CN104894058A - Application of sphingosylphosphorylcholine (SPC) in preparation of medicine for promoting endogenesis heart stem cells to be differentiated into heart lineage cells - Google Patents
Application of sphingosylphosphorylcholine (SPC) in preparation of medicine for promoting endogenesis heart stem cells to be differentiated into heart lineage cells Download PDFInfo
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- CN104894058A CN104894058A CN201510376913.8A CN201510376913A CN104894058A CN 104894058 A CN104894058 A CN 104894058A CN 201510376913 A CN201510376913 A CN 201510376913A CN 104894058 A CN104894058 A CN 104894058A
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Abstract
The invention discloses application of sphingosylphosphorylcholine (SPC) in preparation of a medicine for promoting endogenesis heart stem cells to be differentiated into heart lineage cells, wherein concentration of the sphingosylphosphorylcholine for effectively promoting the endogenesis heart stem cells to be differentiated into the heart lineage cells is 5mu m. The application provided by the invention lays a foundation for research and development of the new medicine for promoting myocardium differentiation, the SPC can also serve as an effective tool, and is used for research on a molecular mechanism of differentiation of the endogenesis heart stem cells.
Description
Technical field
The present invention relates to the new opplication of Sphingosylphosphocholine (sphingosylphosphorylcholine, SPC), particularly relating to Sphingosylphosphocholine in the short endogenous heart differentiation of stem cells of preparation is the application in cardiac linage cell drug.
Background technology
Sphingosylphosphocholine (sphingosylphosphorylcholine, SPC) molecular formula is: C
23h
49n
2o
5p, molecular weight is: 464.62, and structural formula is as follows:
SPC is that one has bioactive sphingophospholipid, is naturally present in blood, is the moiety of high-density lipoprotein (HDL) (HDL) and low-density lipoprotein.SPC, except as except biomembranous moiety, also can exist as regulating the signaling molecule in extracellular and intracellular signal transduction.Have been reported and show, SPC can suppress vascular smooth muscle cell spasm, regulates the apoptosis of vascular endothelial cell, Differentiation and proliferation, shows the important biomolecule of cardiovascular cell active.Such as SPC is in conjunction with reducing the instantaneous heart ischemia of mouse/re-perfusion model cardiac infarct size significantly after HDL, cardioprotection is from the injury in myocardial ischemia and Reperfu-sion and produce anti-inflammatory and Anti-G value.But about the research of SPC in differentiation of stem cells process is very few in published report, though there is report SPC to break up to vascular smooth muscle cell by inducing mesenchymal stem cell, be that there is not been reported both at home and abroad at present for application in cardiac linage cell drug for SPC in the short endogenous heart differentiation of stem cells of preparation.
Summary of the invention
For the deficiencies in the prior art, it is the application in cardiac linage cell drug that the problem to be solved in the present invention is to provide a kind of Sphingosylphosphocholine (SPC) in the short endogenous heart differentiation of stem cells of preparation.
Sphingosylphosphocholine of the present invention (SPC) is the application in cardiac linage cell drug in the short endogenous heart differentiation of stem cells of preparation.
Wherein: effective promotion endogenous heart differentiation of stem cells is that Sphingosylphosphocholine (SPC) concentration of cardiac linage cell is preferably 5 μMs.
The newtype drug developing short Myocardium Differentiation that is applied as of SPC provided by the invention is laid a good foundation.SPC of the present invention is all right as a kind of effective instrument, for the research of the molecular mechanism of the differentiation of endogenous heart stem cell.
In order to understand essence of the present invention better, illustrating it in the short endogenous heart differentiation of stem cells of preparation below in conjunction with the pharmacological evaluation of SPC and the method for Celluar and Molecular Biology and result is the application in cardiac linage cell drug.
1. endogenous Sca-1
+the preparation of cardiac stem cells: extract with magnetic bead sorting method and cultivate the sca-1 of mouse
+cardiac stem cells.
Result shows: the first-generation (P1) cell Sca-1
+positive rate was the 84.8%, five generation (P5) 96.8%, Sca-1
+cell CD34 positive rate about 0.8%, CD45 positive rate is about 3.5-7.3%.Show that the cell that this preparation method extracts is Sca-1
+cardiac stem cells, and non-hematopoietic stem cell.
2. the preparation of SPC medical solution of the present invention: the liquid storage being formulated as 5mg/ml.
3. under inverted phase contrast microscope, timing observes SPC to the impact of cardiac stem cells form.
Result shows: compare with normal group with solvent control group, and the SPC of 5 μMs can promote sca-1
+cardiac stem cells elongates.
4.QPCR detects the change of cardiomyocyte marker thing mRNA level in-site.
Result shows: the marker Gata4 of 5 μMs of remarkable upper centring myocytes of SPC process 2-3 week energy, the expression of Nkx2.5, CTnt, Mef2c etc.
5. Laser Scanning Confocal detects in conjunction with western blot, detects the change of cardiomyocyte marker thing protein level.
Result shows: 5 μMs of SPC can promote the formation of cell internal stress fiber, and raises the protein expression of CTnt.
In addition, 5 μMs of SPC significantly can promote that Cardiac-specific transcription factor GATA4's enters core.
6.QPCR and flow cytometry detect the change of endothelial cell marker thing from mRNA and protein level
Result shows: the expression significantly raising the mRNA level in-site such as marker CD31 and vWF of endotheliocyte 5 μMs of SPC process 2-3 weeks, and the expression of CD31 protein level.
By above-mentioned experiment and result thereof, can draw the following conclusions:
Use the SPC treatment S ca-1 of different concns respectively
+cardiac stem cells 2,3, the result display of 6 weeks, the SPC process sca-1 of 5 μMs
+relative to other concentration, cardiac stem cells 2-3 week significantly can promote that cardiac stem cells is to the cytodifferentiation of cardiac linage.And SPC process promotes after 6 weeks that the effect of differentiation is not clearly.So this specification sheets establish SPC application concentration time 5 μMs, the treatment time be 2-3 week, from 2 weeks, namely just there will be the differentiation of cardiac stem cells.Therefore the present invention is that the mechanism studying endogenous sphingomyelins inducing heart differentiation of stem cells provides theory and experimental basis, the SPC that the present invention relates to can as the effective instrument of one, for the Study on Molecular Mechanism of the differentiation of endogenous heart stem cell, the present invention is simultaneously that the rebirth medicine developed about short Myocardial Regeneration is laid a good foundation, and the Application and Development for clinical medicine provides reliable theoretical foundation.
Accompanying drawing explanation
Fig. 1: be the sca-1 be separated
+cardiac stem cells purity.P1, P5 represent passage number.
Fig. 2: be sca-1
+cardiac stem cells under regular culture conditions, the metamorphosis figure of cell after the SPC process 2-3 week shown under inverted phase contrast microscope.
Wherein: nor: normally cultivation group
Ctr: solvent control group
Spc:5 μM of SPC treatment group
Fig. 3: be SPC process sca-1
+after cardiac stem cells, the change of the mRNA level in-site of cardiac myocytespecific marker.
Wherein:
A.1,2,5 μMs of SPC process cardiac stem cells 3 weeks, QPCR detects myocardial cell transcription factor gata4, the change of Nkx2.5 expression level.Establishing effective induced concentration is 5 μMs.
B. with 5 μMs of SPC process sca-1
+cardiac stem cells 2,3,6 weeks, detects myocardial cell transcription factor gata4, the change of Nkx2.5 expression level again.Establishing effective induction time is 2-3 week.
C. with 5 μMs of SPC process sca-1
+in cardiac stem cells 2-3 week, detect other myocardial cell marker CTnt, the change of the mRNA level in-site of Mef2c.
Fig. 4: be SPC process sca-1
+after cardiac stem cells, cardiac myocytespecific marker is in the change of intracellular distribution and protein level.
Wherein: ctr: solvent control group; Spc:5 μM of SPC treatment group.
A. laser scanning co-focusing microscope binding immunoassay fluorescence chemical shows 5 μMs of SPC to the impact distributed in the cell of cTnt.
B.Western blot shows the expression that 5 μMs of SPC can promote cTnt.Ctr: solvent control group; Spc:5 μM of SPC treatment group.
C. laser scanning co-focusing microscope binding immunoassay fluorescence chemical shows 5 μMs of SPC and promotes that cell specific transcription factor Gata4's enters core.
D. the core rate that enters that core rate is about 20%, SPC treatment group Gata4 that enters of column diagram analysis display control group Gata4 is about 80%.
Fig. 5: be SPC process sca-1
+after cardiac stem cells, endothelial cell specific marker is in the change of mRNA and protein level.
Wherein: nor: normally cultivation group; Ctr: solvent control group; Spc:SPC treatment group.
A.QPCR detects different concns SPC process sca-1
+cardiac stem cells different time is on the impact of CD31 and vWF mRNA level in-site.Establishing effective concentration is 5 μMs, and effective action time is 2-3 week.
B.5 μM SPC process 3 weeks, flow cytomery SPC is on the impact of CD31.
Embodiment
The extraction of embodiment 1Sca-1+ cardiac stem cells and its purity of flow cytomery
1. the C57/BL mouse about 10 week age break neck put to death, disinfect in alcohol rapidly core dirty, cleaning, shred, cut about 1mm
3size.
2. 37 DEG C of trypsin solution digestion 30min.
3. digested and added a little foetal calf serum termination digestion, with the sieved filter of 200 object cell, added the cell of a small amount of cell staining buffer suspension filter, carry out cell counting.
4. by centrifugal for filtrate 350g 8min, with cell staining buffer re-suspended cell, density is made to reach 2 × 10
7/ ml, adds biotin labeled sca-1 antibody in solution., every 1 × 10
7individual cell adds 5 μ l antibody, hatches 20min on ice.
5. the 1xBD IMag of excess volume is used
tXthe cell of buffer washing mark, 350g, centrifugal 8min, and carefully suck supernatant.
6. thorough vortex BD IMag
tXstreptavidin Partides Plus-DM, every 1 × 10
7individual cell adds 25 μ l, and mixing, then hatches 45min for 4 DEG C.
7. 1 × BD IMag is used
tXbuffer, brings up to 20-80 × 10 by label amount
6/ ml.
8. EP pipe is moved on to BD IMag
tXon frame, absorption 8min.(being no more than 1ml), sucking-off supernatant, adds 1xBD IMag again
tXbuffer, resuspended gently, then put on shelf and adsorb 8min.Repeat this step, use substratum suspension cell, cell is moved in 24 orifice plates and cultivate.Choose 1-5 for growth conditions good and to be in the cell of logarithmic phase for subsequent use.
9. to the first-generation, the 5th generation cardiac stem cells carry out flow cytometer detection stem cell purity.Collecting cell, 300g, centrifugal 5min removes waste liquid, with cell staining buffer re-suspended cell, adds PE-Cy5-CD45 antibody, FITC-sca-1+ antibody, PE-CD34 antibody dyes the centrifugal 5min of 30min, 300g on ice, washes twice, finally again add cell staining buffer re-suspended cell, utilize Flow cytometry (the results are shown in Figure 1)
Result shows: the first-generation (P1) cell Sca-1
+positive rate was the 84.8%, five generation (P5) 96.8%, Sca-1
+cell CD34 positive rate about 0.8%, CD45 positive rate is about 3.5-7.3%.Show that the cell that we extract is Sca-1
+cardiac stem cells, and non-hematopoietic stem cell.
The preparation of embodiment 2 SPC liquid storage
The SPC powder compound concentration provided by sigma company is the liquid storage of 5mg/ml, is dispensed in the eppendorf pipe of 1.5ml, dries up with liquid nitrogen ,-20 DEG C of storages.
SPC is to sca-1 for embodiment 3
+the detection of cardiac stem cells form impact
By the sca-1 extracted
+cardiac stem cells kind, in 24 orifice plates, covers with after 7 days, evenly imports in capsule, cultivates, go down to posterity after covering with the IMDM containing somatomedin and 10% foetal calf serum, gets 1-5 for stem cell for experiment.The cell of adherent 24 hours is added the SPC of different concns (1,2,5 μMs) as experimental group.Add ethanol as solvent control group, and be left intact as normal group.Culture condition: 37 DEG C, CO
2cultivate in incubator.
Changed a nutrient solution every three days and continue to add the SPC of described concentration, after cultivating 2-3 week, under inverted phase contrast microscope, (the results are shown in Figure 2) is observed in timing.
Result shows: compare with solvent control group with normal group, and in 5 μMs of SPC process cell 2-3 weeks, cell elongates and attenuates.
The detection of embodiment 4 cardiomyocyte marker thing rna level
By the sca-1 extracted
+cardiac stem cells kind, in 24 orifice plates, covers with after 7 days, evenly imports in capsule, cultivates 24 hours with the IMDM containing somatomedin and 10% foetal calf serum.After cell attachment, abandon waste liquid, with 1 × PBS cleaning, add fresh medium, and add 1 respectively, 2, the SPC of 5 μMs is as experimental group.Add ethanol as solvent control group, and be left intact as normal group.Culture condition: 37 DEG C, CO
2cultivate in incubator.
Changed a nutrient solution every three days and continue to add the SPC of described concentration, adding trizol after cultivating 2-3 week, extract total serum IgE, reverse transcription goes out cDNA, carries out the change (the results are shown in Figure 3) that QPCR detects mRNA level in-site.
Result shows: SPC process 2-3 week of 5 μMs can effective upper centring myocyte marker CTnt, the expression of the mRNA of GATA4, Mef2c, Nkx2.5.**p<0.01。
The detection of embodiment 5 cardiomyocyte marker thing protein level
By the sca-1 extracted
+cardiac stem cells kind, in 24 orifice plates, covers with after 7 days, evenly imports in capsule, cultivates 24 hours with the IMDM containing somatomedin and 10% foetal calf serum.After cell attachment, abandon waste liquid, with 1 × PBS cleaning, add fresh medium, and the SPC adding 5 μMs is as experimental group.Add ethanol as solvent control group.
1. waste liquid is abandoned, after 0.1 × PBS cleans three times, paraformaldehyde with 4% fixes 15 minutes, after cleaning, and lowlenthal serum closes 20 minutes, add primary antibodie 4 DEG C of overnight incubation of CTnt, after 0.1 × PBS cleans three times, add two and resist, hatch 1h for 37 DEG C, after cleaning, observe cardiomyocyte marker thing at laser scanning co-focusing microscope---the changes in distribution of CTnt.
2. extract total protein, western blot detects the protein level of myocardial cell marker CTnt.
3. waste liquid is abandoned, after 0.1 × PBS cleans three times, paraformaldehyde with 4% fixes 15 minutes, after cleaning, and lowlenthal serum closes 20 minutes, add primary antibodie 4 DEG C of overnight incubation of GATA4, after 0.1 × PBS cleans three times, add two and resist, hatch 1h for 37 DEG C, after cleaning, observe cardiomyocyte marker thing at laser scanning co-focusing microscope---the changes in distribution of GATA4.
4. at will choose the picture of 6 GATA4 immunofluorescences, calculate it and enter core rate.(the results are shown in Figure 4).
Result shows: the SPC of 5 μMs can promote the formation of cell internal stress fiber, and the myocardial cell marker CTnT raised.In addition, laser scanning co-focusing microscope binding immunoassay fluorescence chemical shows 5 μMs of SPC and promotes that cell specific transcription factor Gata4's enters core; The core rate that enters that core rate is about 20%, SPC treatment group GATA4 that enters of column diagram analysis display control group GATA4 is about 80%.**p<0.01。
The RNA of embodiment 6 endothelial cell specific marker and the detection of protein level
By the sca-1+ cardiac stem cells kind that extracts in 24 orifice plates, cover with after 7 days, evenly import in capsule, cultivate 24 hours with the IMDM containing somatomedin and 10% foetal calf serum.After cell attachment, abandon waste liquid, with 1 × PBS cleaning, add fresh medium, and add 1,2, the SPC of 5 μMs is as experimental group.Add ethanol as solvent control group.And be left intact as normal group.Culture condition: 37 DEG C, 5%CO
2cultivate in incubator.
1. changed a nutrient solution every three days and continue to add the SPC of described concentration, adding trizol after cultivating 2-3 week, extract total serum IgE, reverse transcription goes out cDNA, carries out the change that QPCR detects mRNA level in-site.
2. 5 μMs of SPC process sca-1+ cardiac stem cells 3 weeks, collecting cell, 300g, centrifugal 5min removes waste liquid, with cell staining buffer re-suspended cell, adds FITC-sca-1
+antibody, PE-CD31 antibody dyes the centrifugal 5min of 30min, 300g on ice, washes twice, finally again adds cell staining buffer re-suspended cell, utilize Flow cytometry (the results are shown in Figure 5).
Result shows: QPCR detects different concns SPC process sca-1
+cardiac stem cells different time is on the impact of CD31 and vWF mRNA level in-site.Establishing effective concentration is 5 μMs, and effective action time is 2-3 week.*p<0.05，**p<0.01。5 μMs of SPC process 3 weeks, flow cytometry shows that SPC promotes the expression of the protein level of endotheliocyte marker CD31.
Claims (2)
1. a Sphingosylphosphocholine (SPC) is the application in cardiac linage cell drug in the short endogenous heart differentiation of stem cells of preparation.
2. apply as claimed in claim 1, it is characterized in that: effectively promotion endogenous heart differentiation of stem cells is Sphingosylphosphocholine (SPC) concentration of cardiac linage cell is 5 μMs.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111868078A (en) * | 2018-03-16 | 2020-10-30 | 斯坦福大学托管董事会 | Reagents and methods for generating and expanding cardiomyocytes using WNT agonists and bioactive lipids |
| CN113662985A (en) * | 2021-09-07 | 2021-11-19 | 李红梅 | Traditional Chinese medicine compound, traditional Chinese medicine effective component composition, compound and application thereof |
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| CN1446108A (en) * | 2000-08-07 | 2003-10-01 | 法商佛梭公司 | Pharmaceutical form comprising a support material and at least a cell regulating factor and/or cell proliferation promoter |
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Patent Citations (2)
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| CN1446108A (en) * | 2000-08-07 | 2003-10-01 | 法商佛梭公司 | Pharmaceutical form comprising a support material and at least a cell regulating factor and/or cell proliferation promoter |
| US20040076601A1 (en) * | 2000-08-07 | 2004-04-22 | Nicole Bru-Magniez | Pharmaceutical form comprising a cell regulating factor and/or a cell proliferation promoter |
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| ALEXANDER KLEGER等: "The bioactive lipid sphingosylphosphorylcholine induces differentiation of mouse embryonic stem cells and human promyelocytic leukaemia cells", 《CELLULAR SIGNALLING》 * |
| EUN SU JEON等: "Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smoothmuscle-like cells through a TGF-β-dependent mechanism", 《JOURNAL OF CELL SCIENCE》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111868078A (en) * | 2018-03-16 | 2020-10-30 | 斯坦福大学托管董事会 | Reagents and methods for generating and expanding cardiomyocytes using WNT agonists and bioactive lipids |
| JP2021518113A (en) * | 2018-03-16 | 2021-08-02 | ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー | Reagents and methods using WNT agonists for generating and proliferating cardiomyocytes, and bioactive lipids |
| CN113662985A (en) * | 2021-09-07 | 2021-11-19 | 李红梅 | Traditional Chinese medicine compound, traditional Chinese medicine effective component composition, compound and application thereof |
| CN113662985B (en) * | 2021-09-07 | 2022-06-24 | 北京中医药大学 | Traditional Chinese medicine composition with effect of promoting directional differentiation of stem cells into myocardial cells, traditional Chinese medicine effective component composition and application thereof |
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