillary spline structure forms experiment
Matrigel experiment is to observe the classical technology of inner skin cell function, especially capillary network Forming ability,
As a result internal new vessels are represented and form activity.For the angiogenesis promoting effect for inquiring into separate sources excretion body, this research will be interior
Chrotoplast is inoculated in the culture plate of Matrigel bed board, and the excretion body of 10 μ g/ml is added in system, counts every high power afterwards for 24 hours
Netted structure number mesh under the visual field.Specifically, being carried out by method reported in the literature:Umbilical-cord endothelial cells are suspended in containing 1% tire ox
In the DMEM culture medium of serum, according to 105/ hole is inoculated in 24 orifice plates of Matrigel bed board.Experiment, which is divided into, does not add excretion body
Negative control group, plus do not stimulate excretion body group, EPO stimulation MSC excretion body group, every group of 3 multiple holes.Document side is pressed in culture afterwards for 24 hours
Method counts.
As a result, it has been found that not stimulating the every visual field reticular structure quantity of group, EPO group is respectively 2.6 ± 0.84,4.6 ± 1.57, carefully
Intracellular growth factor treatment group is significantly higher than untreated fish group (P<0.01).The above results suggest that after EPO is stimulated, what MSC was discharged
Excretion body has stronger angiogenesis promoting ability.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (10)
1. a kind of method of the quantity or performance of the excretion body for improving mescenchymal stem cell secretion, the method includes walking as follows
Suddenly：(1) MSC is suspended in MSC serum free medium, is inoculated in culture dish, overnight incubation keeps cell adherent；(2) removal training
Base is supported, uses α-MEM culture medium instead, and erythropoietin(EPO) (EPO) is added, continues to cultivate 72h；(3) supernatant, centrifugation removal are collected
Cell fragment is collected by centrifugation precipitating, precipitating is suspended in buffer through membrane filtration, i.e., acquisition quantity and performance improve
Excretion body.
2. the method as described in claim 1, the performance refers to that the rush endothelial cell proliferation ability of excretion body and rush blood vessel are new
Raw ability.
3. it is method according to claim 1 or 2, wherein according to 5 × 10 in step (1)6Cell/ware is inoculated in culture dish.
4. method according to claim 1 or 2, wherein the concentration of EPO is 1U/ml in step (2).
5. method according to claim 1 or 2, wherein the centrifugal condition of removal cell fragment is 1500g in step (3),
30min, the centrifugal condition after membrane filtration are 100,000g, are centrifuged 1h.
6. it is method according to claim 1 or 2, precipitating is wherein suspended in KCl containing 5mmol/L, 1mmol/L in step (3)
In the buffer of MgCl2 and 136mmol/L NaCl.
7. method according to claim 1 or 2, the mescenchymal stem cell is people's umbilical cord MSC.
8. erythropoietin(EPO) (EPO) improve mescenchymal stem cell secretion excretion body quantity or aspect of performance application,
Wherein the performance refers to the rush endothelial cell proliferation ability and angiogenesis promoting ability of excretion body.
9. erythropoietin(EPO) (EPO) is in the quantity of excretion body or the reagent of performance that preparation improves mescenchymal stem cell secretion
Application, wherein the performance refers to the rush endothelial cell proliferation ability and angiogenesis promoting ability of excretion body.
10. application as claimed in claim 8 or 9, the mescenchymal stem cell is people's umbilical cord MSC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810769767.9A CN108841787B (en) | 2018-07-13 | 2018-07-13 | Effect of EPO on exosomes released from mesenchymal Stem cells |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810769767.9A CN108841787B (en) | 2018-07-13 | 2018-07-13 | Effect of EPO on exosomes released from mesenchymal Stem cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172103A (en) * | 2019-12-05 | 2020-05-19 | 伯仕利生物科技发展（盐城）有限公司 | Preparation method and application of stem cell exosome stimulated by angelica sinensis extract |
| CN113215100A (en) * | 2021-06-10 | 2021-08-06 | 浙江大学 | Application of small molecule compound MLN4924 in promoting secretion of exosome |
| CN113930392A (en) * | 2020-08-20 | 2022-01-14 | 北京沃森百欧生物科技研究院有限公司 | Mesenchymal stem cell exosome and preparation method and application thereof |
| CN117586955A (en) * | 2024-01-19 | 2024-02-23 | 吉林大学 | Preparation and application of exosomes derived from EPO-stimulated macrophages |
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| CN106282107A (en) * | 2016-08-30 | 2017-01-04 | 章毅 | Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body |
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| CN111172103A (en) * | 2019-12-05 | 2020-05-19 | 伯仕利生物科技发展（盐城）有限公司 | Preparation method and application of stem cell exosome stimulated by angelica sinensis extract |
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| CN113215100A (en) * | 2021-06-10 | 2021-08-06 | 浙江大学 | Application of small molecule compound MLN4924 in promoting secretion of exosome |
| CN117586955A (en) * | 2024-01-19 | 2024-02-23 | 吉林大学 | Preparation and application of exosomes derived from EPO-stimulated macrophages |
| CN117586955B (en) * | 2024-01-19 | 2024-04-19 | 吉林大学 | Preparation and application of EPO-stimulated macrophage-derived exosomes |
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