CN104894058B - Sphingosylphosphocholine is preparing the application that rush endogenous heart stem cell is divided into cardiac linage cell drug - Google Patents
Sphingosylphosphocholine is preparing the application that rush endogenous heart stem cell is divided into cardiac linage cell drug Download PDFInfo
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Abstract
The application that rush endogenous heart stem cell is divided into cardiac linage cell drug is being prepared the invention discloses a kind of Sphingosylphosphocholine；Wherein effectively facilitate endogenous heart stem cell and be divided into Sphingosylphosphocholine (SPC) concentration of cardiac linage cell for 5 μM.The application that the present invention is provided is laid a good foundation to develop the newtype drug of rush Myocardium Differentiation, and the Sphingosylphosphocholine is alternatively arranged as a kind of effective instrument, the research for the molecular mechanism of the differentiation of endogenous heart stem cell.
Description
Technical field
The present invention relates to newly should for Sphingosylphosphocholine (sphingosylphosphorylcholine, SPC)
With, more particularly to Sphingosylphosphocholine prepare promote endogenous heart stem cell be divided into cardiac linage cell drug
Using.
Background technology
Sphingosylphosphocholine (sphingosylphosphorylcholine, SPC) molecular formula is：
C23H49N2O5P, molecular weight is：464.62, structural formula is as follows：
SPC is a kind of sphingomyelins with bioactivity, is naturally occurring in blood, be HDL (HDL) and
The constituent of low-density lipoprotein.SPC, can also be extracellular and thin as adjusting in addition to the constituent as biomembrane
The signaling molecule of intracellular signal transduction is present.Have been reported and show, SPC can suppress vascular smooth muscle cells spasm, adjust blood vessel
The apoptosis of endothelial cell, differentiation and propagation, show the important biomolecule activity to cardiovascular cell.For example after SPC combinations HDL
The instantaneous heart ischemia of mouse/re-perfusion model cardiac infarct size can be significantly decreased, cardioprotection is from myocardial ischemia
With the injury in Reperfu- sion and produce anti-inflammatory and Anti-G value.But relevant SPC is in stem cell in published report
Research in atomization is very few, though have been reported that SPC can be broken up with inducing mesenchymal stem cell to vascular smooth muscle cells, it is right
Not yet appeared in the newspapers both at home and abroad at present in the application that preparation rush endogenous heart stem cell is divided into cardiac linage cell drug in SPC
Road.
The content of the invention
In view of the shortcomings of the prior art, the problem to be solved in the present invention is to provide a kind of Sphingosylphosphocholine
(SPC) application that rush endogenous heart stem cell is divided into cardiac linage cell drug is being prepared.
Sphingosylphosphocholine (SPC) of the present invention promotees endogenous heart stem cell in preparation and is divided into cardiac linage
Application in cell drug.
Wherein：Effectively facilitate the Sphingosylphosphocholine that endogenous heart stem cell is divided into cardiac linage cell
(SPC) concentration is preferably 5 μM.
The application for the SPC that the present invention is provided is laid a good foundation to develop the newtype drug of rush Myocardium Differentiation.The present invention
Described SPC is also used as a kind of effective instrument, the research for the molecular mechanism of the differentiation of endogenous heart stem cell.
Essence for a better understanding of the present invention, gives birth to reference to SPC pharmacological evaluation and cell biology and molecule
The method and result of thing are preparing the application that rush endogenous heart stem cell is divided into cardiac linage cell drug to illustrate it.
1. endogenous Sca-1+The preparation of cardiac stem cells：The sca-1 of mouse is extracted and cultivated with magnetic bead sorting method+Heart
Stem cell.
As a result show：The first generation (P1) cell Sca-1+Positive rate is 84.8%, the 5th generation (P5) 96.8%, Sca-1+Carefully
Born of the same parents CD34 positive rates about 0.8%, CD45 positive rates about 3.5-7.3%.Show that the cell that the preparation method is extracted is Sca-1+'s
Cardiac stem cells, and non-hematopoietic stem cell.
2. the preparation of SPC medical solutions of the present invention：It is formulated as 5mg/ml liquid storage.
3. influences of the SPC to cardiac stem cells form is regularly observed under inverted phase contrast microscope.
As a result show：Compared with solvent control group and normal group, 5 μM of SPC can promote sca-1+Cardiac stem cells are drawn
It is long.
4.QPCR detects the change of cardiomyocyte marker thing mRNA level in-site.
As a result show：5 μM of SPC processing can significantly raise marker Gata4, the Nkx2.5 of cardiac muscle cell in 2-3 weeks,
CTnt, Mef2c etc. expression.
5. Laser Scanning Confocal detection combines western blot, the protein level change of detection cardiomyocyte marker thing.
As a result show：5 μM of SPC can promote the formation of intracellular stress fiber, and raise CTnt protein expression.
In addition, 5 μM of SPC can remarkably promote Cardiac-specific transcription factor GATA4 enter core.
6.QPCR and flow cytometry detect the change of endothelial cell marker from mRNA and protein level
As a result show：5 μM of SPC processing can significantly raise the mRNA water such as the marker CD31 and vWF of endothelial cell for 2-3 weeks
Flat expression, and CD31 protein levels expression.
By above-mentioned experiment and its result, it can be deduced that such as draw a conclusion：
Respectively with the SPC processing Sca-1 of various concentrations+The cardiac stem cells result of 2,3,6 weeks shows, 5 μM of SPC processing
sca-1+Cardiac stem cells can significantly promote cell point of the cardiac stem cells to cardiac linage in 2-3 weeks relative to other concentration
Change.And SPC promotes the effect of differentiation not to be apparent after handling 6 weeks.So this specification established SPC application concentration when
5 μM, processing time is 2-3 weeks, i.e., the differentiation of cardiac stem cells just occurred since 2 weeks.Therefore the present invention is endogenous to study
The mechanism of sphingomyelins inducing heart stem cell differentiation provides theoretical and experimental basis, and SPC of the present invention can conduct
A kind of effective instrument, for the Study on Molecular Mechanism of the differentiation of endogenous heart stem cell, while the present invention has for development
The rebirth medicine for closing rush Myocardial Regeneration is laid a good foundation, and reliable theoretical foundation is provided for the development and application of clinical medicine.
Brief description of the drawings
Fig. 1：For the sca-1 of separation+Cardiac stem cells purity.P1, P5 represent passage number.
Fig. 2：For sca-1+Cardiac stem cells are under regular culture conditions, the SPC processing shown under inverted phase contrast microscope
The metamorphosis figure of cell after 2-3 weeks.
Wherein：nor：Normal culture group
ctr：Solvent control group
spc：5 μM of SPC treatment groups
Fig. 3：It is SPC processing sca-1+After cardiac stem cells, the change of cardiac myocytespecific marker mRNA level in-site.
Wherein：
A.1,2,5 μM of SPC handle cardiac stem cells 3 weeks, QPCR detection cardiac muscle cell's transcription factor gata4, Nkx2.5 tables
Up to horizontal change.Effective induced concentration is established for 5 μM.
B. sca-1 is handled with 5 μM of SPC+Cardiac stem cells 2,3,6 weeks, detect cardiac muscle cell transcription factor gata4 again,
The change of Nkx2.5 expressions.Effective induction time is established for 2-3 weeks.
C. sca-1 is handled with 5 μM of SPC+Cardiac stem cells 2-3 weeks, detect other cardiac muscle cell marker CTnt,
The change of Mef2c mRNA level in-site.
Fig. 4：It is SPC processing sca-1+After cardiac stem cells, cardiac myocytespecific marker distributions in the cell and
The change of protein level.
Wherein：ctr：Solvent control group；spc：5 μM of SPC treatment groups.
A. laser scanning co-focusing microscope combination immunofluorescence shows 5 μM of SPC to cTnt intracellular distribution
Influence.
B.Western blot show that 5 μM of SPC can promote cTnt expression.ctr：Solvent control group；spc：5μM SPC
Treatment group.
C. laser scanning co-focusing microscope combination immunofluorescence show 5 μM of SPC promote cell specific transcriptions because
Sub- Gata4's enters core.
D. cylindricality map analysis shows that the control group Gata4 core rate that enters is about that 20%, SPC treatment groups Gata4 enters core rate about
For 80%.
Fig. 5：It is SPC processing sca-1+After cardiac stem cells, endothelial cell specific marker is in mRNA and protein level
Change.
Wherein：nor：Normal culture group；ctr：Solvent control group；spc：SPC treatment groups.
A.QPCR detection various concentrations SPC processing sca-1+Cardiac stem cells different time is to CD31 and vWF mRNA level in-sites
Influence.It is 5 μM to establish effective concentration, and effective action time is 2-3 weeks.
B.5 μM SPC is handled 3 weeks, influences of the flow cytomery SPC to CD31.
Embodiment
The extractions of embodiment 1Sca-1+ cardiac stem cells and flow cytomery its purity
1. the disconnected neck of the C57/BL mouse of 10 week old or so is put to death, and is disinfected dirty, cleaning of coring in alcohol rapidly, is shredded, cut
About 1mm3Size.
2. 37 DEG C of trypsin solutions digest 30min.
3. digested plus a little hyclone terminates digestion, be sieved through filter with 200 aim cells, add a small amount of cell
The cell of staining buffer suspension filters, carries out cell count.
4. filtrate 350g is centrifuged into 8min, cell is resuspended with cell staining buffer, density is reached 2 × 107/
Ml, the sca-1 antibody of biotin labeling, every 1 × 10 are added into solution7Individual cell adds 5 μ l antibody, and 20min is incubated on ice.
5. the 1xBD IMag of excess volume are usedTXThe cell of buffer washing marks, 350g centrifuges 8min, and carefully suck
Supernatant.
6. thorough vortex BD IMagTXStreptavidin Partides Plus-DM, every 1 × 107Individual cell adds 25 μ
L, is mixed, then 4 DEG C of incubation 45min.
7. with 1 × BD IMagTXBuffer, 20-80 × 10 are brought up to by label amount6/ml。
8. EP pipes are moved on into BD IMagTXOn frame, 8min is adsorbed.(being no more than 1ml), suctions out supernatant, 1xBD is added again
IMagTXBuffer, is gently resuspended, then puts 8min is adsorbed on shelf.The step is repeated, culture medium suspension cell is used, cell is moved
Enter culture in 24 orifice plates.Choose 1-5 good and cell of in exponential phase is standby for growth conditions.
9. to the first generation, the 5th generation cardiac stem cells carry out flow cytometer detection stem cell purity.Collect cell, 300g, centrifugation
5min removes waste liquid, and cell is resuspended with cell staining buffer, adds PE-Cy5-CD45 antibody, and FITC-sca-1+ resists
Body, PE-CD34 antibody dyes 30min, 300g centrifugation 5min on ice, washes twice, cell staining are finally added again
Cell is resuspended in buffer, utilizes Flow cytometry (result is shown in Fig. 1)
As a result show：The first generation (P1) cell Sca-1+Positive rate is 84.8%, the 5th generation (P5) 96.8%, Sca-1+Carefully
Born of the same parents CD34 positive rates about 0.8%, CD45 positive rates about 3.5-7.3%.Show that the cell that we extract is Sca-1+Heart do
Cell, and non-hematopoietic stem cell.
The preparation of the SPC liquid storages of embodiment 2
The liquid storage that the SPC powder compound concentration provided by sigma companies is 5mg/ml, is dispensed into 1.5ml eppendorf
Guan Zhong, is dried up with liquid nitrogen, -20 DEG C of storages.
SPC is to sca-1 for embodiment 3+The detection of cardiac stem cells morphology influence
By the sca-1 extracted+Cardiac stem cells kind is covered with 24 orifice plates after 7 days, in uniform incoming capsule, with containing
The IMDM for having growth factor and 10% hyclone is cultivated, and is passed on after covering with, and takes 1-5 to be used to test for stem cell.Will patch
The SPC that the wall cell of 24 hours adds various concentrations (1,2,5 μM) is used as experimental group.Add ethanol as solvent control group,
And without any processing it is used as normal group.Condition of culture：37 DEG C, CO2Cultivated in incubator.
A nutrient solution was changed every three days and the SPC of the concentration is continuously added into, it is micro- in inverted phase contrast after cultivating 2-3 weeks
Regularly (result is shown in Fig. 2) is observed under mirror.
As a result show：Compared with normal group and solvent control group, 5 μM of SPC handle cell 2-3 weeks, and cell, which is elongated, to attenuate.
The detection of the cardiomyocyte marker thing rna level of embodiment 4
By the sca-1 extracted+Cardiac stem cells kind is covered with 24 orifice plates after 7 days, in uniform incoming capsule, with containing
The IMDM for having growth factor and 10% hyclone is cultivated 24 hours.After after cell attachment, waste liquid is abandoned, with 1 × PBS, plus
Enter fresh medium, and be separately added into 1,2,5 μM of SPC and be used as experimental group.Add ethanol as solvent control group, without doing
Any processing is used as normal group.Condition of culture：37 DEG C, CO2Cultivated in incubator.
A nutrient solution was changed every three days and the SPC of the concentration is continuously added into, culture adds trizol after 2-3 weeks, carries
Take total serum IgE, reverse transcription goes out cDNA, carry out the change that QPCR detects mRNA level in-site (result is shown in Fig. 3).
As a result show：5 μM of SPC processing can effectively raise cardiac muscle cell marker CTnt, GATA4 in 2-3 weeks,
Mef2c, Nkx2.5 mRNA expression.**p<0.01.
The detection of the cardiomyocyte marker thing protein level of embodiment 5
By the sca-1 extracted+Cardiac stem cells kind is covered with 24 orifice plates after 7 days, in uniform incoming capsule, with containing
The IMDM for having growth factor and 10% hyclone is cultivated 24 hours.After after cell attachment, waste liquid is abandoned, with 1 × PBS, plus
Enter fresh medium, and add 5 μM of SPC and be used as experimental group.Add ethanol is used as solvent control group.
1. abandon waste liquid, after 0.1 × PBS three times, 15 minutes, after cleaning, lowlenthal serum are fixed with 4% paraformaldehyde
Closing 20 minutes, adds CTnt 4 DEG C of primary antibody overnight incubation, after 0.1 × PBS three times, adds secondary antibody, 37 DEG C of incubation 1h,
After cleaning, cardiomyocyte marker thing --- CTnt changes in distribution is observed in laser scanning co-focusing microscope.
2. total protein, western blot detection cardiac muscle cell marker CTnt protein level are extracted.
3. abandon waste liquid, after 0.1 × PBS three times, 15 minutes, after cleaning, lowlenthal serum are fixed with 4% paraformaldehyde
Closing 20 minutes, adds GATA4 4 DEG C of primary antibody overnight incubation, after 0.1 × PBS three times, adds secondary antibody, 37 DEG C of incubation 1h,
After cleaning, cardiomyocyte marker thing --- GATA4 changes in distribution is observed in laser scanning co-focusing microscope.
4. the picture of 6 GATA4 immunofluorescences is at will chosen, it is calculated and enters core rate.(result is shown in Fig. 4).
As a result show：5 μM of SPC can promote the formation of intracellular stress fiber, and the cardiac muscle cell marker raised
CTnT.In addition, laser scanning co-focusing microscope combination immunofluorescence show 5 μM of SPC promote cell specific transcriptions because
Sub- Gata4's enters core；Cylindricality map analysis shows that the control group GATA4 core rate that enters is about that 20%, SPC treatment groups GATA4 enters core
Rate is about 80%.**p<0.01.
The endothelial cell specific marker of embodiment 6 RNA and protein level detection
By the sca-1+ cardiac stem cells kind extracted in 24 orifice plates, covered with after 7 days, in uniform incoming capsule, with containing
The IMDM for having growth factor and 10% hyclone is cultivated 24 hours.After after cell attachment, waste liquid is abandoned, with 1 × PBS, plus
Enter fresh medium, and add 1,2,5 μM of SPC and be used as experimental group.Add ethanol is used as solvent control group.It is any without doing
What is handled is used as normal group.Condition of culture：37 DEG C, 5%CO2Cultivated in incubator.
1. changed a nutrient solution every three days and be continuously added into the SPC of the concentration, culture adds trizol after 2-3 weeks,
Total serum IgE is extracted, reverse transcription goes out cDNA, carry out the change that QPCR detects mRNA level in-site.
2. 5 μM of SPC handle sca-1+ cardiac stem cells 3 weeks, collect cell, and 300g, centrifugation 5min removes waste liquid, used
Cell is resuspended in cell staining buffer, adds FITC-sca-1+Antibody, PE-CD31 antibody dyes 30min on ice,
300g centrifuges 5min, washes twice, cell staining buffer is finally added again cell is resuspended, examined using flow cytometry
Survey (result is shown in Fig. 5).
As a result show：QPCR detection various concentrations SPC processing sca-1+Cardiac stem cells different time is to CD31 and vWF
The influence of mRNA level in-site.It is 5 μM to establish effective concentration, and effective action time is 2-3 weeks.*p<0.05, * * p<0.01.5μM
SPC is handled 3 weeks, and flow cytometry shows that SPC promotes the expression of endothelial cell marker CD31 protein level.
Claims (1)
1. a kind of Sphingosylphosphocholine（SPC）Cardiac linage cell medicine is divided into preparing rush endogenous heart stem cell
Application in thing, wherein effectively facilitating the Sphingosylphosphocholine that endogenous heart stem cell is divided into cardiac linage cell
（SPC）Concentration is 5 μM.
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| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (4)
| Title |
|---|
| Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smoothmuscle-like cells through a TGF-β-dependent mechanism;Eun Su Jeon等;《Journal of Cell Science》;20061231;第4994-5005页 * |
| The bioactive lipid sphingosylphosphorylcholine induces differentiation of mouse embryonic stem cells and human promyelocytic leukaemia cells;Alexander Kleger等;《Cellular Signalling》;20071231;第19卷;摘要，第368-370页，Fig1-3 * |
| 内源性心脏干/祖细胞活化的研究进展;郭涛等;《心脏杂志》;20141225;第27卷(第3期);第357-360页 * |
| 神经鞘氨醇磷酸胆碱对人成纤维细胞差异表达基因的筛选;朱明姬等;《中华皮肤科杂志》;20060731;第39卷(第7期);第381-384页 * |
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