A kind of activation method based on colony-stimulating factor 1 to stem cellTechnical field
The present invention relates to the activation field of stem cell, particularly relate to a kind of activation method based on colony-stimulating factor 1 to stem cell.
Background technology
One life entity germinates from a germ cell, go through embryonic stem cell to safeguard to the formation of histoorgan and the ripe individual day after tomorrow, after forming ripe individuality, the quantity of internal stem cell is considerably less, in adult tissue, only the cell of ten thousand/left and right is stem cell, these minimal amount of stem cell will have safeguarded vital effect in the day after tomorrow of life just, except neurocyte lifelong dead except, most adult cell life-spans only have 100-300 days, but Living organism to maintain the several years and even go up life-span of a century and depend on the propagation of stem cell, activation and the potential broken up to other adult cell.
Under normal circumstances, being in the stem cell under quiescent condition, its speed migrating, go back to the nest, breed and being divided into other functioning cells is generally relatively slower.And prior art still can not better solve the propagation of stem cell, activation and break up problem more slowly to other adult cell.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of activation method based on colony-stimulating factor 1 to stem cell, it is intended to solve prior art and can not better solve the propagation of stem cell, activation and break up problem more slowly to other adult cell.
Technical scheme is as follows:
A kind of activation method based on colony-stimulating factor 1 to stem cell, wherein, adopts colony-stimulating factor 1 to stem cell mobilization, propagation and activation.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, described stem cell is hematopoietic stem cell, the step of hematopoietic stem cell mobilization is included by CSF1: collect mice, CSF1 and G-CSF is combined subcutaneous injection, and after three days, tail vein blood is in the anticoagulant tube being added with anticoagulant heparin agent, the quantity of hematopoietic stem cell in peripheral blood after adopting coupling to have the CD34 antibody spike of FITC to mobilize, selected by flow cytometry apoptosis detects CD34 positive cell quantity.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, the step of hemopoietic stem cell proliferation is included by CSF1: the hematopoietic stem cell obtained through CD34 sorting adopts hematopoietic stem cell serum-free medium to cultivate, cultivate after hematopoietic stem cell inoculated and cultured bag, often crossing a few hours gentleness wave and culture bag, whole incubation culture medium is additionally added with CSF1 always；Then estimate hematopoietic stem cell density under microscope, and measure hematopoietic stem cell gross density with mtt assay.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, described stem cell is mescenchymal stem cell, the step that mescenchymal stem cell is bred by CSF1 includes: select between fat, bone marrow and the mescenchymal stem cell in three kinds of histoorgan sources of umbilical cord does Cell culture invitro, mescenchymal stem cell inoculum density controls between 10-20%, culture dish cell attachment material room temperature treatment 1-2 hour or 3-4 DEG C of incubated overnight before inoculation, add culture medium culturing after inoculation；Then a mescenchymal stem cell density was surveyed every 10-12 hour by mtt assay.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, after CSF1 activation, the step of Derived from Mesenchymal Stem Cells lipoblast includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then following steps are adopted to detect: 1) first prepare division culture medium A and B, wherein division culture medium component A includes: basal medium, hyclone, penicillin streptomycin, glutamine, insulin, 3-isobutyl-1-methylxanthine, rosiglitazone, dexamethasone and indomethacin；Division culture medium B component includes: basal medium, hyclone, penicillin streptomycin, glutamine and insulin, prepare complete after mix homogeneously, put refrigerator and keep in Dark Place standby；2) inoculating cell/hole in six orifice plates, continues to cultivate cell；3) growing to after 100% degree of converging until cell, suck old culture medium, every hole adds division culture medium A；4) sucking division culture medium A after three days, every hole adds division culture medium B；5) after 24 hours, sucking division culture medium B, every hole adds division culture medium A；6) rotation adds division culture medium A and B, repeats d-e step 4-5 time；7) it is eventually adding division culture medium B to cultivate 7 days；8) after induction terminates, culture medium is sucked, fixing 30-35 minute of the paraformaldehyde added in every hole；9) 2-3 time is washed with PBS；10) in every hole, add oil red O stain liquid, dye 30-35 minute；11) wash 2-3 time with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics adipose cell.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, after CSF1 activation, the osteoblastic step of Derived from Mesenchymal Stem Cells includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then adopts following steps to detect: 1) first prepare division culture medium, including basal medium, hyclone, penicillin streptomycin, glutamine, vitamin C, β-phosphoglycerol and dexamethasone, mix homogeneously, put refrigerator and keep in Dark Place standby；2) by the gelatin pretreatment of six orifice plates, process with gelatin before inoculating cell, or after addition gelatin, six orifice plates are sealed up, put refrigerator standby；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell in six orifice plates of gelatin pretreatment；Then cultivate in incubator；4) suck culture medium after having cultivated, change Osteoinductive differentiation culture medium；5) an Osteoinductive differentiation culture medium within every 3 days, is changed, induction 2-3 week；6) after induction terminates, suck culture medium, in every hole, add paraformaldehyde fixing 30-35 minute；7) 2-3 time is washed with PBS；8) in every hole, add alizarin red S dyeing liquor, dye 5-10 minute；9) wash 2-3 time with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics osteocyte.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, after CSF1 activation, the step of Derived from Mesenchymal Stem Cells chondroblast includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then adopts following steps to detect: 1) first prepare division culture medium, including basal medium, dexamethasone, ascorbic acid, Insulin-Transferrin-selenium additive, Sodium Pyruvate, proline and TGF-β 3, mix homogeneously, put refrigerator and keep in Dark Place standby；2) by the gelatin pretreatment of six orifice plates；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell in six orifice plates of gelatin pretreatment；4) add into cartilage differentiation culture medium, within 2-3 days, change a not good liquor；5) after cell differentiation 3-4 week, suck culture medium, fix 30-35 minute with paraformaldehyde；6) 2-3 time is washed with PBS；7) in every hole, add A Li Xinlan dyeing liquor, dye 30-35 minute；8) wash 3-4 time with PBS, be subsequently placed under inverted microscope and observe take pictures, the ratio of statistics chondrocyte.
The described activation method based on colony-stimulating factor 1 to stem cell, wherein, CSF1 activation after hematopoietic stem cell the test of the impact of tumor model mouse survival rate is included step: by acute B lymphocyte series leukemia cell line with containing penicillin, streptomycin, inactivation new-born calf serum composition RPMI-1640 complete culture solution, containing CO2Incubator in cultivate cell；With aseptic PBS liquid, concentration of cyclophosphamide is adjusted 10-12mg/mL, to cyclophosphamide described in the lumbar injection of mice, injection 2 days continuously；After 24h, collect and be in the Nalm-6 cell of exponential phase and be centrifuged, be suspended in aseptic PBS liquid, adjust Nalm-6 cell density to 2.5 × 107Individual/mL, carries out tail vein injection to mice, prepares leukemia animal model, and carries out radiotherapy process；Radiotherapy is assisted after processing and is treated with hematopoietic stem cell, adds up the survival rate of mice after 4-5 month.
Beneficial effect: the present invention utilizes CSF1 to stem cell mobilization, propagation and activation, CSF1 is possible not only to promote the in-vitro multiplication of stem cell, may additionally facilitate the mobilization of internal stem cell, propagation and activation, be expected to play a significant role in cell cultivation, disease treatment and anti-ageing healthcare.
Accompanying drawing explanation
Fig. 1 is the CSF1 mobilized effects schematic diagram to CD34 positive cell.
Fig. 2 is the CSF1 proliferation function schematic diagram to hematopoietic stem cell.
Fig. 3 is the CSF1 proliferation function schematic diagram to mescenchymal stem cell.
Fig. 4 is the one-tenth fat ability schematic diagram of mescenchymal stem cell after CSF1 activation.
Fig. 5 is the osteogenic ability schematic diagram of mescenchymal stem cell after CSF1 activation.
Fig. 6 is the one-tenth cartilage ability schematic diagram of mescenchymal stem cell after CSF1 activation.
Fig. 7 is the schematic diagram that after CSF1 activation, tumor model mice survival rate is affected by hematopoietic stem cell.
Detailed description of the invention
The present invention provides a kind of activation method based on colony-stimulating factor 1 to stem cell, and for making the purpose of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
The present invention provides a kind of activation method based on colony-stimulating factor 1 to stem cell, wherein, adopts colony-stimulating factor 1 to stem cell mobilization, propagation and activation.Under normal circumstances, being in the stem cell under quiescent condition, its speed migrating, go back to the nest, breed and being divided into other functioning cells is generally relatively slower.Such as, hematopoietic stem cell is primarily present in bone marrow under normal physiological conditions, and content is very micro-in peripheral blood, under granulocyte colony-stimulating factor (G-CSF) or emergency condition, a large amount of hematopoietic stem cell are mobilized, thus being discharged in peripheral blood, and start hematopoietic stem cell to erythrocyte, immunocyte and hematoblastic atomization.The present invention adopts the colony-stimulating factor 1 (CSF1) can the mobilization of stem cell in effective stimulus body, propagation and activation process.It addition, the present invention adopts CSF1 and G-CSF Combined Treatment mouse model, can effectively strengthen hematopoietic stem cell mobilization efficiency.
The present invention adopts CSF1 as additive, cultivates the propagation that can promote mescenchymal stem cell in vitro, but does not affect the differentiation potential of this kind of stem cell, and this kind of stem cell includes fat stem cell, mesenchymal stem cells MSCs, umbilical cord mesenchymal stem cells.
The present invention adopts CSF1 as activator, acts on that suckling is biological or its corresponding stem cell, including but do not limit to the internal stem cell mobilization in people or mice source or the propagation of ex vivo stem cell.
Preferably, described stem cell is hematopoietic stem cell, the step of hematopoietic stem cell mobilization is included by CSF1: collect mice, CSF1 and G-CSF is combined subcutaneous injection, after three days, tail vein blood is in the anticoagulant tube being added with anticoagulant heparin agent, and the quantity of hematopoietic stem cell in peripheral blood after adopting coupling to have the CD34 antibody spike of FITC to mobilize, selected by flow cytometry apoptosis detects CD34 positive cell quantity.
Preferably, the step of hemopoietic stem cell proliferation is included by CSF1: the hematopoietic stem cell obtained through CD34 sorting adopts hematopoietic stem cell serum-free medium to cultivate, cultivate after hematopoietic stem cell inoculated and cultured bag, often crossing a few hours gentleness wave and culture bag, whole incubation culture medium is additionally added with CSF1 always；Then estimate hematopoietic stem cell density under microscope, and measure hematopoietic stem cell gross density with mtt assay.
Preferably, described stem cell is mescenchymal stem cell, the step that mescenchymal stem cell is bred by CSF1 includes: select between fat, bone marrow and the mescenchymal stem cell in three kinds of histoorgan sources of umbilical cord does Cell culture invitro, mescenchymal stem cell inoculum density controls between 10-20%, culture dish cell attachment material room temperature treatment 1-2 hour or 3-4 DEG C of incubated overnight before inoculation, add culture medium culturing after inoculation；Then a mescenchymal stem cell density was surveyed every 10-12 hour by mtt assay.
Preferably, after CSF1 activation, the step of Derived from Mesenchymal Stem Cells lipoblast includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then following steps are adopted to detect: 1) first prepare division culture medium A and B, wherein division culture medium component A includes: basal medium, hyclone, penicillin streptomycin, glutamine, insulin, 3-isobutyl-1-methylxanthine, rosiglitazone, dexamethasone and indomethacin；Division culture medium B component includes: basal medium, hyclone, penicillin streptomycin, glutamine and insulin, prepare complete after mix homogeneously, put refrigerator and keep in Dark Place standby；2) inoculating cell/hole in six orifice plates, continues to cultivate cell；3) growing to after 100% degree of converging until cell, suck old culture medium, every hole adds division culture medium A；4) sucking division culture medium A after three days, every hole adds division culture medium B；5) after 24 hours, sucking division culture medium B, every hole adds division culture medium A；6) rotation adds division culture medium A and B, repeats d-e step 4-5 time；7) it is eventually adding division culture medium B to cultivate 7 days；8) after induction terminates, culture medium is sucked, fixing 30-35 minute of the paraformaldehyde added in every hole；9) 2-3 time is washed with PBS；10) in every hole, add oil red O stain liquid, dye 30-35 minute；11) wash 2-3 time with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics adipose cell.
Preferably, after CSF1 activation, the osteoblastic step of Derived from Mesenchymal Stem Cells includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then following steps are adopted to detect: 1) first prepare division culture medium, including basal medium, hyclone, penicillin streptomycin, glutamine, vitamin C, β-phosphoglycerol and dexamethasone, mix homogeneously, puts refrigerator and keeps in Dark Place standby；2) by the gelatin pretreatment of six orifice plates, process with gelatin before inoculating cell, or after addition gelatin, six orifice plates are sealed up, put refrigerator standby；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell in six orifice plates of gelatin pretreatment；Then cultivate in incubator；5) suck culture medium after having cultivated, change Osteoinductive differentiation culture medium；6) an Osteoinductive differentiation culture medium within every 3 days, is changed, induction 2-3 week；7) after induction terminates, suck culture medium, in every hole, add paraformaldehyde fixing 30-35 minute；8) 2-3 time is washed with PBS；9) in every hole, add alizarin red S dyeing liquor, dye 5-10 minute；9) wash 2-3 time with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics osteocyte.
Preferably, after CSF1 activation, the step of Derived from Mesenchymal Stem Cells chondroblast includes: cultivate the mescenchymal stem cell of acquisition after CSF1 activation, then following steps are adopted to detect: 1) first prepare division culture medium, including basal medium, dexamethasone, ascorbic acid, Insulin-Transferrin-selenium additive, Sodium Pyruvate, proline and TGF-β 3, mix homogeneously, puts refrigerator and keeps in Dark Place standby；2) by the gelatin pretreatment of six orifice plates；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell in six orifice plates of gelatin pretreatment；4) add into cartilage differentiation culture medium, within 2-3 days, change a not good liquor；5) after cell differentiation 3-4 week, suck culture medium, fix 30-35 minute with paraformaldehyde；6) 2-3 time is washed with PBS；7) in every hole, add A Li Xinlan dyeing liquor, dye 30-35 minute；8) wash 3-4 time with PBS, be subsequently placed under inverted microscope and observe take pictures, the ratio of statistics chondrocyte.
Preferably, CSF1 activation after hematopoietic stem cell the test of the impact of tumor model mouse survival rate is included step: by acute B lymphocyte series leukemia cell line with containing penicillin, streptomycin, inactivation new-born calf serum composition RPMI-1640 complete culture solution, containing CO2Incubator in cultivate cell；With aseptic PBS liquid, concentration of cyclophosphamide is adjusted 10-12mg/mL, to cyclophosphamide described in the lumbar injection of mice, injection 2 days continuously；After 24h, collect and be in the Nalm-6 cell of exponential phase and be centrifuged, be suspended in aseptic PBS liquid, adjust Nalm-6 cell density to 2.5 × 107Individual/mL, carries out tail vein injection to mice, prepares leukemia animal model, and carries out radiotherapy process；Radiotherapy is assisted after processing and is treated with hematopoietic stem cell, adds up the survival rate of mice after 4-5 month.
Describe the present invention below by specific embodiment.
Embodiment 1,
The CSF1 mobilized effects to hematopoietic stem cell
Collecting the mice at 5 monthly ages, every gram of body weight combines subcutaneous injection by 1 microgram CSF1 and 1 microgram G-CSF, and positive control every gram body weight combines subcutaneous injection by 1 microgram G-CSF, and negative control injects isopyknic normal saline.After three days, tail vein blood is in the anticoagulant tube being added with anticoagulant heparin agent, and the quantity of hematopoietic stem cell in peripheral blood after adopting coupling to have the CD34 antibody spike of FITC to mobilize, selected by flow cytometry apoptosis detects CD34 positive cell quantity.Concrete operations are as follows: 1) washed 3 times by cell with PBS；2) dilute antibody according to antibody operation instructions PBS, note lucifuge；3) joining in cell by the antibody diluted, blow and beat gently and uniformly become single cell suspension, lucifuge hatches 30 minutes on ice；4) after having hatched, with 1200 revs/min of low-temperature centrifugations 10 minutes；5) remove supernatant, wash 3 times with PBS；6) by resuspended for cell PBS, lucifuge is noted；7) the resuspended cell streaming pipe with filter screen or strainer filtering are to remove cell mass；8) cell overflow-type cell instrument is detected.That detect it is shown that CSF1 and G-CSF combines mobilization than individually mobilizing with G-CSF, CD34 positive cell ratio increase by 1.8 times, data are the meansigma methods SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 1).
Embodiment 2,
The CSF1 facilitation to hemopoietic stem cell proliferation
The hematopoietic stem cell obtained through CD34 magnetic bead sorting adopts hematopoietic stem cell serum-free medium to cultivate, and hematopoietic stem cell inoculated and cultured bag is placed at 37 DEG C of (5%CO2) cultivate, often cross 12 hours gentle wave and culture bags, whole incubation culture medium is additionally added with 2ng/mLCSF1 always.Under 12 hours microscopes, estimate cell density, after 72 hours, measure cell gross density with mtt assay.Result of study shows, adopt under the described in the invention culture medium condition being added with CSF1, hematopoietic stem cell density ratio improves 3.6 times without the cell density obtained under the condition of culture of CSF1, and data are the meansigma methods SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 2).
Embodiment 3,
The facilitation that mescenchymal stem cell is bred by CSF1
The cellar culture based formulas cultivating mescenchymal stem cell is: DMEM/F12 minimal medium, 10% hyclone (FBS).For comparing the impact on the amplification rate that human stem cell is cultivated of the culture medium described by the present embodiment and the culture medium described by inspection the present embodiment, human stem cell (the ADSC that the present embodiment has been selected between fat, bone marrow and three kinds of histoorgans of umbilical cord are originated, BMSC, UMBSC) cell culture experiments in vitro is done, cell-seeding-density controls between 10-20%, culture dish cell attachment material room temperature treatment 2 hours or 4 DEG C of incubated overnight before inoculation, after inoculation add culture medium culturing (37 DEG C, 5%CO2).A cell density is surveyed by mtt assay every 12 hours.Result of study shows, adopt stem cell media described by the present embodiment to the amplification rate of hematopoietic stem cell compared with conventional blood serum medium, the time reaching cell density saturated is greatly shortened, and data are the meansigma methods SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 3).
Embodiment 4,
The one-tenth fat ability of mescenchymal stem cell after CSF1 activation
Suitably induce mescenchymal stem cell under differentiation condition can be divided into adipose cell in vitro, the one-tenth fat ability of mescenchymal stem cell after cultivating for detection, experimental group is the mescenchymal stem cell cultivating acquisition after CSF1 adds, matched group is be not added with CSF1 to cultivate the mescenchymal stem cell obtained, the source for mesenchymal stem cells cultivated is in umbilicus band, bone marrow and fat, therefore umbilical cord mesenchymal stem cells (UMSC) it is divided into again, mesenchymal stem cells MSCs (BMSC) and fat stem cell (ADSC), this embodiment adopts following steps to detect: 1) first prepare division culture medium A and B, wherein division culture medium component A includes: basal medium, hyclone, penicillin streptomycin, glutamine, insulin, 3-isobutyl-1-methylxanthine, rosiglitazone, dexamethasone and indomethacin；Division culture medium B component includes: basal medium, hyclone, penicillin streptomycin, glutamine and insulin, prepare complete after mix homogeneously, put 4 DEG C of refrigerators and keep in Dark Place standby；2) in six orifice plates, inoculate 105 cells/well, continue to cultivate cell；3) growing to after 100% degree of converging until cell, suck old culture medium, every hole adds 1.5mL division culture medium A；4) sucking division culture medium A after three days, every hole adds 1.5mL division culture medium B；5) after 24 hours, sucking division culture medium B, every hole adds 1.5mL division culture medium A；6) rotation adds division culture medium A and B, repeats d-e step 4 time；7) it is eventually adding division culture medium B and cultivates 7 days (centre needs to change division culture medium B of liquid)；8) after induction terminates, sucking culture medium, 4% paraformaldehyde adding 1mL in every hole fixes 30 minutes；9) 2 times are washed with PBS；10) in every hole, add 1mL oil red O stain liquid, dye 30 minutes；11) wash 3 times with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics adipose cell.Found that, the mescenchymal stem cell of the separate sources obtained after adding CSF1 is relatively not added with the ability of the CSF1 formation adipose cell cultivating the mescenchymal stem cell obtained and keeps completely, and data are the meansigma methods SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 4).
Embodiment 5,
The osteogenic ability of mescenchymal stem cell after CSF1 activation
Suitably induce mescenchymal stem cell under differentiation condition can be divided into osteoblast in vitro, the osteogenic ability of mescenchymal stem cell after cultivating for detection, experimental group is the mescenchymal stem cell cultivating acquisition after CSF1 adds, matched group is be not added with CSF1 to cultivate the mescenchymal stem cell obtained, the source for mesenchymal stem cells cultivated is in umbilicus band, bone marrow and fat, therefore umbilical cord mesenchymal stem cells (UMSC) it is divided into again, mesenchymal stem cells MSCs (BMSC) and fat stem cell (ADSC), the present embodiment adopts following steps to detect: 1) first prepare division culture medium, including basal medium, hyclone, penicillin streptomycin, glutamine, vitamin C, β-phosphoglycerol and dexamethasone, mix homogeneously, put 4 DEG C of refrigerators and keep in Dark Place standby；2) by six orifice plates with the gelatin pretreatment of 0.1%, within before inoculating cell 30 minutes at least in advance, process with gelatin, or after addition gelatin, six orifice plate sealed membranes are sealed up, put 4 DEG C of refrigerators standby；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell (5 × 10 in six orifice plates of 1% gelatin pretreatment4Individual cells/well)；At 37 DEG C, 5%CO2Incubator is cultivated 24 hours；5) suck culture medium after 24 hours, change Osteoinductive differentiation culture medium (1.5mL/ hole)；6) an Osteoinductive differentiation culture medium within every 3 days, is changed, induction 2-3 week；7) after induction terminates, sucking culture medium, 4% paraformaldehyde adding 1mL in every hole fixes 30 minutes；8) 2 times are washed with PBS；9) in every hole, add 1mL alizarin red S dyeing liquor, dye 5-10 minute；9) wash 3 times with PBS, be subsequently placed under inverted microscope and observe take pictures, ratio shared by statistics osteocyte.Result of study shows, the mescenchymal stem cell of the separate sources obtained after adding CSF1 is relatively not added with the osteoblastic ability of shape of the CSF1 mescenchymal stem cell cultivating acquisition and keeps completely, and data are the average SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 5).
Embodiment 6,
The one-tenth cartilage ability of mescenchymal stem cell after CSF1 activation
Suitably mescenchymal stem cell can differentiating cartilage-forming cell under induction differentiation condition in vitro, the one-tenth cartilage ability of mescenchymal stem cell after cultivating for detection, experimental group is the mescenchymal stem cell cultivating acquisition after CSF1 adds, matched group is be not added with CSF1 to cultivate the mescenchymal stem cell obtained, the source for mesenchymal stem cells cultivated is in umbilicus band, bone marrow and fat, therefore umbilical cord mesenchymal stem cells (UMSC) it is divided into again, mesenchymal stem cells MSCs (BMSC) and fat stem cell (ADSC), the present embodiment adopts following steps to detect: 1) first prepare division culture medium, including basal medium, dexamethasone, ascorbic acid, Insulin-Transferrin-selenium additive, Sodium Pyruvate, proline and TGF-β 3, mix homogeneously, put 4 DEG C of refrigerators and keep in Dark Place standby；2) by the gelatin pretreatment of six orifice plates；3) six orifice plates suck unnecessary gelatin liquid before use, then inoculate appropriate cell (5 × 10 in six orifice plates of 1% gelatin pretreatment4Individual cells/well)；4) add into cartilage differentiation culture medium, within 2-3 days, change a not good liquor；5) cell differentiation is after 3 weeks, sucks culture medium, fixes 30 minutes with 4% paraformaldehyde；6) 2 times are washed with PBS；7) addition 1mL A Li Xinlan dyeing liquor in every hole, dyes 30 minutes；8) 3 times are washed with PBS, be subsequently placed under inverted microscope observe take pictures, the ratio of statistics chondrocyte, result of study shows, the mescenchymal stem cell of the separate sources obtained after adding CSF1 is relatively not added with the ability of the CSF1 formation chondrocyte cultivating the mescenchymal stem cell obtained and keeps completely, and data are the average SEM(P < 0.01 of 5 independent experiments) (as shown in Figure 6).
Embodiment 7,
The impact on tumor model mouse survival rate of the hematopoietic stem cell after CSF1 process
Acute B lymphocyte series leukemia cell line (Nalm-6) the inactivation new-born calf serum containing penicillin 100U/mL, streptomycin 100U/mL, 15% percentage by weight is formed RPMI-1640 complete culture solution, at 37 DEG C containing 5%CO2The incubator of saturated humidity (percent by volume) is cultivated, cultivates cell.With aseptic PBS liquid, concentration of cyclophosphamide being adjusted 10mg/mL, to cyclophosphamide described in the lumbar injection of mice (2mg/ is only), injection 2d(d refers to natural law continuously)；After 24h, after collecting the Nalm-6 cell being in exponential phase and carrying out the centrifugal 5min of 1000r/min, it is suspended in aseptic PBS liquid, adjusts Nalm-6 cell density to 2.5 × 107Individual/mL, carries out tail vein injection (5 × 10 to mice6Individual/only), prepare leukemia animal model, and carry out radiotherapy process.Radiotherapy is assisted after processing and is treated with hematopoietic stem cell, namely injects the hematopoietic stem cell 2.5 × 10 expanded by the above process5Individual/only.Adding up the survival rate of animal after 4 months, before result shows hematopoietic stem cell treatment, animal dis motility rate is brought up to 79% by original 18%, and data are the average SEM(P < 0.01 of 10 independent experiments) (as shown in Figure 7).
In sum, a kind of activation method based on colony-stimulating factor 1 to stem cell provided by the invention, the present invention utilizes CSF1 to stem cell mobilization, propagation and activation, CSF1 is possible not only to promote the in-vitro multiplication of stem cell, may additionally facilitate the mobilization of internal stem cell, propagation and activation, be expected to play a significant role in cell cultivation, disease treatment and anti-ageing healthcare.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, it is possible to improved according to the above description or convert, all these improve and convert the protection domain that all should belong to claims of the present invention.