CN105802907B - A kind of cultural method of mesenchymal stem cell - Google Patents
A kind of cultural method of mesenchymal stem cell Download PDFInfo
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- CN105802907B CN105802907B CN201610251223.4A CN201610251223A CN105802907B CN 105802907 B CN105802907 B CN 105802907B CN 201610251223 A CN201610251223 A CN 201610251223A CN 105802907 B CN105802907 B CN 105802907B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The present invention provides a kind of preprocess method of survival ability after raising the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation, this method is that the stable mescenchymal stem cell of the passage acquired by the elderly's marrow is placed in the NAD that joined SRT1720,5mM of 0.5 μm of ol/L+It in the conventional medium of the glutamine of 20mM, pre-processes 24 hours, obtains treated mesenchymal stem cell and be used for cellular transplantation therapy.The beneficial effects of the present invention are embodied in: SRT1720 pretreatment can be by inhibiting the old exogenous apoptosis pathway of human marrow mesenchymal stem cell to inhibit its apoptosis under stressed condition, survival rate after the pretreated mesenchymal stem cell transplantation of SRT1720 in ischemic myocardium increases, and the effect for treating ischemic cardiomyopathy is obviously improved.
Description
(1) technical field
The present invention relates to a kind of preprocess method of survival ability after raising the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation, purports
Survival rate after improving its transplanting, and then improve it for the therapeutic effect of ischemic cardiomyopathy.
(2) background technique
Ischemic cardiomyopathy is to seriously threaten one of killer of human health, and sudden death incidence is high.Most patients although
Positive treatment is received, still inevitably development is heart failure.Though traditional treatment means substantially increase the life of patient
It deposits rate and improves prognosis, but dying cardiac muscle can not be saved, fail to be obviously improved the heart reconstruction after patient's myocardial infarction.
Mesenchymal stem cell causes to close extensively in regenerative medicine field in recent years because it is with self-renewing and Multidirectional Differentiation ability
Note and research.A large amount of clinical research has confirmed its safety and validity on cardiovascular disease therapies, however between marrow
Low survival rate, low field planting rate after mesenchymal stem cells transplanting become the key factor for restricting its curative effect.At the same time, ischemic cardiac
Myopathy takes place mostly in the elderly, and it is still Major cellular sources for transplanting that autologous bone marrow mesenchymal stem cells, which are, thus
Whether age factor, which influences its curative effect also, becomes unavoidable problem.In fact, existing a large amount of research confirms, older individuals
Under its biological character of the mesenchymal stem cell in source such as competence for added value, differentiation capability, anti-stress ability have obviously
Drop, cell of the effect also compared with young individuals source for transplantation treatment obviously weaken.Up to the present, though being mentioned there are many method
Survival rate after the mesenchymal stem cell transplantation of high derived from bone marrow, but change for the mesenchymal stem cell in older individuals source
Being apt to the research of its survival rate and curative effect, there is not been reported.
SIRT1 is a kind of histon deacetylase (HDAC) that NAD+ is relied on, and is extended in anti-aging and service life extensive in phenomenon
Research.Research in recent years has shown that it not only can adjust histone with deacetylation, for nonhistones such as P53, FOXOs, NF-
The Acetylation status of kB etc. also has adjustment effect, and then a series of changes such as cause Apoptosis, oxidative stress, inflammatory reaction
Change.Early-stage study proves that the senile rat mesenchymal stem cell aging state that SIRT1 is overexpressed improves, and the heart is used for after transplanting
The therapeutic effect of flesh infarct improves.The prompt of these results is expressed by medicament adjusting SIRT1 or activity improves the elderly's marrow
A possibility that mescenchymal stem cell therapeutic effect.
SRT1720 is a species specific SIRT1 active agonist, has been widely used in anti-aging, anti-apoptotic, has resisted
The preclinical phase of inflammation, oncotherapy etc. is studied, and shows its huge application potential.That ischemic is clear, ischemic resets and add anoxic, ischemic is clear
Under a variety of external Stress models for adding hydrogen peroxide, all show SRT1720 for the guarantor of old human marrow mesenchymal stem cell
Shield effect, and prove that it inhibits albumen FAIM that exogenous apoptosis pathway is inhibited to play the old people's bone of protection by upregulation of apoptosis
Effect of the bone marrow-drived mesenchymal stem from apoptosis.
(3) summary of the invention
It is an object of the present invention to provide one kind to pre-process old human marrow mesenchymal stem cell with SRT1720 to improve its life
The method for depositing ability, it is intended to improve the survival ability and therapeutic effect after it is transplanted in ischemic myocardial tissue.
The technical solution adopted by the present invention is that:
A method of improving survival ability after the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation, which comprises first will
The stable old human marrow mesenchymal stem cell of secondary culture is in the cells in vitro for containing 0.1~2.0 μm of ol/L SRT1720
It is cultivated 24~48 hours in culture medium, obtains that treated that cell carries out cell transplantation again.The cell injuring model base is normal
The culture medium addition SRT1720 that rule are suitable for mesenchymal stem cell in vitro culture is formed.The elderly referred to for 65 one full year of life
Above people.
Present inventor's discovery, through the pretreated old human marrow mesenchymal stem cell of SRT1720 in vitro ischemic it is clear,
The survival rate that ischemic resets and add anoxic and ischemic is reset and added under the stressed conditions such as hydrogen peroxide all significantly improves.Further research card
Bright SRT1720 be to the improvement of its survival rate inhibit albumen FAIM by upregulation of apoptosis, and then inhibit exogenous apoptosis pathway and
It plays a role.
Preferably, the cell injuring model base composition is as follows: SRT1720 0.1~2.0 μm of ol/L, NAD+3~6mM,
10~30mM of glutamine, fetal calf serum 10%, solvent are low sugar DMEM culture medium.Compared with individually addition SRT1720, the training
Feeding base be remarkably improved old human marrow mesenchymal stem cell hydrogen peroxide stress under survival rate, to rat heart function after transplanting
The improvement result of energy is also more excellent.
The culture is at 37 DEG C, in CO2It is carried out in volumetric concentration 5%, the mixed gas of volume of air concentration 95%.
Preferably, the method is as follows: the old human marrow mesenchymal stem cell in the 3rd generation of subculture is placed in cells in vitro training
It supports in base, at 37 DEG C, in CO2It in volumetric concentration 5%, the mixed gas of volume of air concentration 95%, cultivates 24 hours, obtains
Treated, and cell carries out cell transplantation again；The cell injuring model base composition is as follows: SRT1720 0.5 μm of ol/L, NAD+
5mM, glutamine 20mM, fetal calf serum 10%, solvent are low sugar DMEM culture medium.
The beneficial effects are mainly reflected as follows: it is pre-processed by SRT1720, is remarkably improved the bone in the elderly source
Survival ability of the bone marrow-drived mesenchymal stem in ischemic myocardium, and then improve its effect for treating ischemic cardiomyopathy.
(4) Detailed description of the invention
Fig. 1 is the survival rate under the old human marrow mesenchymal stem cell of SRT1720 pretreatment improvement in vitro stressed condition:
Ctrl represents conventional culture conditions control group, and DMSO represents solvent control group, when different after 0,12,24,48,72 representative pretreatments
Between give stressed condition.
Fig. 2 is that SRT1720 pretreatment can be improved after the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation enters infarcted myocardium of rat
Survival rate: DMSO represents solvent control group, and SRT1720 represents medical preconditioning group, and sry gene represents male sex and determines base
Cause.
Fig. 3 is the heart function that rat can be significantly improved through the pretreated the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation of SRT1720
Can: A is representativeness M type heart hypergraph piece, and B is left ventricular end diastolic diameter；C is left room end systolic diameter, and D is left ventricular ejection point
Number, E are left room short axis shortening rate；Sham represents sham-operation group, and DMEM represents culture medium injection group, and DMSO represents solvent pre-treatment
Group, SRT1720 represent medical preconditioning group.
Fig. 4 is that SRT1720 is pre-processed by inhibiting the old exogenous apoptosis pathway of mesenchymal stem cell to improve it
Survival rate: Ctrl represents routine culture group, and DMSO represents solvent pre-treatment group, and SRT1720 represents medical preconditioning group, EX-527
For SIRT1 specific inhibitor pretreated group.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1:
1. under aseptic condition from suitable gerontal patient (exclude hemopoietic system illness) (plum so-and-so, 85 years old, come from Hangzhou,
I agrees to use Marrow donation for research) bone marrow extraction liquid 10ml in hip replacement, it is placed in preparatory addition
It in the sterile glass vials of 4000IU heparin, mixes well and bone marrow fluid is avoided to solidify, it is next to move to the progress of Laminar Flow Room Biohazard Safety Equipment
Step operation.
2. equivalent low sugar DMEM culture medium (addition 100U/ml penicillin and 100U/ml streptomysin) (is purchased from the U.S.
GIBCO company) be added place bone marrow fluid sterile glass vials in dilute bone marrow fluid, it is thin that marrow is sufficiently dispelled with sterile pipette
Born of the same parents.
3. according to density gradient separation separating bone marrow single nuclear cell.20ml lymph is added in 50ml sterile centrifugation tube
Cell separating liquid (1.077 ± 0.001g/ml of density is purchased from Shanghai Heng Xin chemical reagent Co., Ltd) is light by 20ml bone marrow fluid
Gently it is superimposed upon lymphocyte separation medium surface.In high speed low temperature centrifugal machine, (Heraeus D-37520 is purchased from Germany Thermo
Electron company) in be centrifuged 25 minutes with 900g under the conditions of 4 DEG C.After centrifugation, cell layering, under, it is seen that training
Support base, tunica albuginea layer, lymph separating liquid and red blood cell layer.Intermediate tunica albuginea confluent monolayer cells are collected in another 50ml with 1ml sterile pipette
In centrifuge tube, 20ml low sugar DMEM culture medium is then added, rinsing cell surface remains lymph separating liquid.Under the conditions of 4 DEG C
900g is centrifuged 10 minutes, abandons supernatant, collects bone marrow mononuclear cells.Containing 10% fetal calf serum (being purchased from U.S. GIBCO company)
Low sugar DMEM culture medium be resuspended sedimentation cell, by 2 × 105/cm2Cell density is seeded to 75cm2Grow the cell culture of area
Bottle (is purchased from U.S. Corning company), is placed in 37 DEG C, contains 5%CO2The cell culture of (air 95%, v/v) and saturated humidity
In case (Forma3111 is purchased from U.S. Thermo Fisher company), liquid is changed for the first time after standing 48 hours, it is not adherent thin to remove
Born of the same parents.Hereafter it changes the liquid once within every 4~5 days, covers with 80%~90% or more and pass on.
4. being placed in 37 to carry out primary and passage cell culture containing 10% (v/v) fetal calf serum low sugar DMEM culture medium
DEG C, certain humidity contain 5%CO2Mixed gas (air 95%, v/v) in culture, it is adherent according to mesenchymal stem cell
The characteristic of growth, every 4~5 days replacement cell culture mediums remove non-adherent cell, constantly purifying human marrow mesenchymal stem cell.
Cell can carry out the cellular identifications such as morphology, flow cyctometry when 3 generation of subculture.
5. the 3rd generation human marrow mesenchyme stem cell of subculture cultivated under normal oxygen concentratio replacement is contained 0.5 μm of ol/L's
The NAD of SRT1720,5mM+, the glutamine of 20mM and the low sugar DMEM culture medium of 10% fetal calf serum, continue at 37 DEG C, one
That determines humidity contains 5%CO2Mixed gas (air 95%, v/v) in culture 24 hours after can be used for cell transplantation.
6. the survival ability of application CCK-8 cytoactive detection kit detection human marrow mesenchymal stem cell:
To the pretreated human marrow mesenchymal stem cell of step 5 (to add the cell of DMSO as control), dislocation is in 96
Orifice plate hydrogen peroxide containing 0.5mM (H2O2) low sugar DMEM culture medium in, after culture 3 hours, discard culture medium, every hole, which is added, contains 10 μ
The 110 μ l of low sugar DMEM culture medium of l CCK-8 reagent, after continuing culture 2.5 hours, in detecting 450nm in microwell plate microplate reader
Absorption values at wavelength, and each group survival rate is calculated, as a result as shown in Figure 1.
The result shows that with the NAD of the SRT1720 combination 5mM of 0.5 μm of ol/L+The elderly is pre-processed with the glutamine of 20mM
Mesenchymal stem cell be remarkably improved its hydrogen peroxide stress under survival rate, effect may persist to 3 days or more.
7. the cells survival rate after fluorescence quantitative PCR method detection transplanting:
1 day after cellular transplantation therapy and 3 days rat heart is taken out, liquid nitrogen grinding is taken after mixing to powdered
15mg tissue extracts genomic DNA with complete genome DNA extracts kit (being purchased from TAKARA company of Japan) and is used for quantitative PCR
Reaction.
The primer are as follows:
People's sry gene: upstream primer 5 '-GGTAAGTGGCCTAGCTGGTG-3 ', downstream primer 5 '-
GATCCCGCTTCGGTACTCTG-3'；
Rat 36B4 gene: upstream primer 5 '-CTCACTCCATCATCAATGGATACAA-3 ', downstream primer 5 '-
CAGCCAGTGGGAAGGTGTAGTCA-3’。
Reaction condition are as follows: 95 DEG C preheat 15 seconds, and followed by 95 DEG C are denaturalized 5 seconds, and 60 DEG C are annealed 31 seconds, and 72 DEG C extend 3 seconds, carry out
40 circulations.
Cell relative populations are calculated using Δ Δ Ct method.As a result as shown in Fig. 2, through SRT1720 pretreated old age
1 day and 3 days cell survival rate is distinguished 2.7 times and 1.9 times high than solvent control group after human marrow mesenchymal stem cell transplanting.
8. the improvement situation that cardiac ultrasonic evaluates Cardiac Function in Rat after cell therapy:
Cardiac function is evaluated with through chest cardiac ultrasonic within 28 days after rat cell transplanting, use VisualSonics Vevo
2100 systems, probe sampling frequency are 12.0.With 2% isoflurane (100% oxygen) to rat slight whole body fiber crops in checking process
It is liquor-saturated.Two-dimentional M type ultrasonoscopy is acquired in mitral level.Left ventricular end diastolic dimension (LVEDD) and left ventricular contraction latter stage
Internal diameter (LVESD) at least analyzes 3 independent cardiac cycles.Fractional shortening of the ventricular minor semi axis (LVFS, %) and left ventricular ejection point
Number (LVEF, %) is respectively from the analysis of 2100 system support software of Vevo.As a result as shown in Figure 3.
The results show that Cardiac Function in Rat after the pretreated the elderly's Bone Marrow Mesenchymal Stem Cells Transplantation of SRT1720
Improvement result is substantially better than solvent control group.
9. the expression that Western blot detects intracellular apoptosis-related protein:
To the cell that the pretreated human marrow mesenchymal stem cell of step 5 is collected, dislocation is in 6 orifice plates hydrogen peroxide containing 0.5mM
(H2O2) low sugar DMEM culture medium in, be respectively set the negative control group of routine culture, the pretreated solvent control group of DMSO,
SRT1720 pretreated group, SIRT1 inhibitor EX-527 pretreated group, after culture 3 hours, with RIPA buffer (purchased from China
Green skies Bioisystech Co., Ltd) cracking after, 14,000g centrifugation 30min, collect supernatant freeze it is spare in -80 DEG C.
BCA method (being purchased from U.S. Thermal Fisher company) measurement protein concentration: 40 μ g protein samples are in 15% (v/v)
SDS-PAGE glue Hoefer Mini-Gel system (Amersham Biosciences, purchased from U.S. Piscataway public affairs
Department) electrophoretic separation, it will with Hoefer Transfer Tank (Amersham Biosciences, Piscataway company of the U.S.)
On protein delivery to pvdf membrane (being purchased from U.S. BioRad company), film is placed in buffer, and [Tris buffer salt solution contains 0.1% (v/
V) Tween-20 (TBS-T), 7% (w/v) milk, pH 7.6] 1h is closed at room temperature, corresponding primary antibody (1:1000, purchased from beauty is added
Cell Signaling Technology company of state) it is incubated overnight under the conditions of 4 DEG C；After film is washed with 0.5% (v/v) TBS-T
It is incubated for 1 hour with the anti-rabbit IgG or anti-mouse IgG antibody (being purchased from U.S. Promega company) for combining alkaline phosphatase at room temperature,
It is developed the color with ECL solution after last TBS-T and TBS washing and (is purchased from U.S. Merk Millipore company), in Gel Doc EZ
It is imaged in Imaging System, quantitative analysis is carried out to band with Image Lab software Quantity One software,
Experimental result is shown in Fig. 4.
As the result is shown SRT1720 pretreatment significantly reduce apoptosis-related protein cleaved-caspase 3 and
The expression of cleaved-caspase 8, but do not influence the expression of endogenous apoptosis approach Bcl-2/BAX.
Conclusion: SRT1720 pretreatment can be by inhibiting the old exogenous apoptosis pathway of human marrow mesenchymal stem cell to mention
Its high survival ability, so as to further enhance the curative effect of cell transplantation.
Claims (3)
1. a kind of cultural method of mesenchymal stem cell, it is characterised in that: will be between the stable the elderly's marrow of secondary culture
Mesenchymal stem cells are cultivated 24~48 hours in the cell injuring model base containing 0.1~2.0 μm of ol/L SRT1720, are handled
Cell afterwards；The cell injuring model base composition is as follows: SRT1720 0.1~2.0 μm of ol/L, NAD+3~6mM, glutamy
10~30mM of amine, fetal calf serum 10%, solvent be low sugar DMEM culture medium, it is described old age human marrow mesenchymal stem cell from
65 old man more than one full year of life.
2. the method as described in claim 1, it is characterised in that the culture is at 37 DEG C, in CO2Volumetric concentration 5%, air body
It is carried out in the mixed gas of product concentration 95%.
3. the method as described in claim 1, it is characterised in that the method is as follows: will be between the elderly's marrow in the 3rd generation of subculture
Mesenchymal stem cells are placed in cell injuring model base, at 37 DEG C, in CO2Volumetric concentration 5%, volume of air concentration 95% it is mixed
It closes in gas, cultivates 24 hours, obtain treated cell；The cell injuring model base composition is as follows: 0.5 μ of SRT1720
Mol/L, NAD+5mM, glutamine 20mM, fetal calf serum 10%, solvent are low sugar DMEM culture medium.
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| PCT/CN2016/080303 WO2017181435A1 (en) | 2016-04-20 | 2016-04-27 | Method for improving post-transplant viability of bone marrow mesenchymal stem cell of old adult |
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| CN107630038A (en) * | 2017-09-15 | 2018-01-26 | 浙江大学 | The method of survival ability after raising senile rat Bone Marrow Mesenchymal Stem Cells Transplantation |
| CN107502595A (en) * | 2017-09-15 | 2017-12-22 | 浙江大学 | Improve the method for senile rat mesenchymal stem cells MSCs aging state |
| CN110904038B (en) * | 2019-12-13 | 2023-09-12 | 深圳市蓝思人工智能医学研究院 | Mesenchymal stem cells and application thereof |
| CN113201492A (en) * | 2021-06-22 | 2021-08-03 | 浙江三誉生物科技有限公司 | Culture medium and culture method of bone marrow mesenchymal stem cells |
| CN114763529A (en) * | 2022-05-23 | 2022-07-19 | 温州医科大学附属第一医院 | Application of roxasistat in enhancing survival capability of BMSCs in ischemic environment |
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| US20110269817A1 (en) * | 2008-07-24 | 2011-11-03 | The Regents Of The University Of California | Compositions and methods related to sirt1 function |
| CN101586095B (en) * | 2009-06-08 | 2011-04-27 | 浙江大学 | In-vitro culture medium of myeloid mesenchyma stem cell and use thereof |
| CN102382801B (en) * | 2011-10-26 | 2013-09-18 | 中国人民解放军军事医学科学院野战输血研究所 | Application of protein acetylation enzyme SIRT1 (Silent Mating type Information Regulation 2Homolog1) in promoting proliferation and delaying senility of mesenchymal stem cells |
| CN104958310B (en) * | 2015-05-22 | 2018-09-25 | 大连大学 | Sirt1 activator and its application |
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