CN108624557A - The preparation method and applications of mescenchymal stem cell excretion body - Google Patents
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Abstract
A kind of preparation method of mescenchymal stem cell excretion body uses conditioned medium culture mescenchymal stem cell several hours of serum-free, centrifugation removal cell, after collecting supernatant crude product, then after centrifuging, filtering, after removing remaining cell and cell fragment, the supernatant containing excretion body is made；The polyethylene glycol Centrifugation method DNA used again to the supernatant containing excretion body is made.Excretion body surface made from the method for the present invention reaches specific proteins CD63, can effectively improve the injury of mitochondria of cardiac muscle cell, prevents Apoptosis, improves the function of cardiac muscle cell, is used to prepare the drug for the treatment of myocardial ischemia-reperfusion injury.
Description
Technical field
The present invention relates to a kind of method for producing biological products more particularly to a kind of preparation method of stem cell excretion body,
And preparing the application in treating myocardial ischemia-reperfusion injury medicine.
Background technology
Post-ischemic Reperfusion (ischaemia/reperfusion) damage refer to heart blood supply coronary artery in short-term
Between partly or entirely block after, when blood vessel is unimpeded again, the more serious pathology damage of progressive occurs for former ischemic myocardial tissue
Hinder process, or even serious arrhythmia cordis can occur and cause to die suddenly.Damage is embodied in cardiomyocyte cell death, rhythm of the heart mistake
Often, heart dysfunction eventually leads to heart failure.Cardiac muscle structure function of cell caused by ischemic stage, energetic supersession etc. one
The damaging variation of series, shows more prominent after revascularization.
It is entangled with department of cardiac surgery's extracorporal circulatory system, coronary artery surgery, complicated congenital heart disease and controls art, intracardiac repair under direct vision hand
When art, prosthetic valve replacement, thrombolysis be postoperative and myocardium interior collateral circulating blood volume increases suddenly, myocardial ischemia can all occur
Reperfusion injury afterwards.Its mechanism with the increase of intracellular oxygen radical, calcium overloading, inflammation it is now recognized that mainly make
With related with dysbolism etc..Studies have shown that it is by Ischemia Reperfusion that the mortality with acute myocardial infarction AMI, which is more than half,
Caused by note damage.Ischemic heart disease has very high incidence and case fatality rate in worldwide, clinically most quickly has
The therapy of effect is the timely blood supply for restoring ischemic area, and myocardial damage can be further caused by restoring blood supply.Therefore
Damage is the major issue for being badly in need of solving after how reducing myocardial ischemia.
Placenta mesenchyma stem cell belongs to versatile stem cell, the ability with hyperplasia and Multidirectional Differentiation, suitable micro-
The various kinds of cell such as epithelial stem cell, neural stem cell, myocyte can be divided under environment, to repair the group of impaired or lesion
Organ is knitted, for treating cardiovascular and cerebrovascular disease, the nervous system disease etc..Compared to other stem cells, placenta mesenchyma stem cell
With convenient sources, quantity is sufficient, is easily isolated, cultivates, expands and purifies.
Excretion body (exosome) contains complicated RNA, lipid and protein, the small film bubble (diameter of specific secretion
About 30nm~150nm), other cells can be passed to as signaling molecule to change the function of other cells, participated in immune
Middle antigen presentation, growth and the migration of tumour, tissue damage the physiology courses such as reparation.The excretion body of different cell secretions has
Different constituents and function, can target specific cells or tissue, be a kind of fine targeting drug delivery system.
When having researcher that the stem cell of amplification in vitro culture or cardiac muscle cell are directly injected heart, cardiac muscle can be repaired and lacked
Damage caused by blood Reperfu- sion, improves cardiac function, is the new strategy of cardiac repair after myocardial infarction, but uses cell therapy
Many challenges can be brought, such as：Immunological rejection, cell dedifferentiation growth etc..Discovered in recent years source induced multi-potent stem cell
(iPS) the excretion body of cardiac muscle cell can make damaged heart functionalization again, improve the heart after rat myocardial infarction model so that the heart
Dirty function is restored.Therefore application of the excretion body in treating myocardial ischemia damage receives extensive attention.
Invention content
It is an object of the present invention to provide a kind of preparation methods of mescenchymal stem cell excretion body, and it is dry to obtain mesenchyma
The excretion body of cell origin.
It is another object of the present invention to provide a kind of mescenchymal stem cell excretion bodies to prepare treatment myocardial ischemia again
Application in the drug of perfusion injury.
It is yet a further object of the present invention to provide a kind of mescenchymal stem cell excretion bodies to prepare improvement cardiac function
Application in drug.
A kind of preparation method of mescenchymal stem cell excretion body provided by the invention, includes the following steps：
Using serum-free conditioned medium culture mescenchymal stem cell several hours (such as：72 hours), centrifugation removal is thin
Born of the same parents after collecting supernatant crude product, then centrifuge (such as：2,000g) after, filtering after removing remaining cell and cell fragment, is made
Supernatant containing excretion body；
The polyethylene glycol Centrifugation method DNA used to the supernatant containing excretion body is made.
The preparation method of another kind mescenchymal stem cell excretion body provided by the invention, includes the following steps：
The supernatant after mesenchymal stem cell serum-free culture is collected, using the containing 16% (w/v) Macrogol 6000
A kind of sodium chloride solution and second of sodium chloride solution substep containing 10% (w/v) Macrogol 6000 purify excretion body.
The preparation method of another kind mescenchymal stem cell excretion body provided by the invention, uses the conditioned medium of serum-free
Cultivate mescenchymal stem cell several hours (such as：72 hours), centrifugation removal cell after collecting supernatant crude product, then centrifuges (such as：
2,000g) after, after removing remaining cell and cell fragment, the supernatant containing excretion body is made in filtering；
The first sodium chloride solution (16% containing Macrogol 6000 in equal volume is added to the supernatant containing excretion body
(w/v) Macrogol 6000,1M sodium chloride), place 12 hours or more, centrifugation is (such as：3,500g, 30 minutes), collect precipitation weight
It is suspended from phosphate buffer, isometric second of sodium chloride solution containing Macrogol 6000 is added again, and (10% (w/v) is poly-
Ethylene glycol 6000,1M sodium chloride), place 4 hours or more, centrifugation is (such as：3,500g, 30 minutes), it collects precipitation and is resuspended in phosphoric acid
Buffer solution, excretion body as after purification.
Excretion body surface made from the method for the present invention reaches specific proteins CD63.
Excretion body produced by the present invention can effectively improve the injury of mitochondria of cardiac muscle cell, prevent Apoptosis, improve the heart
The function of myocyte is used to prepare the drug for the treatment of myocardial ischemia-reperfusion injury.
The advantageous effect that technical solution of the present invention is realized：
The present invention collects the excretion body of the Rat Placenta mescenchymal stem cell of purifying amplification in vitro, establishes rat myocardial cell
Oxygen sugar deprivation model is simultaneously incubated with excretion body altogether.Inhibitory rate of cell growth is it is demonstrated experimentally that addition Rat Placenta mesenchyma is dry in advance
The excretion body of cell can significantly reduce oxygen sugar and deprive growth inhibition to rat myocardial cell, improve Bcl-2/Bax albumen ratios
Example, prevents the apoptosis of cardiac muscle cell.Cardiac muscle is inhibited to lack these results indicate that the excretion body of Rat Placenta mescenchymal stem cell has
Blood reperfusion injury protects cardiac muscle cell, can be used for the preparation for the treatment of myocardial ischemia damage drug.
Description of the drawings
Fig. 1 is the Rat Placenta derived mesenchymal stem cells in vitro culture situation observation chart after separation, wherein Figure 1A is initial point
From Primary rat placenta mesenchyma stem cell；Figure 1B is the third generation Rat Placenta mescenchymal stem cell after culture；Fig. 1 C are
By the third generation Rat Placenta mescenchymal stem cell of DAPI nuclear targetings；Fig. 1 D are that third generation Rat Placenta mesenchyma is dry thin
The special molecular CD44 expressions of born of the same parents；
Fig. 2 is the growing state observation chart of the rat myocardial cell after excretion body deprives oxygen sugar；Wherein Fig. 2A is oxygen sugar
Deprive the rat myocardial cell after 8 hours；Fig. 2 B are the rat myocardial cell that is incubated altogether of excretion body after oxygen sugar deprives 8 hours
Growth conditions；Fig. 2 C are that Rat Placenta mescenchymal stem cell excretion body prevents oxygen sugar from depriving the growth for inhibiting rat myocardial cell, *
P ＜ 0.05；
Fig. 3 is that the rat myocardial cell after Rat Placenta mescenchymal stem cell excretion body is deprived with oxygen sugar is incubated altogether, to the heart
The expression figure of the apoptosis-related protein of myocyte；Wherein, Fig. 3 A are the expression of apoptosis-related protein, and Fig. 3 B are quantitative
Analyze the expression of Bcl-2 and Bax.
Specific implementation mode
Below in conjunction with attached drawing detailed description of the present invention technical solution.The embodiment of the present invention is only to illustrate the skill of the present invention
Art scheme and it is unrestricted, although being described the invention in detail with reference to preferred embodiment, those skilled in the art
It should be appreciated that can be modified or replaced equivalently to the technical solution of invention, without departing from the essence of technical solution of the present invention
God and range, should all cover in scope of the presently claimed invention.
1 placenta mesenchyma stem cell of embodiment is separately cultured and its collection of excretion body purifying
(1) Rat Placenta mescenchymal stem cell is separately cultured
Pregnant two weeks Sprague Dawley rats are taken, cervical vertebra detachment is put to death after ether inhalation anesthesia, and the immersion of 75% ethyl alcohol disappears
Poison cuts off pregnant rat abdominal cavity and takes out uterus under the aseptic condition of super-clean bench, cuts off uterus, and denuded amniotic membrane detachment placenta is collected
All Rat Placentas, it is spare after being rinsed with sterile phosphate buffer.
Remaining umbilical cord is removed using tweezers and scissors and amnion tissue, placenta tissue are cut into about 1mm3~2mm3Small tissue blocks,
More fritter later stage digestion effect is better.0.1%I Collagenase Types collagen (Sigma-Aldrich), 0.25% pancreatin is added
(GIBICO), it digesting about 14 hours under the conditions of 37 DEG C, postdigestive mixing liquid is collected after the filtering of 200 mesh stainless steel filtering nets,
It is suspended in DEME/F12 culture mediums (GIBICO), is inoculated in T25 cells (Corning Inc.) culture bottle, DEME/F12 trainings
Base is supported, contains 37 DEG C of 10% fetal calf serum (Hyclone), is cultivated in 5%CO2 incubators, cell growth to about 80% culture bottle bottle
When bottom, 0.25% pancreatin/0.02%EDTA (GIBICO) vitellophag, by 1: 3 passage.Microscopically observation, cell growth shape
State is good, referring to Fig. 1.
(2) identification of Rat Placenta interstital stem cell
The Rat Placenta interstital stem cell of third generation culture, phosphate buffer is taken to wash 3 4% paraformaldehydes and fix 15 points
Clock.Primary antibody uses anti-CD44 monoclonal antibodies (Abcam Co.), secondary antibody to use FITC- sheep anti mouse polyclonal antibodies, routine immunization
Fluorecyte film-making, fluorescence microscope (Leica Microsystems GmbH) are taken pictures.Placenta mesenchyma stem cell specific protein
White normal expression illustrates the certain lineage stem cells of isolated material, and cell purity is more than 90%, referring to Fig. 1.
(3) collection of excretion body
Rat Placenta mescenchymal stem cell is cultivated using the conditioned medium (Biological Industries) of serum-free
72 hours.Culture supernatant is through 500g, 4 DEG C of centrifugations, 15 minutes removal cells, collects the supernatant after centrifugation again through 2000g, 4 DEG C
Centrifugation 20 minutes, 0.45 μm of sterilised membrane filter filtering, removes remaining cell and cell fragment.
Isometric Macrogol 6000/the sodium chloride solution (16% Macrogol 6000+1M sodium chloride) of supernatant addition, 4
DEG C 12 hours.3500g is centrifuged 30 minutes, is collected precipitation and is resuspended in 0.5ml phosphate buffers.The precipitation being collected into is added again
Isometric Macrogol 6000/sodium chloride solution (10% Macrogol 6000,1M sodium chloride), 4 DEG C are incubated 4 hours, 3500g from
The heart 30 minutes collects precipitation and is resuspended in 200 μ l phosphate buffers, excretion body as after purification.
(4) detection excretion body specific proteins CD63
Excretion body sample is taken, its differential protein CD63 is detected using western blot methods.RIPA lysates are used first
(150mM NaCl, 1%NP-40,1% NaTDCs, 0.1% dodecyl sodium sulfate, 50mM Tris-HCl (pH
7.4), 50mM phosphoglyceric acids, 20mM NaF, 20mM EGTA, 1mM dithiothreitol (DTT)s, 1mM Na3VO4, 1mM benzyl sulphonyl
Fluorine) extraction total protein, it is detached through 10%SDS- polyacrylamide gel electrophoresises, electricity goes to pvdf membrane, 5% skimmed milk power room temperature envelope
After closing, it is separately added into diluted primary antibody 1: 1000 (3 monoclonal antibody of anti-CD 6) (Abcam Co.), 4 DEG C, 4 hours, horseradish peroxide
The secondary antibody (sheep anti-mouse igg polyclonal antibody) 1: 5000 (Abcam Co.) for changing enzyme label is incubated at room temperature 1 hour, and ECL is examined after rinsing
It surveys, exposure, development.The results show that differential protein CD63 can be detected by collecting obtained excretion body, illustrate what the present embodiment used
Polyethylene glycol Centrifugation method DNA can isolate and purify to obtain the excretion body of Rat Placenta mescenchymal stem cell.
Application of the embodiment 2 from the excretion body that placenta mesenchyma stem cell is produced in improving cardiac function
(1) foundation of ischemia-reperfusion cardiac muscle cell model
Rat myocardial cell system H9C2 is cultivated with+10% fetal calf serum (Hyclone) of DMEM high glucose mediums (GIBICO)
And it passes on.Three gas incubator oxygen concentrations are adjusted to 1% to prepare anaerobic environment, culture medium is changed to the DMEM without glucose
Culture medium takes out cell under the conditions of Anoxia in different time, and observation cytomorphology changes under inverted microscope,
CCK-8 colorimetric determination cell survival rates, compare growth inhibition ratio.The result shows that under the conditions of Anoxia, rat myocardial cell
Growth in conspicuousness inhibit (p ＜ 0.05), referring to Fig. 2.
(2) effect of the excretion body to rats Myocardial Ischemia-reperfusion Injury cell
Experiment packet：Control group (high glucose medium is normally cultivated), excretion body group, OGD groups (oxygen sugar deprives group) and OGD groups
+ excretion body group (excretion body additive amount 0.65g).Cytomorphology variation is observed under inverted microscope, CCK-8 colorimetric determinations are thin
Born of the same parents' survival rate, compares growth inhibition ratio.Cell protein is extracted, western blot methods detect idiocrasy Apoptosis correlation egg
White caspase3, Bcl-2 and Bax albumen (detection antibody is purchased from Abcam Co.).The result shows that in the case where being incubated altogether with excretion body,
The growth inhibition ratio of the rat myocardial cell of Anoxia condition is eased, and cell growth rate is significantly improved (p ＜
0.05).Western blot the result shows that, the ratio of the rat myocardial cell Bcl-2/Bax of Anoxia condition declines, and indicates
Mitochondrial damages can cause Intramitochondrial cromoci to overflow, cause later stage Apoptosis.The rat of Anoxia condition
For cardiac muscle cell after excretion body is incubated altogether, the ratio and normal cell of Bcl-2/Bax is approximate.This shows Rat Placenta stem cell
Excretion body cardiac muscle cell can be protected in the injury of mitochondria of Anoxia condition, prevent Apoptosis, can be applied to treat
Myocardial ischemia-reperfusion injury, referring to Fig. 3.
Claims (11)
1. a kind of preparation method of mescenchymal stem cell excretion body, it is characterised in that：
Using conditioned medium culture mescenchymal stem cell several hours of serum-free, it is thick to collect supernatant for centrifugation removal cell
After product, then after centrifuging, after removing remaining cell and cell fragment, the supernatant containing excretion body is made in filtering；
The polyethylene glycol Centrifugation method DNA used again to the supernatant containing excretion body is made.
2. a kind of preparation method of mescenchymal stem cell excretion body, it is characterised in that：
The supernatant after mesenchymal stem cell serum-free culture is collected, using the first chlorine of the Macrogol 6000 containing 16w/v%
Change second of sodium chloride solution substep purifying excretion body of sodium solution and Macrogol 6000 containing 10w/v%.
3. a kind of preparation method of mescenchymal stem cell excretion body, it is characterised in that：
Using conditioned medium culture mescenchymal stem cell several hours of serum-free, it is thick to collect supernatant for centrifugation removal cell
After product, then after centrifuging, after removing remaining cell and cell fragment, the supernatant containing excretion body is made in filtering；
The first sodium chloride solution containing Macrogol 6000 in equal volume is added to the supernatant containing excretion body, places 12 hours
More than, centrifugation is collected precipitation and is resuspended in phosphate buffer, isometric second of chlorine containing Macrogol 6000 is added again
Change sodium solution, place 4 hours or more, centrifugation collects precipitation and is resuspended in phosphate buffer, excretion body as after purification；
The first described sodium chloride solution Macrogol 6000 containing 16w/v%；
Second of sodium chloride solution Macrogol 6000 containing 10w/v%.
4. the preparation method of mescenchymal stem cell excretion body according to claim 3, it is characterised in that the first described
Sodium chloride solution contains 1M sodium chloride.
5. the preparation method of mescenchymal stem cell excretion body according to claim 3, it is characterised in that described second
Sodium chloride solution contains 1M sodium chloride.
6. the preparation method of mescenchymal stem cell excretion body according to claim 3, it is characterised in that the item of the centrifugation
Part is 3,500g, 30 minutes.
7. the preparation method of the mescenchymal stem cell excretion body according to one of claim 1~6, it is characterised in that obtained
Excretion body surface reaches specific proteins CD63.
8. a kind of excretion body of expression specificity PROTEIN C D63, it is characterised in that by the method system described in one of claim 1~7
.
9. drug is made in the injury of mitochondria for improving cardiac muscle cell as active constituent in excretion body according to claim 7
In application.
10. excretion body according to claim 7 is as active constituent in the medicine for preparing treatment myocardial ischemia-reperfusion injury
Application in object.
11. a kind of drug, it is characterised in that contain the excretion body described in claim 7.
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| CN109777770A (en) * | 2019-02-01 | 2019-05-21 | 中国农业科学院特产研究所 | A kind of excretion body and the preparation method and application thereof |
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