CN101407790B - Processing method for enhancing human medulla ossium mesenchyma stem cell paracrine ability - Google Patents
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Abstract
The invention provides a processing method for enhancing the paracrine capacity of human mesenchymal stem cells for improving the curative effect of transplanting the human mesenchymal stem cells for repairing tissue damage. The method comprises the following steps: the stable human mesenchymal stem cells after subculture are anoxygenously cultured for 20 to 30 hours and then cultured under normal oxygen and used for cell transplanting. The processing method has the beneficial effects as follows: the paracrine capacity of the human mesenchymal stem cells can be effectively improved and a plurality of cell growing factors and angiogenic growth factors, erythropoietin and the receptors thereof can be improved, thereby improving the curative effect of transplanting the human mesenchymal stem cells for repairing tissue damage through the anoxygenous culturing.
Description
(1) technical field
The present invention relates to a kind of treatment process that strengthens human medulla ossium mesenchyma stem cell paracrine ability, transplant the curative effect of repair tissue damage to improve human marrow mesenchymal stem cell.
(2) background technology
The damage of cellular transplantation therapy repair tissue is present emerging medical field focus.Cell therapy is a kind of means of supplementing out economy to traditional medicine, for the treatment of some refractory disease provides new hope.Stem cell be a kind of can self-replacation infinite multiplication, under specific inductive condition to progenitor cell with germinal layer or other germinal layer cytodifferentiation.According to different tissue sources, stem cell can be divided into embryonic stem cell and adult stem cell, and the latter is distributed in the most tissues.Because adult stem cell is transplanted no ethnics Problem and tumorigenicity, is more suitable for " seed cell " in gene and cell engineering.Mescenchymal stem cell derives from mesoblastema.Can obtain from a lot of separate tissue, as liver, fetus and Cord blood, amniotic fluid and fatty tissue, but research at most and the most facile be marrow (account for＜0.01%) at whole BMNCs.
The evaluation of human marrow mesenchymal stem cell mainly comprises: 1. in normal condition of in vitro culture adherent growth, be spindle shape or trilateral; 2. fluidic cell is learned the mesenchymal stem cells MSCs that detects more than 95% and is expressed CD105, CD73 and CD90, and do not express CD45, CD34, CD14 (or CD11b), CD19 (or CD79-α), human leucocyte antigen (HLA) HLA-DR surface molecular (less than 2% positive expression rate); 3. have many differentiation potentials.A large amount of experimentation on animalies show that mesenchymal stem cells MSCs have its special advantages and good security in the repair tissue damage, and human marrow mesenchymal stem cell is transplanted and can significantly be improved behind the acute myocardial infarction heart function and delay remodeling ventricle.Further discover, the human marrow mesenchymal stem cell of transplanting directly is divided into cardiac-like muscle cell, vascular endothelial cell and smooth muscle cell, the participation new vessel forms, the more important thing is that transplanted cells passes through a large amount of cytokine of paracrine and somatomedin, anti-apoptotic and angiogenesis promoting, the survival rate of increase transplanted cells.Have research to think, after the Bone Marrow Stem Cells Transplantation can part survival propagation only account for 1/3 of transplanted cells certainly will can improve the curative effect of Transplanted cells because improve transplanted cells in the survival rate and the paracrine ability of local organization.
(3) summary of the invention
The object of the invention provides a kind of treatment process that strengthens human medulla ossium mesenchyma stem cell paracrine ability, transplants the curative effect of repair tissue damage to improve human marrow mesenchymal stem cell.
The technical solution used in the present invention is:
A kind of treatment process that strengthens human medulla ossium mesenchyma stem cell paracrine ability, described method is: will cultivate stable human marrow mesenchymal stem cell anoxic cultivation through going down to posterity after 20～30 hours, and recover normal oxygen again and cultivate to be used for Transplanted cells.Described anoxic is cultivated and is not meant having and cultivates in the presence of the oxygen, can under the atmosphere of nitrogen, carbonic acid gas or nitrogen and carbon dioxide mix gas, cultivate, except atmosphere was different with conventional cultivation, other culture condition all can carry out with reference to the condition of in vitro culture of conventional human marrow mesenchymal stem cell.
Anoxic pre-treatment energy activated cell endogenous protection mechanism starts the synthetic of some expression of gene and stress protein, strengthens the cell anti-anoxia ability.Discover, anoxic pre-treatment (sublethal dose) can improve synthetic a large amount of cell growth factor and the angiogenic growth factors of mesenchymal stem cells MSCs, comprise HIF-1 α (HIF-1 α), blood vessel synthetic chemical-1, vascular endothelial cell factor acceptor Flk-1, erythropoietin and acceptor thereof.Anoxic pre-treatment energy inducing bone mesenchymal stem cell expressing promoting existence albumen P105, the NF-κ B P65 of subunit and P50, inhibitor of apoptosis protein Bcl-2 and Bcl-xL reduce the expression that apoptosis is carried out PROTEIN C aspase-3.The pretreated Bone Marrow Mesenchymal Stem Cells Transplantation of anoxic can reduce transplanted cells and cardiac cellular apoptosis to the acute myocardial infarction part, promotes more angiogenesis, dwindles myocardial infarction area and improves heart function.
Concrete, described method is: before Transplanted cells, just the human marrow mesenchymal stem cell in the 3rd generation of subculture is after anoxic is cultivated 20～30 hours in low sugar DMEM substratum under 37 ℃, recover 37 ℃ of normal oxygen and cultivate more than 2 hours (it is comparatively stable to cultivate 24 hour cells usually), the human marrow mesenchymal stem cell after obtaining to handle is used for Transplanted cells.
Concrete, described anoxic is cultivated and is: at CO
2 Volumetric concentration 5%, N
2Cultivate in the mixed gas of volumetric concentration 95%.Described normal oxygen is cultivated and is: at CO
2 Volumetric concentration 5%, O
2Cultivate in the mixed gas of volumetric concentration 95%.
Concrete, described method is as follows: before Transplanted cells, with human marrow mesenchymal stem cell under 37 ℃, at CO
2 Volumetric concentration 5%, N
2In the mixed gas of volumetric concentration 95%, anoxic was cultivated after 24 hours in low sugar DMEM substratum, recovered 37 ℃ of normal oxygen and cultivated 24 hours, and the human marrow mesenchymal stem cell after obtaining to handle carries out Transplanted cells again.For improving human medulla ossium mesenchyma stem cell paracrine ability, this step operation can repeat.
Beneficial effect of the present invention is mainly reflected in: cultivate by anoxic, can effectively improve the paracrine ability of human marrow mesenchymal stem cell, can improve synthetic a large amount of cell growth factor and the angiogenic growth factors of mesenchymal stem cells MSCs, erythropoietin and acceptor thereof, and then improve the curative effect that human marrow mesenchymal stem cell is transplanted the repair tissue damage.
(4) description of drawings
Fig. 1 cultivates synoptic diagram for cell under different oxygen concentrations; 1 is pneumatic accumulator, and 2 is CO
2Bottle, 3 is N
2Bottle, 4 is the oxygen detection monitoring device, and 5 is cell culture incubator, and 6 is gas valve;
Fig. 2 is human marrow mesenchymal stem cell morphology performance under the phase microscope;
Fig. 3 cultivates for anoxic stimulates human marrow mesenchymal stem cell secretory cell somatomedin and angiogenic growth factor at double;
Fig. 4 cultivates inducing human mesenchymal stem cells expressing promoting existence albumen P105, the NF-κ В P65 of subunit and P50 for anoxic, and inhibitor of apoptosis protein Bcl-2 and Bcl-xL reduce the expression that apoptosis is carried out PROTEIN C aspase-3.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
1. the posterior superior iliac spine from suitable adult patients (getting rid of the hemopoietic system illness) extracts bone marrow fluid 30～50ml under the aseptic condition, be positioned in the aseptic vial that adds the 4000IU heparin in advance, fully mixing avoids bone marrow fluid to solidify, and moves to the Laminar Flow Room Biohazard Safety Equipment and carries out next step operation.
2. equivalent low sugar DMEM substratum (adding 100U/ml penicillin and 100U/ml Streptomycin sulphate, available from U.S. GIBCO company) is added in the aseptic vial of placing bone marrow fluid and dilute bone marrow fluid, fully dispel medullary cell with aseptic pipettor.
3. according to the density gradient separation separating bone marrow single nuclear cell.In the aseptic centrifuge tube of 50ml, add 20ml lymphocyte separation medium (density is 1.077 ± 0.001g/ml, available from Shanghai permanent letter chemical reagent company limited), the 20ml bone marrow fluid is superimposed upon the lymphocyte separation medium surface gently.(Heraeus D-37520 is available from German Thermo Electron company) was with centrifugal 25 minutes of 900g under 4 ℃ of conditions in high speed low temperature centrifugal machine.Behind centrifugal the finishing, the cell layering, from down to down, visible substratum, tunica albuginea layer, lymph parting liquid and red corpuscle layer.Collect middle tunica albuginea confluent monolayer cells in another 50ml centrifuge tube with the aseptic pipettor of 1ml, add 20ml low sugar DMEM substratum then, the residual lymph parting liquid of rinsing cell surface.900g is centrifugal 10 minutes under 4 ℃ of conditions, abandons supernatant, collects BMNC.To contain the resuspended sedimentation cell of low sugar DMEM substratum of 10% foetal calf serum (available from U.S. GIBCO company), by 2 * 10
5/ cm
2Cell density is seeded to 75cm
2The Tissue Culture Flask (available from U.S. Coming company) of growth area is positioned over 37 ℃, contains 5%CO
2With in the cell culture incubator (Forma3111 is available from U.S. Thermo Fisher company) of saturated humidity (referring to Fig. 1), leave standstill after 48 hours and change liquid first, to remove not attached cell.After this changed liquid once, and covered with more than 80%～90% and go down to posterity in per 4～5 days.
4. carry out former generation and subculture cell cultures to contain 10% foetal calf serum low sugar DMEM substratum, be positioned over 37 ℃, certain humidity contain 5%CO
2Mixed gas in normal oxygen cultivate, according to the characteristic of mesenchymal stem cells MSCs adherent growth, changed cell culture medium in per 4～5 days, remove non-adherent cell, constantly the purifying human marrow mesenchymal stem cell.Subculture during the 3rd generation cell can carry out cells such as morphology, fluidic cell and identify.
5. subculture the 3rd generation human marrow mesenchyme stem cell that normal oxygen concentratio is cultivated down moves to and carries out the anoxic pre-treatment in the hypoxia culture box.The cell growth reached about 80% fusion before anoxic was cultivated, and cell is changed low sugar DMEM substratum.In 37 ℃, saturated humidity contain 5% (v/v) CO
2With 95% (v/v) N
2Anoxic is cultivated in the mixed gas, and anaerobic treatment is carried out in the ProOx-C-chamber system (available from U.S. Biospherix, Redfield company) of an energy accuracy controlling oxygen concn, and setting oxygen concn is 0.5%, and the anoxic time is 24 hours.Anaerobic treatment finishes the normal oxygen cultivation of back recovery and (contains 5%CO
2, 95% air, v/v) after 2 hours, carry out immunoblotting and detect.
6. for improving anti-apoptosis of human marrow mesenchymal stem cell and paracrine ability, can carry out above-mentioned the 3rd step operation repeatedly.
7. immunoblotting detects Intracellular growth factor HIF-1 α (HIF-1 α), blood vessel synthetic chemical-1 (Ang1), the vascular endothelial cell factor (VEGF) and acceptor Flk-1 thereof, erythropoietin (EPO) and acceptor albumen such as (EPOR) thereof and anti-apoptotic genes expression cleavedcaspase-3, bcl-2, bcl-xL, P60, the proteic expression of P65 change.
The cell of collecting RIPA buffer (50mM HEPES, pH7.3,1%sodiumdeoxycholate, 1%Triton X-100,0.1%SDS, 150mM NaCL, 1mM EDTA, 1mM Na3VO4,1mM NaF) after the cracking, 14, the centrifugal 30min of 000g, it is frozen standby in-80 ℃ to collect supernatant liquor.BCA method (Bicinchoninic Acid Assay is available from U.S. Sigma company) is measured protein concentration.40 μ g protein samples are at 6-15%SDS-PAGE gradient glue Hoefer Mini-Gel system (Amersham Biosciences, available from U.S. Piscataway company) electrophoretic separation, with Hoefer Transfer Tank (AmershamBiosciences, U.S. Piscataway company) albumen is transferred on the pvdf membrane (available from U.S. BioRad company), film places damping fluid [Tris buffer salt solution, contain 0.1%Tween-20 (TBS-T), 7% milk, pH7.6] seal 2h under the room temperature, add corresponding one anti-(1:1000, available from U.S. Santa Cruz Biotechnology company) overnight incubation under 4 ℃ of conditions, mouse anti β-actin (1:1000 is available from state Santa Cruz Biotechnology company) is as sample contrast on the albumen; Film is with hatching 2 hours with the anti-rabbit igg or the anti-mouse IgG antibody (available from U.S. Promega company) of combined alkali acid phosphatase under the room temperature of 0.5%TBS-T washing back, develop the color (available from U.S. Sigma company) with BCIP/NBT solution in last TBS-T and TBS washing back, the scanning back is used
CS8.0 software analysis signal, experimental result is seen Fig. 2～Fig. 4.
Conclusion: anoxic is cultivated can not change the cell growthhabit, anoxic is cultivated can stimulate human marrow mesenchymal stem cell to secrete HIF-1 α (HIF-1 α) at double, blood vessel synthetic chemical-1, vascular endothelial cell factor acceptor Flk-1, erythropoietin and acceptor thereof, energy inducing human mesenchymal stem cells expressing promoting existence albumen P105, the NF-κ В P65 of subunit and P50, inhibitor of apoptosis protein Bcl-2 and Bcl-xL, reduce apoptosis and carry out the expression of PROTEIN C aspase-3, hence one can see that, and anoxic is cultivated can improve the curative effect of Transplanted cells.
Claims (3)
1. treatment process that strengthens human medulla ossium mesenchyma stem cell paracrine ability, described method comprises:
To cultivate the anoxic cultivation in low sugar DMEM substratum of stable human marrow mesenchymal stem cell through going down to posterity recovered normal oxygen again and cultivates to be used for Transplanted cells after 20～30 hours; Described anoxic is cultivated: at CO
2Volumetric concentration 5%, N
2Cultivate in the mixed gas of volumetric concentration 95%, described normal oxygen is cultivated and is: at CO
2Cultivate in the mixed gas of volumetric concentration 5%, volume of air concentration 95%.
2. the method for claim 1, it is characterized in that described method is: with the human marrow mesenchymal stem cell in the 3rd generation of subculture after anoxic is cultivated 20～30 hours in low sugar DMEM substratum under 37 ℃, recover 37 ℃ of normal oxygen and cultivate more than 2 hours, the human marrow mesenchymal stem cell after obtaining to handle is used for Transplanted cells.
3. the method for claim 1 is characterized in that described method is as follows: before Transplanted cells, human marrow mesenchymal stem cell is cultivated in anoxic under 37 ℃, in low sugar DMEM substratum
After 24 hours, recover 37 ℃ of normal oxygen and cultivated 24 hours, the human marrow mesenchymal stem cell after obtaining to handle.
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| EP3428272A4 (en) * | 2016-03-10 | 2019-11-27 | KintaroCellsPower Co., Ltd. | Method for manufacturing culture product liquid |
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| US20160095885A1 (en) * | 2014-10-01 | 2016-04-07 | WibiWorks Therapeutics, Inc. | Induction Medium & Methods for Stem Cell Culture & Therapy |
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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 |
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