CN108715831A - Application of the mescenchymal stem cell conditioned medium in the cellular damage for improving cardiomyocytes induced by hypoxia and reoxygenation - Google Patents
Application of the mescenchymal stem cell conditioned medium in the cellular damage for improving cardiomyocytes induced by hypoxia and reoxygenation Download PDFInfo
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Abstract
The invention discloses application of the mescenchymal stem cell conditioned medium in the cellular damage for improving cardiomyocytes induced by hypoxia and reoxygenation.Belong to biomedicine field.The mescenchymal stem cell conditioned medium is prepared by the following method：（1）：Obtain mescenchymal stem cell；（2）The incubation step in complete medium（1）The mescenchymal stem cell of middle gained；（3）Second generation mescenchymal stem cell is passed on, with 1*106It is a to be inoculated in 75cm2In culture bottle, cultivating 24 hours, abandon culture supernatant, PBS is washed 2 times, and the fresh cell culture mediums for containing 1% fetal calf serum of 10ml are added, and the basal medium of the cell culture medium is the basal medium of purpose cell, at 37 DEG C, 5%CO2It is cultivated for 24 hours under environment；（4）Supernatant is taken to filter, filtrate is mescenchymal stem cell conditioned medium.Mescenchymal stem cell conditioned medium obtained in the present invention can improve the various kinds of cell damage of cardiomyocytes induced by hypoxia and reoxygenation.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to mescenchymal stem cell conditioned medium is improving anoxia _ reoxygenation
Application in the cellular damage of induction.
Background technology
Ischemic disease is clinically very common, and for the organ or tissue of ischemic, it is to have the most to restore blood supply in time
The therapy of effect.But restore also cause even more serious damage while blood supply, referred to as ischemical reperfusion injury (lacks
The cellular damage of oxygen/reoxygenation induction).Restore blood supply in time, although can be difficult fundamentally to restore with patients in remission
Damaging cells quantity improves prognosis.
Mescenchymal stem cell is a kind of multipotent adult stem cells, can in vitro under specific inductive condition, be divided into fat,
Cartilage, bone, cardiac muscle, nerve etc., due to deriving from a wealth of sources, the advantages that being limited without ethics, using being very much extensive.Some researches show that,
Mescenchymal stem cell has the organ damages such as the heart, liver, lung, kidney, brain certain therapeutic effect.The therapeutic effect of mescenchymal stem cell
Depend on the cell factor of its paracrine.Mescenchymal stem cell conditioned medium for stem cell, be more easy to obtain,
Easy to use and acellular immunogenicity and oncogenicity.
The document of cell conditioned medium application is reported for work numerous, but its collection method there is no unified standard.It establishes and optimizes
The collection method of mescenchymal stem cell conditioned medium is conducive to the repeatability and result reliability that increase scientific experiment.
Invention content
In order to solve the problems in the existing technology, the present invention is experimentally confirmed the CMC model of mescenchymal stem cell
Base can be effectively improved the damage of the various kinds of cell of cardiomyocytes induced by hypoxia and reoxygenation (H/R), therefore, mescenchymal stem cell conditioned medium
The potential function become with treatment ischemia/reperfusion injury venereal disease.
In order to achieve the above object, technical scheme is as follows：
In a first aspect, the present invention provides mescenchymal stem cell conditioned medium in the cell damage for improving cardiomyocytes induced by hypoxia and reoxygenation
Application in wound, the preparation method of the mescenchymal stem cell conditioned medium, includes the following steps：
(1) mescenchymal stem cell is obtained；
(2) mescenchymal stem cell obtained by complete medium in incubation step (1)；
(3) second generation mescenchymal stem cell is passed on, with 1*106Cell inoculation is in 75cm2In culture bottle；Culture 24 hours
Afterwards, culture supernatant is abandoned, PBS is cleaned 2 times；The fresh cell culture mediums for containing 1% fetal calf serum of 10mL, the cell culture medium is added
For purpose cell be free of serum and dual anti-basal medium, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours；
(4) supernatant is taken to filter, filtrate is mescenchymal stem cell conditioned medium.
Preferably, the source for mesenchymal stem cells described in above-mentioned steps (1) is in tissues such as marrow, fat, umbilical cord, placentas,
It is preferably derived from the mescenchymal stem cell (BMSC) of marrow.
Preferably, the filtering described in above-mentioned steps (4) is the membrane filtration for selecting 0.22 μm.
Preferably, above application is that the conditioned medium of gained is added to aim cell as protective ingredient to train completely
It supports in base, added ratio is 50%.
Compared with prior art, effect of the invention is：
1, mescenchymal stem cell conditioned medium is applied to improve the cellular damage of cardiomyocytes induced by hypoxia and reoxygenation for the first time by the present invention
In, it was demonstrated that it can be effectively improved the damage of the various kinds of cell of cardiomyocytes induced by hypoxia and reoxygenation, such as：Mescenchymal stem cell conditioned medium energy
The survival rate for enough improving damaged cell, inhibits the Apoptosis of cardiomyocytes induced by hypoxia and reoxygenation, reduce lactic dehydrogenase emission levels to
The vigor of cell is increased, cardiac muscle cell's mitochondrial membrane potential is stablized.
2, by the way that a certain number of second generations are added in the preparation method of mescenchymal stem cell conditioned medium of the invention
Mescenchymal stem cell is to 75cm2In culture bottle, optimizes the collection method of mescenchymal stem cell conditioned medium, be conducive to increase section
Grind the repeatability and result reliability of experiment.
Description of the drawings
Fig. 1 is the myocyte survival rate figure that mescenchymal stem cell conditioned medium increases cardiomyocytes induced by hypoxia and reoxygenation；
Fig. 2 is the H9c2 apoptosis rate figures (A that mescenchymal stem cell conditioned medium reduces H/R inductions：Streaming is thin
Born of the same parents' art detects apoptosis rate, B：Apoptosis rate statistical chart)；
Fig. 3 is the H9c2 cardiomyocyte viability figures that mescenchymal stem cell conditioned medium increases H/R inductions；
Fig. 4 is H9c2 cardiac muscle cell's mitochondrial membrane potential figure that mescenchymal stem cell conditioned medium stablizes H/R inductions
(A：Super-resolution confocal detection mitochondrial membrane potential in anoxic, B：Mitochondrial membrane potential statistical chart)；
Fig. 5 is that mescenchymal stem cell conditioned medium (CM) lives to in-vitro simulated cerebral ischemia re-pouring microglia BV2
The influence diagram of property；
OGD4h/R6h is simultaneously reoxygenation 6 hours of oxygen sugar stripping degree 4 hours；
Fig. 6 is that light microscopic observation mescenchymal stem cell conditioned medium (CM) is thin to the small glue of in-vitro simulated cerebral ischemia re-pouring
The influence diagram of born of the same parents' BV2 forms；
Fig. 7 is mescenchymal stem cell conditioned medium to in-vitro simulated cerebral ischemia re-pouring microglia BV2 apoptosis
Influence diagram (A：Apoptosis by Flow Cytometry rate, B：Apoptosis rate statistical chart)；
Fig. 8 is mescenchymal stem cell conditioned medium (CM) to in-vitro simulated cerebral ischemia re-pouring microglia BV2 breasts
The influence diagram of acidohydrogenase (LDH) release；
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.【Embodiment 1】Between fill
The preparation of matter stem cell conditioned medium
Rat abdominal cavity anesthesia is put to death, and is impregnated 10 minutes through 75% alcohol, is detached rat long bone of limbs and is cleaned 3 through PBS
It is secondary.Cut off long bone both ends, exposure ossis；Bone marrow irrigation liquid is drawn with 5mL syringes and rinses ossis, collects liquid to 15mL
In centrifuge tube；1000rpm is centrifuged 6 minutes, abandons supernatant, and cell is resuspended；With 1*104/cm2Cell density be seeded in culture bottle,
Appropriate mescenchymal stem cell complete medium (MEM- α low sugar, 10%FBS, 1% is dual anti-) is added；37 DEG C are placed in, 5%CO2Training
It supports and is cultivated 2-3 days in case；Culture medium is replaced to remove non-attached cell；Attached cell is continued culture to cell fusion degree to reach
80% or so；After 0.25% trypsin treatment cell 2-3 minutes, cell is centrifuged and be resuspended, is seeded to two 75cm2Culture
In bottle；Continue to cultivate cell to the second generation.By 1 × 106Second generation mescenchymal stem cell is inoculated in 75cm2In culture bottle, culture 24
Culture supernatant is abandoned after hour, PBS is cleaned 2 times, and the fresh culture mediums for containing 1% fetal calf serum of 10mL are added, and (basal medium is mesh
Cell culture medium), continue at 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.Collect culture supernatant and with 0.22 μm of filtration membrane
Filtering collects filtrate and supplements serum-concentration to 10%, as mescenchymal stem cell conditioned medium, preserved for -80 DEG C after packing.
【Embodiment 2】Improve the experiment of cardiomyocytes induced by hypoxia and reoxygenation myocardial cell injury
1, method：
(1) H9c2 cell lines culture
H9c2 cardiac muscle cell's (Shanghai Cell Bank of the Chinese Academy of Sciences) is inoculated in the culture dish of a diameter of 10cm, with containing
10% fetal calf serum and 1% dual anti-DMEM in high glucose culture medium, 37 DEG C, 5%CO2、21O2It is cultivated in % incubators, waits for that cell is given birth to
When length is fused to 70-80% degrees of fusion, with 0.25% trypsin digestion and passes on, be inoculated in 96 orifice plates and 6 orifice plates, take logarithm
Growth period cell is for testing.
(2) structure of cell hypoxia/reoxygenation model：
After cardiac muscle cell grows to exponential phase in normal incubator, when anoxic, old culture medium is discarded, PBS washes 2
Time, it changes fresh sugar-free DMEM (contain 1% dual anti-, serum-free) into, places in three gas incubators that (parameter is adjusted to：37 DEG C, 5%
CO2, 1%O2) culture 8h after, the complete medium that anoxic liquid is changed into fresh cardiac muscle cell is (dual anti-containing 10%FBS and 1%
DMEM in high glucose), cultivated for 24 hours under regular culture conditions.Anoxia _ reoxygenation+conditioned medium group：It is prepared in Example 1
Conditioned medium, room temperature dissolve.When cell reoxygenation is handled with 50% volume ratio by the complete of conditioned medium and cardiac muscle cell
Culture medium mixes, culture experiment cell.
By the cell survival rate of CCK8 methods two groups of cells of detection, the bis- dyeing of AnnexinV/PI, flow cytomery is thin
Born of the same parents' apoptosis rate, LDH kit detection cell vigor, super-resolution confocal detection cardiac muscle cell's mitochondrial membrane potential.
(3) CCK-8 detects H9c2 cardiac muscle cell's relative survival rate
By cell with 3 × 104A/mL is inoculated in 96 well culture plates, then carries out anoxic 8h processing.When reoxygenation, fill
Mescenchymal stem cell conditioned medium (addition of volume ratio 50%) is added in matter stem cell conditioned medium group, and reoxygenation is for 24 hours.Processing
After, CCK8,10 holes μ L/ is added, 37 DEG C of incubation 2h measure the absorbance at 450nm, as a result with absorbance in microplate reader
(OD values) indicates.Cell activity (OD values)=experimental group OD values-blank well OD values.
(4) the bis- dye methods of Annexin V-FITC/PI, Apoptosis by Flow Cytometry
By cardiac muscle cell with 2.5 × 105A/hole is inoculated in 6 well culture plates, then carries out anoxic 8h processing.When reoxygenation,
Mescenchymal stem cell conditioned medium (addition of volume ratio 50%), reoxygenation is added in mescenchymal stem cell conditioned medium group
24h.After being disposed, first upper layer culture medium is collected in 2ml EP pipes, the trypsin digestion cell for being free of EDTA with 0.25%
3min, and be collected into corresponding EP pipes.800r/min centrifuges 5min, cell is washed 1 time with the PBS of precooling, with 400 μ l 1X
Annexin V combination liquid suspension cells.5 μ l Annexin V-FITC dyeing liquors are added in cell suspending liquid, gently after mixing
It is incubated 15 minutes under the conditions of being protected from light in 2-8 DEG C.Then it is added after 7 μ l PI dyeing liquors that gently mixing is incubated under the conditions of being protected from light in 4 DEG C
5 min.Flow cytomery is used immediately.The FITC positives are apoptotic cell (the sum of right upper quadrant and right lower quadrant).
(5) cell conditioned medium lactic dehydrogenase (LDH) vitality test
By H9c2 cardiac muscle cell with 3 × 104A/mL is inoculated in 96 well culture plates, then carries out anoxic 8h processing.It is multiple
When oxygen, mescenchymal stem cell conditioned medium (addition of volume ratio 50%) is added in mescenchymal stem cell conditioned medium group, multiple
Oxygen is for 24 hours.After being disposed, cell conditioned medium is collected.It executes in strict accordance with specification program, is finally measured at 450nm in microplate reader
The absorbance (OD values) in each hole calculates supernatant LDH contents by standard curve.
(6) super-resolution confocal detection cardiac muscle cell mitochondrial membrane potential
Normally culture is to after exponential phase by H9c2 cardiac muscle cell, after anoxic 8h, the BMSC-CM of BMSC-CM groups when reoxygenation
It is classified as 1 with the ratio of cardiac muscle cell's complete medium (serum for being added a concentration of 10%):1, simple reoxygenation group gives cardiac muscle cell
Complete medium (final concentration of 10%) of serum, reoxygenation for 24 hours after, after JC-1 dyeing, the burnt analysis mitochondrial membrane of super-resolution copolymerization
Current potential.As a result it is indicated with red, green fluorescence intensity rate.
Fig. 1's -4 the results show that anoxia _ reoxygenation+conditioned medium group cell survival rate and cell viability be apparently higher than it is scarce
Oxygen/reoxygenation group cell, apoptosis rate are significantly lower than anoxia _ reoxygenation group cell, and conditioned medium can stablize myocardial cell membrane electricity
Position, difference have conspicuousness.
【Embodiment 3】Improve the glial cell damages experiment of cardiomyocytes induced by hypoxia and reoxygenation
1, method：
(1) BV2 cell lines culture：
BV2 is applied to as the replacement cell of microglia in this experiment, and by BV2 cells, (Chinese Academy of Sciences Shanghai is thin
Born of the same parents library) it is placed in DMEM in high glucose/F12 culture mediums (Gibico companies) containing 10% inactivated fetal bovine serum (56 DEG C, 30min processing)
In the culture bottle of 25cm2.The culture in 37 DEG C, 5%CO2, incubator (95% air) waits for that cell growth is fused to 80% left side
Old culture solution is abandoned on the right side, and PBS is cleaned 2 times, is digested 2min with 0.25% pancreatin, cell passage is carried out, when cell growth is in logarithm
It is tested when the phase.
(2) hypoxia-reoxygenation model structure and packet transaction
By BV2 cells progress glycosyloxy deprive reoxygenation (Oxygen Glucose Deprivation/Reoxygenation,
OGD/R it) handles.
Three gas incubator parameters are adjusted to 37 DEG C, 94%N in advance before modeling2, 5%CO2, 1%O2State.Take logarithmic growth
Phase BV2 cell discards old culture medium, and PBS is softly cleaned 2 times, changes sugar-free culture-medium into, places in hypoxia culture box, first carries out
Then anoxic 4h processing changes sugar-free culture-medium into fresh complete medium (the DMEM/F12 high glucose mediums of 10%FBS)
After be put into 37 DEG C, 5%CO2, 95% air jet flow case reoxygenation handle 6 hours.Anoxia _ reoxygenation+conditioned medium group：Example
The conditioned medium prepared in 1, after room temperature dissolves.It by conditioned medium and is tested with 50% volume ratio when cell reoxygenation is handled
The complete medium of cell (Deiter's cells) mixes, culture experiment cell.
Evaluate cell survival rate, the cell activity of anoxia _ reoxygenation+conditioned medium group and simple anoxia _ reoxygenation group
(LDH), apoptosis rate and cell phenotype.
(3) CCK-8 detects BV2 cell activity
By cell with 1 × 105A/mL is inoculated in 96 well culture plates, then carries out anoxic 4h processing.When reoxygenation, fill
Mescenchymal stem cell conditioned medium (50%), reoxygenation 6h is added in matter stem cell conditioned medium group.After being disposed, it is added
CCK8,10 holes μ L/, 37 DEG C of incubation 2h are measured the absorbance at 450nm in microplate reader, are as a result indicated with absorbance (OD values).Carefully
Cytoactive (OD values)=experimental group OD values-blank well OD values.
(4) the bis- dye method Apoptosis by Flow Cytometries of Annexin V-FITC/PI
By cell with 2.5 × 105A/mL is inoculated in 24 well culture plates, then carries out anoxic 4h processing.When reoxygenation,
Mescenchymal stem cell conditioned medium (50% than row), reoxygenation 6h is added in mesenchymal stem cells conditioned medium group.After being disposed,
First upper layer culture medium is collected in 2ml EP pipes, with 0.25% trypsin digestion cell 2min, and is collected into corresponding EP pipes.
4 DEG C, 1500r/min, 5min is centrifuged, cell is washed 1 time with the PBS of precooling, suspended with 400 μ l 1X Annexin V combination liquid
Cell.5 μ l Annexin V-FITC dyeing liquors are added in cell suspending liquid, are incubated under the conditions of being gently protected from light in 2-8 DEG C after mixing
It educates 15 minutes.Then it is added after 7 μ l PI dyeing liquors that gently mixing is incubated 5min under the conditions of being protected from light in 4 DEG C.Fluidic cell is used immediately
Instrument detects.The FITC positives are apoptotic cell (the sum of right upper quadrant and right lower quadrant).
(5) cell conditioned medium lactic dehydrogenase (LDH) vitality test
By BV2 with 1 × 105A/mL is inoculated in 96 well culture plates, then carries out anoxic 4h processing.When reoxygenation, mesenchyma
Mescenchymal stem cell conditioned medium is added in stem cell conditioned medium group, then reoxygenation 6h.After being disposed, collect on cell
Clearly.It is executed in strict accordance with by specification program, the absorbance (OD values) in each hole at 450 nm is finally measured in microplate reader, by standard
Curve calculates supernatant LDH contents.
Fig. 5's -8 the results show that anoxia _ reoxygenation+conditioned medium group cell activity, cell survival rate are apparently higher than merely
Anoxia _ reoxygenation group, apoptosis rate are significantly lower than anoxia _ reoxygenation group.
Claims (5)
1. application of the mescenchymal stem cell conditioned medium in the cellular damage for improving cardiomyocytes induced by hypoxia and reoxygenation, which is characterized in that
The preparation method of the mescenchymal stem cell conditioned medium, includes the following steps：
（1）Obtain mescenchymal stem cell；
（2）The incubation step in complete medium（1）The mescenchymal stem cell of middle gained；
（3）Second generation mescenchymal stem cell is passed on, with 1*106Cell inoculation is in 75cm2In culture bottle；After culture 24 hours, abandon
Culture supernatant, PBS are cleaned 2 times；The fresh cell culture mediums for containing 1% fetal calf serum of 10 mL, the base of the cell culture medium is added
Basal culture medium be purpose cell basal medium, 37 DEG C, 5% CO2Under the conditions of cultivate 24 hours；
（4）Supernatant is taken to filter, filtrate is mescenchymal stem cell conditioned medium.
2. application according to claim 1, which is characterized in that step（1）The source for mesenchymal stem cells in marrow,
Fat, umbilical cord, placenta.
3. application according to claim 2, which is characterized in that step（1）The source for mesenchymal stem cells is in marrow.
4. according to claim 1-3 any one of them applications, which is characterized in that step（4）The filtering is selection 0.22
μm membrane filtration.
5. application according to claim 4, which is characterized in that be added the conditioned medium of gained as protective ingredient
Into aim cell complete medium, added ratio is 50%.
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Cited By (3)
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| CN109486748A (en) * | 2018-12-27 | 2019-03-19 | 佛山科学技术学院 | A kind of preparation method of placenta mesenchyma stem cell conditioned medium |
| CN113106056A (en) * | 2020-02-25 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Method for inducing differentiation of astrocytes by umbilical cord mesenchymal stem cells |
| CN115125198A (en) * | 2022-07-11 | 2022-09-30 | 湖北沃德利派生物科技有限公司 | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486748A (en) * | 2018-12-27 | 2019-03-19 | 佛山科学技术学院 | A kind of preparation method of placenta mesenchyma stem cell conditioned medium |
| CN113106056A (en) * | 2020-02-25 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Method for inducing differentiation of astrocytes by umbilical cord mesenchymal stem cells |
| CN115125198A (en) * | 2022-07-11 | 2022-09-30 | 湖北沃德利派生物科技有限公司 | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof |
| CN115125198B (en) * | 2022-07-11 | 2024-01-26 | 湖北沃德利派生物科技有限公司 | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof |
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