CN103060271A - Application of naringin in promoting proliferation and differentiation of neural stem cell - Google Patents
Application of naringin in promoting proliferation and differentiation of neural stem cell Download PDFInfo
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- CN103060271A CN103060271A CN2011103236243A CN201110323624A CN103060271A CN 103060271 A CN103060271 A CN 103060271A CN 2011103236243 A CN2011103236243 A CN 2011103236243A CN 201110323624 A CN201110323624 A CN 201110323624A CN 103060271 A CN103060271 A CN 103060271A
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Abstract
The invention belongs to the field of regenerative medicine and pharmacology, relates to an application of a double hydrogen flavonoid small molecule compound, and particularly relates to a new application of naringin in promoting proliferation and differentiation of a neural stem cell. The invention is characterized by relating to an application of the naringin in medicament for neural stem cell transplantation in the treatment of neurodegenerative diseases, an application of the naringin in repairing damaged nerve tissues and preparation a function-reconstructable nerve nutriment and cell differentiation agent, and an application of the naringin in constructing an efficient medicament screening model and applying the efficient medicament screening model to preliminary screening and evaluation of the medicament efficacy. The invention confirms the new pharmacological activity of the naringin, establishes an application foundation in stem cell transplantation (SCT), and provides new thoughts for developing new medicaments with independent intellectual property rights.
Description
Technical field
The invention belongs to regenerative medicine and area of pharmacology, relate to a kind of purposes of Flavanones micromolecular compound, particularly naringin is promoting the neural stem cell in-vitro multiplication and is inducing new purposes aspect the differentiation.
Background technology
Naringin (Naringin) claim again naringin, naringin, aurantiin, mainly is present in the pericarp and pulp of rutaceae natsudaidai, tangerine, orange.Naringin also is the main effective constituent of herbal medicine Rhizome of Fortune's Drynaria, the dried immature fruit of citron orange, Fructus Aurantii, dried tangerine peel.In each kind of plant there be than big difference with the difference in kind, the place of production naringin content, common immature fruit content is higher.
The chemical structural formula of naringin is as follows:
Molecular formula: C
27H
32O
14
Molecular weight: 580.54
The preparation of naringin: take Pummelo Peel as raw material.Get Pummelo Peel 20 grams, add 400mL distilled water, direct heating extracted 1 hour; Suction filtration is got residue while hot, adds 300mL distilled water, direct heating 30 minutes; Merging filtrate is placed, suction filtration, and drying gets the naringin crude product, and the water recrystallization must be made with extra care naringin.
Pharmacological action: naringin is a kind of flavanone compounds.Owing to do not have conjugation fully between A ring and the B ring, so at 282nm strong ultraviolet absorption peak is arranged, make naringin show various biological activity and pharmacological action.Have report to show that rat skin lower injection 100mg/kg naringin has obvious anti-inflammatory action, the naringin of 200mg/L concentration has very strong restraining effect to vesicular stomatitis virus.Naringin also can reduce blood viscosity, reduce the formation of thrombus, and the effect of analgesia, calmness and stronger increase laboratory animal choleresis is arranged.Naringin also has desensitization and antianaphylaxis, blood circulation promoting spasmolysis, improves the performance of local microcirculation and nutrition supply, to promoting drug excretion, releasing Streptomycin sulphate the infringement of the 8th pair of cranial nerve, the toxic side effect of alleviation Streptomycin sulphate is had unique curative effect.
Have no so far both at home and abroad naringin is being promoted the neural stem cell in-vitro multiplication and inducing research report aspect the differentiation.
Summary of the invention
The invention discloses a kind of new purposes of naringin, find that naringin has the effect of the cell proliferation of nerve cord of remarkable promotion vitro culture, make it continue to keep vegetative state, and can be divided under optimum conditions the neurone of function, can be used for neural stem cells transplantation, treat nerve degenerative diseases, and can be used as neurenergen and the cell differential agent of neurotization and reconstruction.
An object of the present invention is to utilize naringin induced nerve stem cells vitro directed differentiation, the neurone that function is arranged that obtains can be used for making up high efficiency medicaments sifting model, and utilizes this model to carry out preliminary screening and the evaluation of medicine effect.
Technical scheme of the present invention comprises following content:
(1) Isolation and culture of neural stem cell and evaluation
Select the execution of craning one of 14 days (E14d) mouse of embryo, the Embryonic mesencephalon tissue is taken out in aseptic technique, makes the isolated cell suspension.In serum-free DMEM/F12 substratum, adopt the suspension culture mode to cultivate, add EGF and bFGF in the nutrient solution.After 7～10 days former culture formed " neural stem cell ball " is separated the cultivation of going down to posterity.Getting s-generation cell cultures identified after 7～10 days.Adopt immunocytochemistry to identify neural stem cell: the cell of Nestin staining examine neural stem cell, GFAP staining examine astroglia cell, TuJ1 staining examine neurone, GalC staining examine oligodendrocyte and BrdU dye marker propagation.
(2) naringin is to the proliferation of the neural stem cell of vitro culture
With the neural stem cell that naringin directly acts on former generation or goes down to posterity, experiment is divided into five groups: control group, 5x10
-7Mol/L naringin group, 5x10
-6Mol/L naringin group, 5x10
-5Mol/L naringin group, 5x10
-4Mol/L naringin group.In external cultivate respectively 4 days and 7 days after microscopically observe, adding the neural stem cell ball showed increased of cultivating under the naringin condition, statistical analysis is when the concentration of naringin surpasses 5x10
-6When mol/L was above, the formation number of neural ball was apparently higher than control group.CCK-8 result shows that also the cell survival rate of naringin group is all apparently higher than control group.
(3) to induce the cell differentiation of nerve cord of vitro culture be the neurone that function is arranged to naringin
The neural ball that experiment adopts following two kinds of conditions to cultivate: normal cultivation group (without the neural ball of any processing), naringin propagation group (the neural ball after naringin is processed).The neural stem cell ball of cultivating under the different condition is blown and beaten into single cell suspension respectively in division culture medium (DMEM/F12+10% foetal calf serum), being inoculated in poly-l-lysine processed in 24 orifice plates of circle slide, the equal adherent growth of visible cell after 24 hours, after adding the naringin processing, continue to cultivate 7 days, detect neuronic specific marker thing with immunocytochemistry, the expression of the specific marker thing of star spongiocyte and the specific marker thing of oligodendrocyte, and counting is divided into neurone, the percentage of star spongiocyte and oligodendrocyte.The result shows that it is in good condition to add the neure growth that forms after the cell differentiation of nerve cord of naringin, and neuronic number is more than control group.And for the neural ball of propagation after being processed by naringin, it is divided into the neuronic ratio of function apparently higher than the neural ball of normal cultivation, the differentiation capability that shows the neural stem cell of propagation after naringin is processed is not subject to obvious impact, and more is conducive to the direction differentiation of neuralward unit.
Effect of the present invention and benefit are that the drug effect to naringin has been carried out strict demonstration on the cell aspect, and the large number of biological information that wherein contains has been carried out further investigated, and advantage is remarkable:
1. the present invention has opened up the new purposes of naringin, and namely naringin has the effect of significantly short cell proliferation of nerve cord, makes it continue to keep vegetative state, and can be divided under optimum conditions the neurone of function.
2. naringin can be used for the Neural Stem Cells ' Transplantation nerve degenerative diseases, also can be used as neurenergen and the cell differential agent of neurotization and reconstruction.The cellular replacement therapy nerve degenerative diseases is in the experimental study stage at present, and the research of naringin will help the deep development in this field.
3. to adopt a kind of traditional Chinese medicine monomer become to assign to propagation and the directed differentiation of induced nerve stem cells be the neurone that function is arranged in the present invention, substitute cell or the dead cell of damaged, for the preventive and therapeutic effect of Chinese medicine provides material base, the new drug that has an independent intellectual property right for the modernization of Chinese medicine, for exploitation of illustrating of Pharmacodynamical mechanism is offered reference on the other hand.
4. the present invention utilizes the neurone that function is arranged that naringin induced nerve stem cells vitro directed differentiation obtains, and is further used for making up high efficiency medicaments sifting model, and the new drug development that can be the treatment nerve degenerative diseases provides detection platform.
Description of drawings
Fig. 1 is the impact of naringin vitro culture of the present invention " neural ball " propagation, is the displaing micro photo figure of s-generation Culture of neural stem cells after 4 days of former culture.A is control group among the figure; B is 5X10
-5Mol/L naringin group.
Fig. 2 be naringin of the present invention on the impact of the neural stem cell viability of vitro culture, be that naringin promotes the dose-effect figure of cell proliferation of nerve cord.Each experimental group of the numeral of axis of abscissa among the figure (wherein 0: control group; 1:5x10
-7Mol/L naringin group; 2:5x10
-6Mol/L naringin group; 3:5x10
-5Mol/L naringin group; 4:5x10
-4Mol/L naringin group), length axis represents the cell proliferation percentage (* P＜0.05, expression is with the significant difference between the control group) of neural stem cell.
Embodiment
Describe specific embodiments of the invention in detail below in conjunction with technical scheme and accompanying drawing.
Separation and Culture and the evaluation of embodiment 1 external neural stem cell
(1) separation and the cultivation of the neural stem cell in midbrain source
Get the execution of craning one of E14d mouse, the embryo is taken out in aseptic technique, separates Embryonic mesencephalon tissue, be digested to unicellular after, trypan blue is counted, with 2x10
5Individual/mL is inoculated in 37 ℃, 5%CO in the T25 culturing bottle
2Suspension culture, stem cell media are DMEM/F12 (1: 1), 2%B27, bFGF (10ng/mL), EGF (20ng/mL).Amount was changed liquid in per 3～4 days half, cultivated number and the size of observing the suspension cell ball of growth after the week.Collect neural ball, after machinery is blown and beaten into single cell suspension, place and continue under the similarity condition to cultivate, determine whether go down to posterity depending on growing state.
(2) continuous passage of cell subclone and induce differentiation
Take out single larger neural stem cell ball (diameter is about about 150 μ m) with pasteur pipet from culturing bottle, machinery is blown and beaten into single cell suspension, is inoculated in the stem cell media number of observation of cell growing state and neural stem cell ball.After seven days, from culturing bottle, take out the soft machinery piping and druming of single larger stem cell ball (DMEM/F12+10% foetal calf serum) in division culture medium with pasteur pipet again, disperse as far as possible neural ball, and be inoculated in poly-l-lysine processed the circle slide 24 orifice plates in, continue to cultivate 7 days, amount was changed liquid in 3～4 days half.
(3) evaluation of neural stem cell
The neural stem cell ball of former culture, after going down to posterity, be seeded in the culture dish of 35mm that the bottom scribbles poly-lysine, cultivate after 3 days, Paraformaldehyde 96 with 4% (prepares with 0.1M PBS, pH 7.2～7.4, fixing 30min after the 0.1M PBS flushing 3 times, carries out neural stem cell marker Nestin and the cytochemical staining of BrdU antibody mediated immunity.
Experimental result: in former culture neural stem cell after 7～9 days, can obtain in a large number not of uniform size, the clone ball that is suspension growth.The cultivation of going down to posterity after the former generation single clone ball mechanical separation can form new cell clone (subclone).These clone balls show that through the dyeing that is positive of Nestin and BrdU Identification of the antibodies they are neural stem cell, have the division growth ability.
(1) experimental design and drug treating
Experiment is divided into five groups, is respectively control group, 5x10
-7Mol/L naringin group, 5x10
-6Mol/L naringin group, 5x10
-5Mol/L naringin group, 5x10
-4Mol/L naringin group.The s-generation neural stem cell of former culture is collected, with 2x10
5The concentration of individual/mL is inoculated in 24 orifice plates, and every hole 500uL processes for every group and establishes 6 multiple holes, cultivates respectively the survival rate of observing afterwards number and the cell of neural ball in 4 days and 7 days.
(2) detection method of cell proliferation of nerve cord
1. neural ball count method: the s-generation neural stem cell of former culture is respectively after cultivating 4 days and 7 days under the effect of different concns naringin, double-blind method is counted the formation number of neural ball, 10 visuals field are counted in every hole at random under the 10x20 fluorescent microscope, and its mean value is calculated in every group of 6 holes.Relatively use non-matching Student ' s T-test between group.
2. CCK-8 method: the proliferation rate of cell adopts the CCK-8 colorimetric determination.Control group be normal control and blank (normal control is for adding the medicine group, blank for experimental port parallel, except not containing the identical control wells of other condition of extracellular).CCK-8 colorimetry ultimate principle is: containing its chemical name of WST-8[in this reagent is: 2-(2-methoxyl group-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-and 2H-tetrazolium list sodium salt], it is reduced to the yellow Jia Za dyestuff with high water soluble by the desaturase in the cell mitochondrial under the effect of electron carrier 1-methoxyl group-5-toluphenazine methyl-sulfate.The quantity of the first Za thing that generates is directly proportional with the quantity of viable cell, carries out colorimetric in microplate reader, the power of the large I reflection cell metabolic activity of the OD value of examining.Every hole adds 50uLCCK-8 (final concentration is about 0.5mg/mL) in 5%CO during detection
2, cultivated 3 hours for 37 ℃, measure 450nm with microplate reader, reference wavelength is the OD value of 630nm, calculates the proliferation rate of cell.
The proliferation rate of cell=(OD
Medicine feeding hole-OD
Medicine feeding hole is blank)/(OD
Control wells-OD
Control wells is blank) x100%
Experimental result: neural stem cell is after cultivating 4 days and 7 days under the naringin effect, and microscopically is observed, compared with the control, and the quantity showed increased of neural ball.Statistical analysis, naringin is at 5x10
-6～5x10
-4In the concentration range of mol/L, can obviously promote the propagation of neural ball.5x10
-4The naringin effect of mol/L is after 4 days, and the neural nodule number order of propagation is 3.1 times (seeing Fig. 1) of normal control, also is significantly improved with the viability of like cell, and the proliferation rate of cell has reached 200 ± 8.3% (seeing Fig. 2) of contrast.
(1) naringin is to the differentiation of inducing of neural stem cell
With the s-generation neural stem cell ball of former culture with through 5x10
-4The neural stem cell ball of the naringin effect of mol/L after 7 days collected, and blows and beats into single cell suspension respectively in division culture medium (DMEM/F12+10% foetal calf serum), with 2x10
5The concentration of individual/ml is inoculated in poly-l-lysine and processed in 24 orifice plates of slide, and every hole 500uL processes for every group and establishes six multiple holes.The equal adherent growth of visible cell after 24 hours is added different concns naringin (5x10
-6, 5x10
-5, 5x10
-4Mol/L) after the processing, culture plate is placed saturated humidity, 5%CO
2, 37 ℃ of incubators continue to cultivate 7 days.
(2) Immunofluorescence dyeing
After cultivating end, take out the cover glass in each hole of 24 well culture plates.Paraformaldehyde 96 with 4% (prepares with 0.1M PBS, pH 7.2～7.4) fixing 30min, 0.01M after the PBS flushing 3 times, each group is carried out Immunofluorescence dyeing, detects the expression of the specific marker thing (GalC) of the specific marker thing (GFAP) of neuronic specific marker thing (TuJ1), star spongiocyte and oligodendrocyte.
(3) cell counting
Under the 10x20 fluorescent microscope, the cell of the Immunofluorescence dyeing TuJ1 positive is counted (with 0.25mm
2The zone is as tally's area).Every group of cover glass that 6 culture of neural stem cells neural are arranged chosen 4 tally's areas at random on each cover glass, the percentage of counting cells sum and stained positive cell count and stained positive cell.The percentage of same method counting GFAP and GalC stained positive cell count and stained positive cell.
Experimental result: naringin can promote effectively that cell differentiation of nerve cord is the neurone that function is arranged.It is in good condition to add the neure growth that forms after the cell differentiation of nerve cord of naringin, the naringin experimental group of different concns, cell differentiation of nerve cord is that neuronic percentage all is higher than control group (P＜0.05), but does not have significant difference between the naringin experimental group of different concns.And for the neural ball of propagation after being processed by naringin, it is divided into the neuronic ratio of function apparently higher than the neural ball of normal cultivation.The differentiation capability that shows the neural stem cell of propagation after naringin is processed is not subject to obvious impact, and more is conducive to the direction differentiation of neuralward unit.
Although the present invention promoting the neural stem cell in-vitro multiplication and inducing the purposes aspect the differentiation to describe as example take naringin, this description and not meaning that is construed as limiting the present invention.With reference to description of the invention, other distortion of the cell of other kind and embodiment all can be expected for those skilled in the art.Therefore, claim restricted portion and spirit under such distortion can not break away from.
Claims (2)
1. a naringin is promoting the neural stem cell in-vitro multiplication and is inducing purposes aspect the differentiation, make the neural stem cell of naringin effect vitro culture, induce its propagation and be divided into the neurone of function, it is characterized in that: the application in Neural Stem Cells ' Transplantation nerve degenerative diseases medicine; Repair injured nerve tissue and the neurenergen of reconstruction and the application in the cell differential agent in preparation; Make up high efficiency medicaments sifting model, and utilize this model in the preliminary screening of medicine effect and the application in estimating.
2. a kind of naringin according to claim 1 is characterized in that promoting the neural stem cell in-vitro multiplication and inducing purposes aspect the differentiation naringin concentration that the vitro culture neural stem cell is used is 5x10
-6～5x10
-4Mol/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109498653A (en) * | 2017-09-12 | 2019-03-22 | 中国科学院苏州纳米技术与纳米仿生研究所 | Promote composition, its application and the evaluation method of cerebral injury neural restoration |
| CN110938599A (en) * | 2019-12-30 | 2020-03-31 | 南昌诺汇医药科技有限公司 | Culture method of neural stem cells and application thereof |
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| CN101104845A (en) * | 2007-04-16 | 2008-01-16 | 大连理工大学 | Use of protocatechuic acid in accelerating nerve stem cell multiplication in vitro and inducement differentiation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109498653A (en) * | 2017-09-12 | 2019-03-22 | 中国科学院苏州纳米技术与纳米仿生研究所 | Promote composition, its application and the evaluation method of cerebral injury neural restoration |
| CN110938599A (en) * | 2019-12-30 | 2020-03-31 | 南昌诺汇医药科技有限公司 | Culture method of neural stem cells and application thereof |
| CN110938599B (en) * | 2019-12-30 | 2020-09-29 | 马旭艳 | Culture method of neural stem cells and application thereof |
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