CN103146646A - Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells - Google Patents
Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a preparation for mobilizing mesenchymal stem cells and a method for separating the mesenchymal stem cells. The preparation for mobilizing the mesenchymal stem cells comprises CoCl2, wherein the CoCl2 is a hypoxia mimetic agent, and the dosage is in a range of 5-20mg/kg. The CoCl2 and AMD3100 are used in a combined manner. The dosage of the AMD3100 is 5mg/kg. According to the method for separating the mesenchymal stem cells by using the preparation, the preparation is used for actuating the mesenchymal stem cells to be mobilized, enter peripheral blood and then be separated. The method comprises the following separation steps of: sampling the periphery blood, separating mononuclear cells by using a lymphocyte separating medium, performing resuspension by using a DMEM (dulbeccos modified eagle medium) containing 20% of fetal bovine serum, inoculating to a culture flask, culturing for 7 days, and changing a culture solution, thus obtaining the mesenchymal stem cells of the peripheral blood on the 10th day. The mesenchymal stem cells of the peripheral blood highly express CD90, CD29 and CD44, do not express CD45 and CD34, and have the capacities of in vitro bone formation, fat formation and cartilage differentiation formation. The preparation is high in MSCs (mesenchymal stem cells) mobilization efficiency, and an effective preparation and an effective method are provided for tissue engineering and regenerative medicine.
Description
Technical field
The invention belongs to biological technical field, relate generally to a kind of preparation and separation method of mobilizing mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) has powerful multiplication capacity and the adult stem cell of multi-lineage potential.Owing to can being divided into the tissues such as bone, cartilage, fat, muscle, tendon, nerve under certain condition, MSCs has become organizational project and the very potential seed cell of regenerative medicine.The MSCs potential applicability in clinical practice is wide: treatment bone, joint, Tendon Defection and damage; Promote neurocyte, myocardial cell, liver cell regeneration; Hematopoiesis support promotes hemopoietic stem cell to implant; Graft versus host disease (GVH disease) (GVHD) after prevention and treatment hematopoietic stem cell transplantation; Immune modulating treatment autoimmune disorder disease etc.
Marrow is the abundantest source of MSCs, present clinical trial with use, from donor or self extract a certain amount of marrow, Isolation and culture is the main path that obtains MSCs.The feasible easy grasp of this method, but also have some shortcomings: cell derived is limited, culturing step is loaded down with trivial details, amplification needs length, the non-humanization of nutrient solution additive, pollution problem, tumorigenesis risk etc. of time to required cell concentration.The foundation of mobilizing method can be avoided the shortcomings of transplantation treatment after vitro culture in the MSCs body, for new way is opened up in the clinical application of MSCs.
Mobilize (Mobilization) to refer to that stem cell is entered the process of peripheral circulation by the fundamental weave site.Stem cell mobilization is very potential methods for the treatment of diseases, has been widely used at present clinical as hematopoietic stem cell mobilization.Mescenchymal stem cell is mobilized and to be referred to use mobilization agent or adopt the mobilization measure to impel in marrow MSCs to discharge and move to peripheral blood.Effectively MSCs mobilizes and can avoid the long shortcoming such as window golden hour of easily missing of infusion of therapeutic exists after vitro culture cell contamination, incubation time.In the MSCs body, mobilization is transplanted incomparable low wound and high efficiency after having vitro culture, and is therefore very meaningful to the research of mobilizing method in the MSCs body.Many investigators also once attempted adopting granulocyte colony-stimulating factor (G-CSF) to mobilize MSCs, but result is disappointing.Still lack at present effective and clinical feasible MSCs mobilization agent.
In early-stage Study, we find that initiatively hypoxia-inducible factor-1 alpha (HIF-1 α) is key factor (the Stem Cells Dev. 2,011 20 (11): 1961-71) during mescenchymal stem cell is mobilized.We have set up the animal model that hypoxia inducible rat MSCs mobilizes, the kinetics of mobilizing by detecting hypoxia inducible MSCs, we find that the short-term anoxic can mobilize rat MSCs, mobilize through identifying the immunophenotype and skeletonization, the one-tenth fat that enter peripheral blood MSCs, become the cartilage ability substantially similar to derived from bone marrow MSCs.In further Mechanism Study, we have confirmed that HIF-1 α plays pivotal role in hypoxia inducible MSCs mobilizes, and HIF-1 α downstream passages CXCL12 α (SDF-1 α) and receptor CXCR 4 thereof participate in the MSCs mobilization.HIF-1 is a heterodimer that includes HIF-1 α and two subunits of HIF-1 β.HIF-1 β is the common subunits of many transcription factors, is constitutive character and expresses in cell.HIF-1 α is adjusting subunit and the active subunits of HIF-1.The physiologically active of HIF-1 depends primarily on the active of HIF-1 α subunit and expresses.The adjusting that HIF-1 alpha expression amount increases is level after protein translation mainly, namely by increasing the stability of albumen, suppresses it and degrades to realize.When cell was in normal oxygen condition, under the effect of prolyl hydroxylase (prolyl hydroxylase, PHD), in the HIF-1 alpha molecule, 402 and 564 s' proline residue was by hydroxylation.By the HIF-1 α of hydroxylation can with tumor suppressor protein von Hippel Lindau (VHL) combination, the latter can be identified the ubiquitin degraded occurs.In this degradation pathway, PHD is the enzyme that plays a crucial role.PHD belongs to Fe
2+, the dioxygenase superfamily that relies on of 2-oxoglutaric acid, be the rate-limiting enzyme of HIF-1 α DeR.The PHD inhibitor can be stablized the expression of HIF-1 α.Research is found, iron chelating agent cobalt chloride (CoCl
2) can suppress the activity of PHD, and suppress the hydroxylation of HIF-1 α, reduce the degraded of HIF-1 α under normal oxygen condition, be called as " anoxic simulant ".
SDF-1/ CXCR4 axle plays an important role in mobilizing in the stem cell body.Stroma cell derivative factor (SDF-1) is the CXC type chemokine that is produced by marrow stromal cell, the CXC cytokine receptor 4(CXCR4 of family) be unique acceptor of SDF-1 known today.Studies confirm that, SDF-1 is subjected to transcription factor HIF-1 direct regulation and control at endotheliocyte.AMD3100 is the small molecular antagonists of SDF-1 α/CXCR4 axle, can effectively block the interaction of SDF-1 α and CXCR4 and CXCR4 is not had agonism.
Although according to bibliographical information and our early-stage Study result as can be known HIF-1/SDF-1 α-CXCR4 axle play a crucial role in MSCs mobilizes, still lack at present a kind of effective MSCs mobilization preparation, still lack feasible MSCs mobilizing method.
Summary of the invention
In order to overcome the deficiencies in the prior art, the inventor finds anoxic simulant cobalt chloride (CoCl first
2) can induce rat MSCs to mobilize to peripheral blood, and CoCl
2The inhibitor AMD3100 of associating CXCR4 can significantly improve mobilization efficient.The invention provides a kind of preparation and separation method of mobilizing mescenchymal stem cell.
A kind of preparation for the mescenchymal stem cell mobilization comprises CoCl
2
Described CoCl
2Be the anoxic simulant, dosage range 5-20mg/kg.
Described CoCl
2Unite use with AMD3100.
Described AMD3100 dosage is 5 mg/kg.
A kind of method of utilizing described preparation separating mesenchymal stem cell is utilized described preparation to impel the mescenchymal stem cell mobilization to enter peripheral blood and is then separated.
Described separating step is as follows: take peripheral blood, separate mononuclearcell with lymphocyte separation medium, the DMEM substratum of 20% foetal calf serum is resuspended with containing, and is seeded to culturing bottle, cultivates and changes liquid after 7 days, obtains the peripheral blood mescenchymal stem cell on the 10th day.
Described peripheral blood mescenchymal stem cell high expression level CD90, CD29, CD44 do not express CD45, CD34; Have external skeletonization, become fat, become the cartilage differentiation ability.
Beneficial effect of the present invention: have higher MSCs and mobilize efficient, for organizational project and regenerative medicine etc. provides a kind of effective preparation and method.
Description of drawings
Fig. 1 is cobalt chloride (CoCl
2) induce rat MSCs to mobilize to peripheral blood, and CoCl
2The inhibitor AMD3100 of associating CXCR4 can significantly improve mobilization efficient.
Fig. 1 A: each treatment group rat peripheral blood CFU-F number.
In figure, CFU-F: fibroblast sample colony.NS group: physiological saline treatment group.CoCl
2Group: CoCl2 10mg/kg/d * 7d treatment group.The AMD:AMD3100 treatment group gave respective volume physiological saline in namely the 1st ~ 7 day, gave AMD3100 5mg/kg on the 8th day.AMD+ CoCl
2: AMD3100 unites CoCl
2Group first gives CoCl2 10mg/kg/d * 7d, the 8th day abdominal injection AMD3100 5mg/kg.* represent to compare with the NS group P＜0.05.* represents to compare with the AMD3100 group, P＜0.05.# represents CoCl
2Group is compared, P＜0.05.
Figure 1B: each treatment group rat peripheral blood CD45
-CD90
+The ratio schematic diagram of cell.
In figure, NS group: physiological saline treatment group.CoCl
2Group: CoCl
210mg/kg/d * 7d treatment group.The AMD:AMD3100 treatment group gave respective volume physiological saline in namely the 1st ~ 7 day, gave AMD3100 5mg/kg on the 8th day.AMD+ CoCl
2: AMD3100 unites CoCl
2Group first gives CoCl
210mg/kg/d * 7d, the 8th day abdominal injection AMD3100 5mg/kg.
Fig. 2 is the immunophenotype of mobilized peripheral blood source MSCs.
In figure, mobilized peripheral blood source MSCs high expression level CD90, CD29, CD44, and do not express CD45, CD34.
Fig. 3 is that mobilized peripheral blood source MSCs can be to skeletonization, one-tenth fat, one-tenth cartilage differentiation.
The 14th day light microscopic photo (100 *) of Fig. 3 A:PB-MSCs Osteoinductive differentiation.
The 14th day Von Konssa coloration result figure (100 *) of Fig. 3 B:PB-MSCs Osteoinductive differentiation.
Fig. 3 C:PB-MSCs becomes fat to induce the 21st day light microscopic photo (200 *) of differentiation.
Fig. 3 D:PB-MSCs becomes fat to induce the 21st day oil red O stain result schematic diagram (200 *) of differentiation.
Fig. 3 E:PB-MSCs becomes the 21st day light microscopic photo of chondrocyte induction differentiation.
Fig. 3 F:PB-MSCs becomes the 21st day Toluidine blue staining coloration result of chondrocyte induction differentiation.（400×）。
Embodiment
The present invention is described further below in conjunction with embodiment and accompanying drawing.Concrete MSCs mobilizes technological method as follows:
1. CoCl2 pulvis (be purchased from traditional Chinese medicines group chemical pharmacy company limited) is made into physiological saline the solution that concentration is 4mg/ml, AMD3100(is purchased from Sigma company) be made into the solution of 1mg/ml with physiological saline.Take rat as experimental subjects, CoCl
2Mobilization group: give rats by intraperitoneal injection CoCl every day
2Solution 10mg/kg; CoCl
2Associating AMD3100 mobilization group: first give CoCl
210mg/kg/d * 7d, the 8th day abdominal injection AMD3100 5mg/kg; Set up corresponding control group.
2. gather and respectively organize the rat peripheral blood, adopt the fibroblast-like cells colony to form two kinds of methods of culture method (CFU-F method) and Flow cytometry and detect the quantity of respectively organizing peripheral blood MSCs, confirm CoCl
2And CoCl
2The mobilized effects of associating AMD3100 to MSCs.
3. the peripheral blood of mobilizing out is become the cultivation of going down to posterity of fiber-like colony, by the expression of its surface markers of Flow cytometry CD44, CD29, CD90, CD34, CD45, identify immunophenotype.
4. the peripheral blood of mobilizing out is become the cultivation of going down to posterity of fiber-like colony, become fat, skeletonization, the differentiation of one-tenth chondrocyte induction, identify the Multidirectional Differentiation ability of mobilized peripheral blood source MSCs.
1. reagent preparation
(1) CoCl
2: take 400mg CoCl
2Pulvis adds 10ml physiological saline, is made into the storage liquid of 40mg/ml, packing after 0.22 μ m filter filters ,-20 ℃ of preservations.During animals administer, storage liquid is transferred to 4mg/ml with 10 times of normal saline dilutions with concentration.
(2) the AMD3100:5g pulvis adds 5ml physiological saline, is made into the solution of 1mg/ml, and 0.22 μ m filter filters packing ,-20 ℃ of preservations.4 times of normal saline dilutions are used for the mouse administration to 1.25mg/ml.
2. animal grouping
(1) for detecting CoCl
2And CoCl
2The mobilized effects of associating AMD3100 to MSCs is divided into 4 groups with rat, 5 every group, is respectively: 1. the physiological saline group is the NS group: give rats by intraperitoneal injection physiological saline every day (with CoCl
2Same volume), totally 7 days; 2. CoCl
27d group: give abdominal injection CoCl every day
2Solution 10mg/kg, totally 7 days; 3. AMD3100 organizes: gave respective volume physiological saline on the 1st ~ 7 day, the 8th day abdominal injection AMD3100 5mg/kg, the punctual peripheral blood that gathers after 1 hour; 4. CoCl
2Associating AMD3100 group: first give CoCl
210mg/kg/d * 7d, the 8th day abdominal injection AMD3100 5mg/kg, the punctual peripheral blood that gathers after 1 hour.
3. the rat peripheral blood cells is extracted
Each treatment group arrives respective handling after the time, gets respectively peripheral blood mononuclear cell and carries out CFU-F method detection MSCs quantity and Flow cytometry CD45
-CD90
+The cell mass ratio.
4% Chloral Hydrate 10ml/kg intraperitoneal injection of anesthesia SD rat is got blood 8 ~ 10ml with the 10ml syringe from postcaval vein, and lymphocyte separation medium separates mononuclearcell (MNCs), draws the tunica albuginea layer, after PBS washs 3 times, and counting.
4. CFU-F method
Contained MNCs in the 6ml peripheral blood is resuspended with the LG-DMEM substratum that 5ml contains 20% FBS, 1% mycillin, and being inoculated in floorage is 12.5cm
2The plastic culture bottle in, be placed in 37 ℃ and contain 5%CO
2Incubator is cultivated.Cultivate and change liquid after 7 days, observe under inverted microscope, counting peripheral blood CFU-Fs in the time of the 10th day.To be inoblast sample form, growth intensive, the colony of 50 cells is designated as CFU-F.
The adherent i.e. amplification of MSCs forms a CFU-F, and based on this theory, the CFU-F culture method has become the classical way that detects peripheral blood MSCs quantity.CFU-F method detected result is found: give CoCl
210mg/kg/d * 7d, rat peripheral blood CFU-Fs quantity significantly increases (2.37 ± 0.19 vs.1.27 ± 0.08 CFU-Fs/ml, P＜0.05) than control group.Compare with the physiological saline control group, in AMD3100 group peripheral blood, CFU-Fs quantity there is no obvious increase.Compare with alone CoCl2 group, CoCl2 associating AMD3100 can significantly increase MSCs and mobilize efficient (3.83 ± 0.32 vs.2.37 ± 0.19 CFU-Fs/ml, P＜0.05), referring to Figure 1A.Applicant research is found: CoCl
210mg/kg gives can increase in 7 days the quantity of rat peripheral blood MSCs.CoCl
2Associating CXCR4 inhibitor AMD3100 can increase MSCs and mobilize efficient.
5. CD45 in Flow cytometry rat peripheral blood and marrow
-CD90
+The cell mass ratio
After counting, peripheral blood mononuclear cells of rats is adjusted cell concn to 10 with PBS
7/ ml.Add 100 μ l and adjust rear cell to 1.5ml EP pipe, monochromatic and double-colored homotype control group are set simultaneously.The every pipe 5 μ l of streaming antibody staining: CD90-PE, the every pipe 2 μ l of CD45-FITC.After lucifuge incubated at room 30min, PBS washing 2 times adds the streaming pipe, upper machine testing after resuspended with 400 μ lPBS.
Flow cytometry is found: CoCl
210mg/kg gives can make CD45 in peripheral blood in 7 days
-CD90
+The cell mass ratio raises, referring to Figure 1B; And sum increases (2.93 ± 0.40 vs. 1.46 ± 0.16 * 104/ml P＜0.05), sees table.CoCl
2After the AMD3100 coupling, CD45 in the rat peripheral blood
-CD90
+The cell mass ratio is than CoCl
2Group further raises, referring to Figure 1B; Quantity also obviously increases (4.71 ± 0.11 vs. 2.93 ± 0.40 * 104/ml, P＜0.05), sees the following form 1.This result has also further confirmed CoCl
210mg/kg gives can induce in 7 days rat MSCs to mobilize.CoCl
2Associating CXCR4 inhibitor AMD3100 can increase MSCs and mobilize efficient.
Table 1 each treatment group peripheral blood mononuclear cells of rats quantity and CD45
-
CD90
+
Cell mass changes
*Expression is compared with the NS group, P＜0.01;
﹠amp;Expression is compared with the AMD3100 group, P＜0.01;
△Expression and CoCl
2Group compares, P＜0.05.
6. flow cytometry peripheral blood and marrow CFU-Fs Immunophenotyping
Peripheral blood or derived from bone marrow CFU-Fs are expanded to P2 generation, 0.25% pancreas enzyme-EDTA digestion, and the centrifugal 6min of 1000r/min, after the PBS washed twice, counting.Regulate concentration to 5 * 10
5Cell/100 μ l are divided into 7 pipes.2 pipe Control add respectively FITC and the contrast of PE homotype, and other 5 pipes add respectively CD44-FITC 2 μ l, CD29-PE 2 μ l, CD90-PE 5 μ l, CD45-FITC 2 μ l, CD34-PE 5 μ l.After temperature is hatched 30min, PBS washs 2 times.With the 400 resuspended streaming pipes that are placed in of μ l PBS, upper machine testing, Cell-Quest software analysis result.
Flow cytometry mobilized peripheral blood source MSCs(PB-MSCs) immunophenotype, result shows that it is mark CD45(0.20 ± 0.10% that PB-MSCs does not express hematopoiesis), CD34(0.37 ± 0.38%), and positive expression CD44 (93.80 ± 2.62%), CD29(99.67 ± 0.42%) and CD90(98.50 ± 2.18%), referring to Fig. 2.PB-MSCs is similar to the expression of derived from bone marrow MSCs surface antigen, through identifying, meets the MSCs immunophenotype.
7. Current concepts in vitro osteogenesis of bone is identified
Peripheral blood or derived from bone marrow CFU-Fs are expanded to P2 generation, 0.25% pancreas enzyme-EDTA digestion.With cell with 10,000/cm
2Be inoculated in 6 orifice plates every group of 3 holes.Be placed in 37 ℃ and contain 5%CO
2Incubator was cultivated after 2 days, changed nutrient solution, added Osteoblast Differentiation induced liquid (LG-DMEM of 10% FBS contains 0.1 μ M dexamethasone, 10mM β-phospho-glycerol, 50 μ M vitamins Cs), changed liquid, and cultivated observation of cell form under inverted microscope 14 days in every 3 days.
The Osteoblast Differentiation result is identified in Von Kossa dyeing: blot nutrient solution in culture plate, fix 15 min with 4% paraformaldehyde, distilled water rinsing 2 times, after naturally drying, every hole adds 2% silver nitrate solution dyeing 5min of the new preparation of 2ml, after deionized water fully washs, be placed in the lower 15 ~ 30min of ultraviolet ray, distilled water rinsing 2 times, 5% Sulfothiorine is processed 2min, distilled water rinses 2 times, and Microscopic observation is taken pictures.
Mobilized peripheral blood source MSCs(PB-MSCs) inducing differentiation the 14th day, more calcium salt secretion namely appears in cell surface, and referring to Fig. 3 A, Von Konssa dyeing all is positive, referring to Fig. 3 B.These results suggest that PB-MSCs has the Osteoblast Differentiation ability.
8. become the fat directed differentiation to identify
Derived from peripheral blood CFU-Fs is expanded to P2 generation, 0.25% pancreas enzyme-EDTA digestion.With cell with 20,000/cm
2Be inoculated in 6 orifice plates every group of 3 holes.Be placed in 37 ℃ and contain 5%CO
2Incubator is cultivated, and when the nearly 90%-100% of cell merges, adds into fat induction liquid (containing 45ml LG-DMEM substratum and 5ml fat differentiation additive), changes liquid, and cultivates observation of cell form under inverted microscope 21 days in every 3 days.
Oil red O stain is identified into the fat differentiated result: blot nutrient solution in culture plate, PBS washs 1 ~ 2 time gently, and 4% paraformaldehyde is 15min fixedly.After 60% isopropyl alcohol, every hole adds freshly prepared oil red O dye liquor 2ml, and dyeing 10min uses 60% isopropyl alcohol 2 times after blotting dye liquor again, and the deionization distilled water rinses 2 times, and Microscopic observation is taken pictures.
Through inducing differentiation 21 days, can be observed mobilized peripheral blood source MSCs(PB-MSCs) all occur the high refractivity fat that cluster distributes in endochylema and drip, referring to Fig. 3 C, be orange through oil red O stain, referring to Fig. 3 D.
9. become the cartilage directed differentiation to identify
Peripheral blood or derived from bone marrow CFU-Fs are expanded to P2 generation, 0.25% pancreas enzyme-EDTA digestion.The PBS washed twice is with cartilage differentiation liquid (serum-free HG-DMEM contain 1 * contain Sodium.alpha.-ketopropionate Regular Insulin-Transferrins,iron complexes-selenium, 0.1 μ M dexamethasone, 200 μ M vitamins Cs) re-suspended cell, 5 * 10
5/ pipe is inoculated in the 15ml centrifuge tube, every pipe liquor capacity 0.5ml, 500g 6min is centrifugal, adds at last TGF-β 1(10ng/ml), TGF-β 3(10ng/ml).Be placed in 37 ℃ and contain 5%CO
2Incubator changed liquid once, and added fresh cytokine at every turn in every 3 days.Cultivate and observed the formation of chondrocyte's agglomerate on the 21st day.
Toluidine blue staining is identified into the cartilage differentiation result: the cartilage differentiation cell mass takes out tissue block with the fixing 24h of 4% paraformaldehyde, wraps with the silk stocking pouch, and silk thread fastens.Tissue dewatering is put respectively and is organized agglomerate in following solution: 50% ethanol 1h, 70% ethanol 15min, 80% ethanol 15min, 90% ethanol 15min and dehydrated alcohol 40min.Transparency of organization is put respectively agglomerate in dimethylbenzene and alcohol geometric ratio mixed solution 20min and pure dimethylbenzene 20min.Waxdip, embedding, tissue slice (5mm/ sheet) then, the tissue slice oven dry is spent the night.Then dewax to water, put respectively and organize agglomerate in following solution: dimethylbenzene 30min, dimethylbenzene and alcohol geometric ratio mixed solution 5min, dehydrated alcohol 10min, 95% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min and 50% ethanol 5min, last deionization washing 5min.1% toluidine blue dye liquor dyeing 10 ~ 15 minutes, flowing water rinses, and after conventional mounting, Microscopic observation is taken pictures.
We adopt external high-density micelle culture method and Toluidine blue staining to the mobilized peripheral blood MSCs(PB-MSCs that originates) one-tenth cartilage differentiation potential assess.Found that: PB-MSCs was through external one-tenth chondrocyte induction differentiation 21 days, and cell mass breaks up front obvious increase, referring to Fig. 3 E.After the cell micelle carried out the processing such as embedding, section, Toluidine blue staining detected the expression of the acid glycosaminoglycan of chondrocyte's characteristic secretory product.Coloration result shows: it is painted that the rear cell of PB-MSCs differentiation all is metachromatism, and namely extracellular matrix is dyed to purple, and nucleus is mazarine, referring to Fig. 3 F.Above result shows: PB-MSCs has into cartilage differentiation potential.
Comprehensive the above results can draw to draw a conclusion: (1) CoCl
210mg/kg * 7 day can induce rat MSCs to mobilize; (2) CoCl
210mg/kg * 7 day associating AMD 3100 5mg/kg * 7 times can increase MSCs and mobilize efficient; (3) to have the immunophenotype similar to bone marrow MSCs be high expression level CD90, CD29, CD44 to mobilized peripheral blood source MSCs, and do not express CD45, CD34.Mobilized peripheral blood source MSCs have with the similar Multidirectional Differentiation ability of bone marrow MSCs can be to skeletonization, becomes fat, become chondrocyte induction to break up.
For detecting CoCl
2To the effective concentration that MSCs mobilizes, we adopt the CFU-F method to study and give rats by intraperitoneal injection CoCl every day
2Solution 5mg/kg, 20mg/kg totally 7 days, the quantity of rat peripheral blood CFU-Fs, result shows: CoCl
25mg/kg * 7 day, CoCl
220mg/kg * 7 day all have mobilized effects to rat MSCs, and peripheral blood CFU-F all increases than the physiological saline control group, but mobilize efficient lower than CoCl
210mg/kg * 7 day group.Concrete outcome is: CoCl
25mg/kg * 7 day group vs. physiological saline group (2.02 ± 0.12 vs.1.27 ± 0.08 CFU-Fs/ml, P＜0.05), CoCl
220mg/kg * 7 day group vs. physiological saline group (1.68 ± 0.08 vs.1.27 ± 0.08 CFU-Fs/ml, P＜0.05).
Claims (7)
1. one kind is used for the preparation that mescenchymal stem cell is mobilized, and it is characterized in that, it comprises CoCl
2
2. preparation according to claim 1, is characterized in that, described CoCl
2Be the anoxic simulant, dosage range 5-20mg/kg.
3. preparation according to claim 1, is characterized in that, described CoCl
2Unite use with AMD3100.
4. preparation according to claim 3, is characterized in that, described AMD3100 dosage is 5 mg/kg.
5. a method of utilizing the described preparation separating mesenchymal of claim 1 or 2 stem cell, is characterized in that, utilizes described preparation to impel the mescenchymal stem cell mobilization to enter peripheral blood and then separate.
6. method according to claim 5, it is characterized in that, described separating step is as follows: take peripheral blood, separate mononuclearcell with lymphocyte separation medium, resuspended with the DMEM substratum that contains 20% foetal calf serum, be seeded to culturing bottle, cultivate and change liquid after 7 days, obtained the peripheral blood mescenchymal stem cell on the 10th day.
7. method according to claim 6, is characterized in that, described peripheral blood mescenchymal stem cell high expression level CD90, CD29, CD44 do not express CD45, CD34, has external skeletonization, becomes fat, becomes the cartilage differentiation ability.
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| CN106264688A (en) * | 2016-08-01 | 2017-01-04 | 北京臻惠康生物科技有限公司 | It is set with for Endometrium collection |
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| CN108135941A (en) * | 2015-08-19 | 2018-06-08 | 儿研所儿童医学中心 | For treating the composition of graft versus host disease(GVH disease) and method |
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| CN102864123A (en) * | 2011-07-06 | 2013-01-09 | 北京大学第三医院 | Acquisition method of peripheral blood mesenchymal stem cells and application thereof |
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| WO2009074807A2 (en) * | 2007-12-12 | 2009-06-18 | Imperial Innovations Limited | Methods |
| CN102864123A (en) * | 2011-07-06 | 2013-01-09 | 北京大学第三医院 | Acquisition method of peripheral blood mesenchymal stem cells and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108135941A (en) * | 2015-08-19 | 2018-06-08 | 儿研所儿童医学中心 | For treating the composition of graft versus host disease(GVH disease) and method |
| CN106264688A (en) * | 2016-08-01 | 2017-01-04 | 北京臻惠康生物科技有限公司 | It is set with for Endometrium collection |
| WO2018023827A1 (en) * | 2016-08-01 | 2018-02-08 | 北京臻惠康生物科技有限公司 | Protective fluid for endometrial stem cell |
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