CN101273983B - Use of neurocyte differentiated from stem cell induced by isobavachin - Google Patents
Use of neurocyte differentiated from stem cell induced by isobavachin Download PDFInfo
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- CN101273983B CN101273983B CN2008100614012A CN200810061401A CN101273983B CN 101273983 B CN101273983 B CN 101273983B CN 2008100614012 A CN2008100614012 A CN 2008100614012A CN 200810061401 A CN200810061401 A CN 200810061401A CN 101273983 B CN101273983 B CN 101273983B
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Abstract
The invention provides a single type nerve cell which is formed by in vitro directed differentiation of sobavachin-induced stem cells (comprising embryonic stem cells, neural stem cells and bone marrow mesenchymal stem cells). The single type nerve cell is applied in the preparation of drugs used for stem cell transplantation therapy of neurodegenerative diseases, in the preparation of cell differentiation agents used for repairing and reconstructing of damaged nerve cells and is also applied in the construction of a efficacy screening and evaluation model. The invention develops a new usage of the isobavachin, provides a physical basis for the prevention and treatment functions of the isobavachin traditional Chinese medicine and also provides a basis for the drug regulation and control regenerative medicine or the tissue engineering.
Description
Technical field
The invention belongs to cytobiology and area of pharmacology, the purposes that relates to a kind of isopentene group Chinese medicine monomer chemical compound, be specifically related to the purposes of isobavachin aspect the in-vitro directed differentiation of induced dry-cell, can be used for the short cell differential agent of regenerative medicine of Drug therapy neurodegenerative diseases and reconstruction injured nerve tissue.
Background technology
Fructus Psoraleae is the fruit of legumes psoraleae (Psoralea corylifolial), has the function of reinforcing the kidney and supporting YANG, controlling nocturnal emission with astringent drugs reducing urination, warming spleen and stopping diarrha, hemostasis hidroschesis, helping inspiration to relieve asthma.The chemical constituent of Fructus Psoraleae contains compositions such as Coumarins, flavonoid, monoterpene phenols and volatile oil, saponin, polysaccharide, lipoid.The flavone compound of separation and purification has isobavachin (isobavachin), neobavaisoflavone (neobavaisoflavone), corylifolinin (isobavachalcone), 6-(3-methylbut-2-enyl)coumestrol. (psoralidin).Modern pharmacological research shows that Fructus Psoraleae has multiple physiologically active, comprises the following aspects:
1. cardiovascular system effect: zoopery shows that Fructus Psoraleae has the coronary artery dilator effect, can excited heart, improve the cardiac work rate, but that myocardial oxygen consumption increases is not obvious.
2. antibacterial action: staphylococcus aureus is had inhibitory action.
3. genitourinary system effect: energy tonifying YANG controlling nocturnal emission with astringent drugs is a medicine commonly used of controlling sexual impotence and premature ejaculation, has astringent or styptic treatment for spontaneous sweating effect.
4. to the effect of smooth muscle: can make and exsomatize and intestinal tube excitement on the throne, the Cavia porcellus uterus then is lax, the effect of diastole bronchial smooth muscle.
5. function of increasing leukocyte: zoopery shows has facilitation to granulocytic growth, and the leukopenia that can watch for animals and cause behind the injection cyclophosphamide.
6. immunoregulation effect: humoral immunization is had certain regulating action.Still can regulate nerve and blood system, promote bone marrow hematogenesis, promote phagocytic function, enhance immunity and endocrine function, thereby the effect of performance anti-senility life-prolonging.
7. liver protection effect: the good curing effect is arranged to cause the mouse liver infringement with hydrocortisone.
The in-vitro directed neural cell model that is divided into of stem cell has been used for embryo's neurodevelopment and regenerative medicine research.The drug regulation cell micro-environment can be used for studying stem cell directional differentiated neuron and glial cell phenotype and differentiation signal path Study on Mechanism, also can be in order to screen short Neural Differentiation " medicine sample " micromolecular compound.The previous work of the present patent application person's research group finds, isopentene group chemical compound 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one has that remarkable inducing mouse embryonic stem cell is in-vitro directed to be divided into the effect of neurocyte phenotype.Do not see that in addition other is the report of neurocyte about drug-induced differentiation of stem cells.In view of the chemical structural formula of isobavachin and 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one similar; all belong to the isoamylene radical chromocor compounds; demand further inquiring into isoamylene radical chromocor analog active and machine-processed aspect performance Neural Differentiation and neuroprotective urgently; be intended to for new structure chemical compound control neurological sexually revises, drug regulation regenerative medicine or organizational project provide foundation.
Do not see that so far domestic and international monomeric compound, especially isobavachin induced dry-cell directed differentiation to Fructus Psoraleae extract is neurocyte Study on Effect report.
The isolating isopentene group structural formula of compound of Fructus Psoraleae is:
The isobavachin neobavaisoflavone
The 6-(3-methylbut-2-enyl)coumestrol. corylifolinin
Summary of the invention
An object of the present invention is to provide that isobavachin induced dry-cell (comprising embryonic stem cell, neural stem cell, mesenchymal stem cells MSCs) is in-vitro directed to be divided into the application of single type neurocyte in preparation cellular replacement therapy neurodegenerative diseases medicine.
Another object of the present invention provides the neurocyte of the in-vitro directed differentiation acquisition of isobavachin energy induced dry-cell (comprising embryonic stem cell, neural stem cell, mesenchymal stem cells MSCs) and uses in the short cell differential agent of preparation reconstruction injured nerve tissue.
A further object of the present invention provides isobavachin and can use in making up medicine efficacy screening and evaluation model by the in-vitro directed differentiation acquisition of induced dry-cell (comprising embryonic stem cell, neural stem cell, mesenchymal stem cells MSCs) neurocyte.
Isobavachin of the present invention can be realized by following steps by the in-vitro directed differentiation acquisition of induced dry-cell (comprising embryonic stem cell, neural stem cell, mesenchymal stem cells MSCs) neurocyte:
Main 4-/4+ the method for classics that adopts is induced differentiation culture.In the culture dish interior surface, the upset back forms hanging drop to embryonic stem cell (ES cell), places incubator 3 days by about 900/30 μ l cell inoculations.(embryoid bodies, hanging drop EBs) change in the culture dish that fills 10ml differentiation culture liquid, continue suspension culture 1 day will to form embryoid body.Added in second day and cultivated altogether by reagent thing isobavachin and ES cell embryoid body (Ebs) suspension 4 days.Again EBs is changed in the Tissue Culture Plate that overlays poly-ornithine.The index of estimating neural like cell be occur cell process length be cell space diameter 3-5 doubly more than.
The present invention's drug effect to isobavachin on the cell aspect has been carried out strict demonstration, and the large number of biological information of wherein containing has been carried out preliminary discussion, and following usefulness is specifically arranged:
1. the present invention has opened up the new purposes of isobavachin, and promptly induced dry-cell (comprising embryonic stem cell, neural stem cell, mesenchymal stem cells MSCs) directed differentiation is the single type neurocyte.
2. isobavachin can be used as the short cell differential agent of cellular replacement therapy neurodegenerative diseases and reconstruction injured nerve tissue.The cellular replacement therapy neurodegenerative diseases is in the experimentation stage (containing clinical and experimental study) at present, and the intervention of the short cell differential agent isobavachin of expectation will help the research in this field.
3. one aspect of the present invention is clear and definite possesses the composition isobavachin of pharmacologically active, and for the Chinese herb prevention effect provides material base, the new drug that has an independent intellectual property right for modernization of Chinese medicine exploitation of illustrating of drug effect mechanism is offered reference on the other hand.
Description of drawings
Fig. 1 is the short Neural Differentiation effect of isoamylene radical chromocor compounds isobavachin.
Fig. 2 is a flow cytometry detection by quantitative nestin positive neurons precursor ratio.
The cell positive that Fig. 3 induces the ES cell differentiation to become for isobavachin is expressed neurocyte hypospecificity albumen.
Fig. 4 induces the neurocyte β-tubulinIII of ES cell differentiation and neurogliocyte specificity glial fibrillary acidic protein (GFAP) mRNA for isobavachin and expresses.
The specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.Should be understood that in addition following preferred specific embodiments only illustrates, but not limit the scope of the invention by any way.
Embodiment 1: the in-vitro directed research that is divided into neurocyte of isobavachin inducing embryo stem cell (embryonic stem cell, ES cell)
1.ES the cell support is cultivated:
Mouse ES cells D3 cell strain (ES-D3 cell) is in basal medium (containing high sugared DMEM, non essential amino acid, 10% hyclone, leukaemia inhibitory factor), by 1 * 10
6The density of individual/ml is inoculated on the feeder layer cells (l cell), the cultivation of going down to posterity in 2～3 days.
2. isobavachin induces the ES cell directional to be divided into the cultivation of neurocyte:
The trypsinization of ES-D3 cell, be made into single cell suspension with division culture medium (contain high sugared DMEM, non essential amino acid, 10% hyclone, do not contain leukaemia inhibitory factor), be inoculated on the culture dish cover inner surface by about 600/30 μ L, the lid upset is formed hanging drop, put in the incubator and cultivated 3 days.After this (embryoid bodies, hanging drop EBs) change in the culture dish that fills the 10mL division culture medium, continue suspension culture 1 day will to form embryoid body.Adding isobavachin in second day cultivated 4 days altogether with the suspension of ES cell embryoid body.Afterwards, embryoid body is transferred in 96 orifice plates that overlay poly-ornithine and laminin, every hole adds 100 μ L ES cell differentiation culture medium (DMEM/F12+N2/B27+1% hyclone) and isobavachins again.Begin this moment with inverted microscope observation of cell every day, to catch the initial time that neural like cell (length of cell process is more than 5 times of cell space diameter) occurs.
Referring to Fig. 1, by the embryoid body of above experiment acquisition, if neural like cell (length of cell process is more than 5 times of cell space diameter) can occur, then with this index as the directed differentiation neuroblast.Observe by phase contrast microscope, it is very obvious that the result shows that isobavachin induces the ES cell directional to be divided into the neurocyte effect, compare with the solvent control group, when itself and ES cell embryoid body are hatched 8-10 days altogether, just there is 80% above embryoid body neural like cell, One's name is legion to occur, the neurocyte projection is closeer, interconnect and reticulate, around embryoid body, sprawl distribution, slowly to remote extension.A among Fig. 1: solvent control (low power), B: solvent control (high power), C: isobavachin 0.1mol/L (low power), D: isobavachin 0.1mol/L (high power).
Embodiment 2: immunofluorescence experiment identifies that it is neural precursor that isobavachin is induced ES cells in vitro directed differentiation
The ES cell differentiation is early stage, and the triploblastica structure is in the continuous differentiation, and wherein the formation of neural precursor is that cell differentiation is the committed step of neurocyte.Nestin (nestin) is the marker protein of neural precursor, and we investigate the difference of breaking up neural precursor under the early stage isobavachin inducing action as sign.The result shows, at 8+0 days, nestin immunostaining positive cell had than evident difference after isobavachin is induced, and flow cytometry detection by quantitative result shows, isobavachin can significantly increase the ES cell differentiation and next nestin positive neurons precursor ratio, referring to Fig. 2.Fig. 2 is that isobavachin can significantly increase the ES cell differentiation and next nestin positive neurons precursor ratio chart, and (the positive contrast of tretinoin RA) wherein Fig. 2 A is streaming testing result figure; Fig. 2 B is the quantitative analysis bar diagram of A figure.
Embodiment 3: immunofluorescence experiment identifies that it is neurocyte that isobavachin is induced ES cells in vitro directed differentiation
Collect 8+6 days isobavachin of differentiation and induce the group cell sample, with the fixing 10min of pure cold methanol, reuse Ox blood serum sealing treatment 30min, adding one then resists: (1) neurogliocyte specific proteins monoclonal mouse anti-GFAP, dilution in 1: 200, (2) neuronal cell specific proteins monoclonal mouse anti-β-tubulin III, dilution in 1: 100,4 ℃ of overnight incubation.Adding two in second day resists: FITC-Conjugated goat anti-Mouse IgG or Rhodamine-Conjugated goatanti-Rabbit IgG, dilution in 1: 1000 is hatched 1.5h for 37 ℃.Use fluorescence microscope, Taking Pictures recording after the no fluorescence glycerol mounting.
Referring to Fig. 3, the result shows, isobavachin was induced the ES cell differentiation 8+6 days, have male neurogliocyte of neurogliocyte specificity glial fibrillary acidic protein (GFAP) and neuronal cell specificity tubulin (the male neuronal cell of β-TubulinIII), wherein see so that β-TubulinIII positive cell more, present red fluorescence, be distributed in the clone edge more, cell space is circular, oval, projection be one to several, length scale differs, and its elongated protrusion is connected with each other to join and is woven into the netted of complexity, and they may be the neuronal cells of different developmental phases; A few cell is expressed neurogliocyte mark GFAP, presents green fluorescence.A among Fig. 3: β-tubulin III positive cell (redness), B:GFAP positive cell (green).
It is neurocyte that embodiment 4:RT-PCR experimental identification isobavachin is induced ES cells in vitro directed differentiation
Collect differentiation d 8+6 and induce group and solvent control group cell, extract total mRNA according to the Trizolreagent description by isobavachin.Primer sequence and experiment condition following (table 1):
Table 1. primer sequence and PCR reaction condition
The PCR reaction system: 94 ℃ of degeneration 30s, 72 ℃ are extended 60s, and β-tubulinIII, GFAP, β-actin annealing temperature are respectively 58 ℃, 58 ℃, 55 ℃, circulate respectively 40,40,30 to take turns, and last circulates back 72 ℃ and extends 10min again, 4 ℃ of cooling preservation.PCR product 1.5% agarose gel electrophoresis is observed and photographic analysis with gel imaging system.
Referring to Fig. 4, experimental result shows that induce the ES cell differentiation in the time of 8+6 days through isobavachin, β-tubulin III and GFAP gene have expression, and expression and the apparent in view increase of solvent control group.
Embodiment 5: (bone mesenchymal stemcells, BMSCs) directed differentiation is the research of neurocyte to isobavachin inducing bone mesenchymal stem cell
BMSCs has self replication and two characteristics of multidirectional differentiation potential of stem cell, can derive from ectoderm and mesoblastic histo-differentiation to multiple connective tissue and part, comprising neurocyte.
1.BMSCs separation and Culture: get rat femur, tibia bone marrow places centrifuge tube, micro pipette is inhaled repeatedly and is blown bone marrow, form dispersive single cell suspension, add the PERCOLL separating medium of preparation, density is 11073g/mL, centrifugal 2000r/min, as seen middle behind the 20min have the thick white layer of the about 1～2mm of one deck, and its composition is mainly MSCs, carefully draws this one deck with suction pipe, after reuse PBS is centrifugal, by 1 * 10
4Individual/mL cell density is seeded in the DMEM culture medium that contains 10%FBS and cultivates.
2. will reach the BMSCs in the 3rd, 5,7 generations according to 0.4 * 10
6/ ml is inoculated in preparation cell climbing sheet in six orifice plates that are placed with the disinfection cap slide in advance.When cell reaches 70%～80% fusion, abandon culture fluid, add after the L-DMEM culture fluid contain bFGF and 20%FCS induces 24h in advance, change culture fluid, PBS washes twice, add the variable concentrations isobavachin more respectively BMSCs is induced differentiation, adopt immunocytochemistry to detect the specific expressed albumen of neurocyte.
Embodiment 6: the isobavachin inducing nerve stem cell orienting is divided into the research of neurocyte
Neural stem cell (neural stem cells, NSCs) be the cell that nervous system keeps multiplication capacity and differentiation potential throughout one's life, can be by symmetry or asymmetric division, generate the daughter cell that new stem cell and differentiation potential reduce gradually, finally generate neuron, astrocyte and oligodendrocyte.
Get pregnant 13 days tire Mus brains, with Mechanical Method it is prepared into single cell suspension under aseptic condition, add and contain B27, the DMEM/F12 culture fluid of bFGF and insulin is cultivated.NSC cultivated after 7～10 days, formed neural ball, was the suspension growth.Collect the NSCs colony, behind the centrifugal 5min of 800r/min, be inoculated in behind the counting in advance in 6 orifice plates of handling with poly-D-lysine, the DMEM/F12 differentiation culture liquid that adding contains the variable concentrations isobavachin carries out inducing culture.Adopt immunocytochemistry to detect the specific expressed albumen of neurocyte.
Isobavachin is that this process of neurocyte has remarkable induction of differentiation to cell differentiation of nerve cord.It is the novel differentiation agent of inducing of action target spot with the nerve that isobavachin is expected to become a kind of.Because in isobavachin induced dry-cell directed differentiation is to be rich in the neurocyte process to grow the dependency bio information, can provide a lot of target spots to use for evaluating drug effect, the index that therefore neurodevelopment dependency special gene and protein expression etc. can be able to be reflected the neurocyte function, by technology such as PCR, SABC, Western Blot the drug effect of isobavachin is carried out deep evaluation, to prove the facilitation of isobavachin to nerve growth and function improvement.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The sequence that the present invention relates to
＜110〉Zhejiang University
＜120〉isobavachin induced dry-cell directed differentiation is the purposes of neurocyte
<160>6
<210>1
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to β-actin mRNA sequential design detects the forward primer sequence
<400>1
TGACGGGGTCACCCACACTGTGCCCATCTA?30
<210>2
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to β-actin mRNA sequential design detects the downstream primer sequence
<400>2
CTAGAAGCATTTGCGGTGGACGATGGAGGG?30
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to β-tubulinIII mRNA sequential design detects the forward primer sequence
<400>3
GGTGTCCGAGTACCAGCAGT 20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to β-tubulinIII mRNA sequential design detects the downstream primer sequence
<400>4
GAAGAGCACCAGAGACCCAG 20
<210>5
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to GFAP mRNA sequential design detects the forward primer sequence
<400>5
AAGCTCCAAGATGAAACCAACCTGA?25
<210>6
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PCR according to GFAP mRNA sequential design detects the downstream primer sequence
<400>6
GCAAACTTAGACCGATACCACTC 23
Claims (4)
1. the purposes of an isobavachin aspect the in-vitro directed differentiation of induced dry-cell, it is characterized in that, at the in-vitro directed single type neurocyte that is divided into, as the application in the short cell differential agent of preparation treatment neurodegenerative diseases, described stem cell is meant mouse embryo stem cell, neural stem cell or mesenchymal stem cells MSCs when stem cell transplantation.
2. the purposes of an isobavachin aspect the in-vitro directed differentiation of induced dry-cell, it is characterized in that, as the application in the short cell differential agent of preparation reconstruction injured nerve tissue, described stem cell is meant mouse embryo stem cell, neural stem cell or mesenchymal stem cells MSCs when stem cell transplantation.
3. the purposes of an isobavachin aspect the in-vitro directed differentiation of induced dry-cell is characterized in that, uses in making up medicine efficacy screening and evaluation model, and described stem cell is meant mouse embryo stem cell, neural stem cell or mesenchymal stem cells MSCs.
4. according to the application of the arbitrary described isobavachin of claim 1-3, it is characterized in that: described isobavachin can the in-vitro directed differentiation of induced dry-cell obtain the single type neurocyte, realizes by following steps:
(1) adopt classical 4-/4+ method to induce differentiation culture, mouse embryo stem cell is pressed 900/30 μ l cell inoculations in the culture dish interior surface, the upset back forms hanging drop, placed incubator 3 days, (2) hanging drop that will form embryoid body changes in the culture dish that fills 10m1 differentiation culture liquid, continued suspension culture 1 day, added in second day and cultivated altogether by reagent thing isobavachin and embryoid body suspension 4 days, (3) more established embryoid body is changed in the Tissue Culture Plate that overlays poly-ornithine, the index of estimating neural like cell be occur cell process length be cell space diameter 3-5 doubly more than.
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