CN103127164A - Application of k-carrageenan oligosaccharides capable of adjusting activated states of gitter cell - Google Patents
Application of k-carrageenan oligosaccharides capable of adjusting activated states of gitter cell Download PDFInfo
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Abstract
The invention discloses application of k-carrageenan oligosaccharides capable of adjusting activated states of a gitter cell. The k-carrageenan oligosaccharides is used for the gitter cell in a quiescent condition, can promote cell multiplication and improve cell viability, and can further effectively prevent lipopolysaccharide from excessively activating activity of the gitter cell, and reduce damage of peripheral nerve cells because of excessive multiplication of the gitter cell and release of inflammatory factors. The k-carrageenan oligosaccharides can be used for preparing medicine for treatment of nervous system disease and prevention of the nervous system disease which are mediated by the gitter cell. The k-carrageenan oligosaccharides can also be used for improving immunity of organisms by promoting multiplication of immune cells such as the gitter cell. As an agent for treatment of the nervous system disease and an agent for prevention of the nervous system disease, the application of the k-carrageenan oligosaccharides capable of adjusting the activated states of the gitter cell can be added to health food for preventing the nervous system disease and improving the immunity of the organisms and drugs for preventing the nervous system disease and improving the immunity of the organisms, and can also be added to any liquid food and solid food.
Description
Technical field
The present invention relates to a kind of kappa-carrageenin oligose of scalable microglial activation state in the application of medicine and field of health care food, especially in the purposes of the aspects such as anti-autoimmune systemic disease, ischemia resisting reperfusion injury, infection and anti-chronic inflammatory reaction, reach as health food in the purposes aspect the raising immunity of organisms.
Background technology
Carrageenan (Carrageenan) is the polysaccharide of a kind of sulfur acid group of extracting from red algae.Kappa-carrageenan is to be repeated to form by 3,6-inner ether-α-D-galactose and 4-sulphuric acid-β-D-galactose unit.Have research to find that sulfated polysaccharide has relatively unique biological activity, Carrageenan sugar is with respect to polysaccharide, and structure is homogeneous more, and the molecular weight less is soluble, is easilier utilized by body, also is easier to separation and purification, is convenient to analyze.Research in recent years shows, Carrageenan sugar has the various active such as immunomodulating, antitumor, antioxidation, antiviral, anticoagulation.At present, the main method of polysaccharide degraded has chemical degradation, mechanical degradation and enzymatic degradation.Additive method is arranged relatively, and enzymatic degradation has the advantages such as product is single-minded, reaction condition is gentle, power consumption is few, is with a wide range of applications.
Carrageenan sugar can be used as a kind of immunomodulator, under different experiment conditions, can optionally show promotion and inhibitory action to macrophage, Carrageenan sugar has regulating action preferably to mice with radiation injury T cell function and hypotype, promote or induce relevant cell factor to play a role, improve body's immunity, radiation damage is had obvious protective action.The biological activity of Carrageenan sugar is relevant to the quantity of its sulfate group and position, and Carlucci etc. studies show that to only have sulfate group position and the quantity of Carrageenan sugar suitable, just show antiviral activity.And the antiviral dominant mechanism of Carrageenan sugar be itself with negative charge can with virus with positive charge zone combination, thereby stop viral specific region and Cell binding, suppress virus infected cell, play antiviral effect.Studies show that, Carrageenan sugar also has outstanding performance aspect immunomodulating.Carrageenan sugar can suppress the growth of mouse S 180 sarcoma, and mice H22 hepatocarcinoma is also had obvious growth inhibited effect, but the growth of the weight of animals is not but had obvious inhibitory action; Carrageenan sugar can also increase the weight of immune organ Thymus and spleen, and the spleen of raising tumor-bearing mice is thymus index particularly, and this phenomenon shows that it has immunological enhancement.In addition, Carrageenan sugar has significant regulating action to T cell function and the hypotype of mice with radiation injury.Carrageenan sugar can also suppress the propagation of broiler chicken intestinal, promotes the intestinal microvillus growth promoter, improves immunocompetence and fertility performance.
Microglia (microglia, MG) be the main immunocyte of central nervous system, its effect is main similar to macrophage, as central nervous system's main immunocyte, under normal circumstances, microglia remains static, its form is " branched ", the microglia form that activates is " shaft-like " or " circle shape ", and the activated form microglia is " amoebiform ", has phagocytic function.The activation microglia is found around the denatured neurons of many nervus centralis diseases such as the pathological changes such as Alzheimer, parkinson disease, amyotrophic lateral sclerosis and multiple sclerosis, participating in downright bad neuronic removing, is the effector lymphocyte of neuronal degeneration, necrosis.Immunoreation is a kind of important defense mechanism of body, can remove exotic antigen material and the interior damaged cell of body etc. by some inflammatory factor mediation, central nervous system disease is often with a large amount of necrosis and the apoptosis of neuronal cell, and a large amount of microglias that activate of gathering around these necrotic lesions, and the cytokine of its secretion can be detected, the cytotoxic factor of its release also will cause further damage to neuronal cell.There are some researches show, microglia existing Nutrition in vivo also has cytotoxicity.The inflammatory reaction of this microglia mediation is considered to the mechanism that multiple nervous system disease occurs or worsens.Microglia is main effects cell in the inflammation that causes such as nervous system primary disease or infection, can produce cytokine profiles, as NO, tumor necrosis factor (TNF-α), interleukin-6 (IL-6) etc., promote generation and the development of inflammatory reaction, resist former substance reaction very rapid, the physical factors such as electromagnetic radiation, microwave also can make the MG activation.Under normal circumstances, owing to having some intrinsic polyphenoils in body, the ROS that produces can kill and wound the exotic disease pathogenic microorganism but can not damage body by auxiliary body, yet can produce excessive ROS in inflammatory cell, the oxidative stress that this ROS brings out can cause the damage of cell and tissue.And the antioxidant activity of Carrageenan sugar may have certain inhibitory action to the burst size of the ROS of inflammatory cell.When the central nervous system is inflamed when reaction, the microglia of tranquillization is activated, and the mononuclear cell of invading with blood circulation is engulfed nervous tissue's fragment of degeneration jointly.After the damage recovery from illness, they revert to again microglia.
The pathology damage of the multiple disease of brain of infective agent wide participation, its mechanism of action are mainly by activating microglia, induce microglia to express nitricoxide synthase (NOS) and activation nadph oxidase, generating a large amount of O
2-And NO, both further react and generate the stronger material-peroxynitrite of toxicity.At pathogenic infection in earlier stage, microglia can pass through the antigen presentation activated T cell, thus the inflammatory reaction of mediation body.Infect the later stage, a large amount of cytotoxic factor because microglia discharges as NO, TNF-α etc., can cause excessive damage to neurocyte.At nervous system, NO participates in the pathological process of growth promoter, synapse transmission, neural plasticity, learning and memory and some neurodegenerative diseases.Studies show that in recent years, microglial activation plays a crucial role in the immunoreation that central nervous system infection causes.Because the central nervous system has blood brain barrier as first immunity defence line, therefore generally pathogenic microorganism is difficult to enter the central nervous system by the blood circulation approach.But under pathologic condition, some pathogenic microorganism materials can enter by blood brain barrier, and microglia can be activated rapidly, produces a series of inflammatory factors, cause that immunoreation is to remove antigen.Microglia is activated, breeds by a large amount of after viral infection as antigen presenting cell important in brain, and cell surface major histocompatibility antigen (MHC) is expressed to be increased, the performance antigen presentation.Suppress or reduce microglia to NO and O
2-Generation, should be the effective ways that the protection oligodendroglia precursor is avoided infective agent infringement in theory.
Suitably suppress the effect of microglia in central nervous system disease, limit it to neuronic damaging action, help to slow down some chronic therapeutic effect that carries out the process of disease and high acute central nervous system disease is provided.By suitable route of administration, kappa-carrageenin oligose can suitably suppress because lipopolysaccharide (LPS) stimulates the microglia that is activated, but do not affect the immunological effect of himself, suppress excessive immunoreation to the injury of body, thereby reach the effect of health care.Because kappa-carrageenin oligose is by extracting in natural red algae, make through after enzymolysis, safety is easy to get relatively, have broad application prospects, and microglia all participates in immunoreation in various diseases, be the potential target of Treatment of Central Nervous System Diseases and prevention, kappa-carrageenin oligose be applied to microglia will obtain gratifying effect.
At present, the pathogenesis of common central nervous system disease is studied clear not yet fully, but microglia plays vital effect in its course of disease, microglia can be engulfed impaired cell debris on the one hand, remove antigenic substance, on the other hand, the cytotoxic factor that discharges after microglia is activated can increase the weight of the damage of neurocyte again.It is the cell that to breed due to the neuron majority, in case being difficult to self, damage repairs, therefore, suitably suppress the effect of microglia in central nervous system disease, limit it to neuronic damaging action, help to slow down some chronic therapeutic effect that carries out the process of disease and high acute central nervous system disease is provided.Originally studies show that, by suitable route of administration, kappa-carrageenin oligose can suitably suppress because LPS stimulates the microglia that is activated, but does not affect the immunological effect of himself, suppress excessive immunoreation to the injury of group, thereby reach the effect of health care.
Summary of the invention
In the present invention, the structural formula of kappa-carrageenin oligose is by 3,6-inner ether-α-D-galactose and 4-sulphuric acid-β-D-galactose unit repeats to form, the degree of polymerization is the mixture of 2-10, it is characterized in that containing sulfate group as main functional group, its pivotal role aspect the excessive activation that suppresses microglia and raising immunity of organisms.
A kind of application of regulating the kappa-carrageenin oligose of microglial activation state, kappa-carrageenin oligose acts on the microglia of quiescent condition, can promote the propagation of cell, improves cell viability.Simultaneously, kappa-carrageenin oligose also can effectively suppress the activity through LPS excessive activation microglia.The main realization is, the hyper-proliferative that suppresses microglia, reduce the ability that it discharges inflammatory factor, as NO, TNF-α, IL-10 etc., therefore can weaken the damage of the peripheral nerve-cell that the release due to the hyper-proliferative of microglia and inflammatory factor causes.Although this explanation kappa-carrageenin oligose can make the microglia vigor strengthen, obviously be weaker than the pathological state that the exotic antigen such as LPS causes, and can control the microglia vigor by dosage.Therefore, nervous system disease can be treated and prevent to kappa-carrageenin oligose.
The present invention is to be the clinical method for the treatment of and preventing the nervous system disease of microglia mediation that provides, and kappa-carrageenin oligose can be used for preparing the medicine of the nervous system disease for the treatment of and preventing to be mediated by microglia.The nervous system disease of microglia mediation comprises the diseases such as Alzheimer, parkinsonism, epilepsy, ischemical reperfusion injury and infection.
The present invention can promote the propagation of the immunocytes such as microglia, is used for improving immunity of organisms.
The present invention is as treatment and prevent nervous system disease preparation and immunomodulator can add the prevention nervous system disease to and improve in the health food and medicine of immunity of organisms, also can add in the middle of various liquid and solid food.
Description of drawings
The inverted microscope observed result of Fig. 1 kappa-carrageenin oligose on the impact of microglia propagation.The microglia of state of activation is as shown in black arrow.A: Normal group; The B:LPS group; C, D:KCOS, DKCOS independent role group; E, F: protect in advance (12h) through KCOS, DKCOS, LPS activation group (12h); After G, H:LPS activate (12h), KCOS, DKCOS effect group (12h).a，P＜0.05vs.control；b，P＜0.05vs.LPS?group；c，P＜0.05vs.KCOS?or?DKCOS?group。
The mtt assay testing result of Fig. 2 kappa-carrageenin oligose to the microglia effect of vigor.A: protect in advance (12h) through KCOS, DKCOS, LPS activation group (12h); After B:LPS activates (12h), KCOS, DKCOS effect group (12h); C:KCOS, DKCOS independent role group.a，P＜0.05vs.control；b，P＜0.05vs.LPS?group；c，P＜0.05vs.KCOS?or?DKCOS?group。
Fig. 3 kappa-carrageenin oligose discharges the Griess method testing result of NO amount impact on microglia.A:KCOS, DKCOS protect (12h) in advance, LPS activation group (12h); B: microglia discharges the ability of NO.a，P＜0.05vs.control；b，P＜0.05vs.LPS?group；c，P＜0.05vs.KCOS?or?DKCOS?group。
Fig. 4 kappa-carrageenin oligose discharges the ELISA method testing result of TNF-α amount impact on microglia.a，P＜0.05vs.control；b，P＜0.05vs.LPS?group；c，P＜0.05vs.KCOS?or?DSK?group。
Fig. 5 kappa-carrageenin oligose discharges the ELISA method testing result of IL-10 amount impact on microglia.a，P＜0.05vs.control；b，P＜0.05vs.LPS?group；c，P＜0.05vs.KCOS?or?DKCOS?group。
Fig. 6 detects FITC-KCOS (100 μ g/ml) and microglia combination with laser co-focusing.
Microglia combination after Fig. 7 detects Carrageenan sugar (100 μ g/ml) and activates 30min through LPS (1 μ g/ml) with laser co-focusing.
Fig. 8 detects FITC-KCOS (100 μ g/ml) and the microglia combination that activates through LPS (1 μ g/ml, 30min) with laser co-focusing.
Fig. 9 Flow cytometry LPS (1 μ g/ml) is on the impact of FITC-KCOS (100 μ g/ml) with the microglia combination.A, green (groupl): after LPS effect 15min, add FITC-KCOS combined effect 60min; A, red (group2): after LPS effect 15min, remove FITC-KCOS effect 60min after LPS; B: fluorescence intensity quantitative analysis.a：P＜0.01VS?control；b：P＜0.01VS?group1。
The specific embodiment
The invention will be further elaborated below in conjunction with embodiment:
Embodiment 1: the impact of kappa-carrageenin oligose (KCOS) on microglia propagation situation
1.1, kappa-carrageenin oligose is on the inverted microscope observed result of microglia propagation impact
Cell culture condition: IMDM (sigma, USA) culture medium contains 10% calf serum, 100 μ g/ml streptomycins and 100IU/ml penicillin, 5%CO
2, 37 ℃, pass every three days once generation.Test the previous day, N9 is changed liquid.Clean 3 times with PBS, 0.125% trypsinization, piping and druming, making cell density with the IMDM culture medium that contains 10% hyclone is 5 * 10
4The cell suspension of individual/mL.In 96 well culture plates, every hole adds 200 μ l cell suspension, after cultivating 12h, the careful culture fluid of drawing, PBS cleans, and experimental group every hole adds KCOS and the DKCOS (the desulfurization acid groups derivant of kappa-carrageenin oligose) of variable concentrations, makes its final mass concentration be respectively 100 μ g/L.After cultivating 12h, discard culture medium, add every hole to add to contain 1 μ g/L LPS fresh culture 200 μ l, blank is for dosing not and process without LPS, and positive control is through LPS processing 12h but not dosing.Experiment in contrast, experimental group activates 12h with LPS in advance, then processes respectively with kappa-carrageenin oligose and derivant thereof, and whether drug level and processing time are constant, observe oligosaccharide and to the effect of microglia be and LPS competes bind receptor.
Result: enzymatic degradation product and desulfurization acid product are carried out the sulfate assay, kappa-carrageenan sulfate group content is 20% left and right, after the enzymatic degradation that this laboratory makes, KCOS sulfate group content is 21.72% (± 2.14), illustrate that enzymatic degradation does not obviously destroy oligosaccharide structure, the DKCOS sulfate group content that obtains after the desulfurization acid treatment is 12.92% (± 1.85).The microglia that is in quiescent condition is fusiformis or branched, (as accompanying drawing 1A), and the microglia that is activated is ellipse or amoebiform, (as accompanying drawing 1B).As processing 12h through LPS again through KCOS and DKCOS difference pretreatment 12h, be in the microglia that activates shape and obviously reduce (as accompanying drawing 1C, D).Activate through LPS as microglia, then process respectively through KCOS and DKCOS, the quantity that is in the microglia of state of activation does not obviously reduce (as accompanying drawing 1E, F).Process respectively 12h with KCOS and DKCOS separately, although there is the microglia of some to be activated, quantity is than LPS group few (as accompanying drawing 1G, H).
1.3, kappa-carrageenin oligose is to the mtt assay testing result of microglia effect of vigor
Cell processing procedure such as example 1.2, experimental group every hole add KCOS and the DKCOS of variable concentrations, make its final mass concentration be respectively 10,50,100,200 μ g/L, 4h before stopping cultivating, and every hole adds MTT (O.5%) 20 μ l.After cultivating end, the careful suction abandoned supernatant, and every hole adds 150 μ l dimethyl sulfoxide, vibration is dissolved purple crystal fully, survey the light absorption value (measuring wavelength 490nm, reference wavelength 630nm) in each hole on microplate reader, represent with percentage ratio after result is compared with the blank group.
Result: LPS is the important component part of gram-negative bacteria cell wall, but the inducing immune cells activation, and the microglia multiplication capacity through activating obviously strengthens.As shown in accompanying drawing 2A; LPS can effectively activate microglia; cell viability is improved; and through KCOS and DKCOS pre-protection respectively; can effectively suppress the cell viability that LPS causes increases, and this depression effect demonstrates the concentration dependence, although and the oligosaccharide derivative of removing sulfate group has inhibitory action; but action effect weakens relatively, illustrates that oligosaccharide may be relevant with sulfate group to the inhibitory action of cell viability.But through the microglia that LPS activates, KCOS and DKCOS can not weaken its state of activation (as accompanying drawing 2B).Through KCOS and the DKCOS microglia vigor enhancing of individual processing respectively, but be weaker than LPS group (as accompanying drawing 2C), illustrate that kappa-carrageenin oligose can improve immunity of organisms, and the raising of this ability can't make the microglia overactivity, causes the further damage of body.
Embodiment 2: the impact of kappa-carrageenin oligose on the cytokine of microglia secretion
2.1 kappa-carrageenin oligose discharges the Griess method testing result of NO amount impact on microglia
NO in vivo or very easily be oxidized to NO in aqueous solution
2-, under acid condition, NO
2-With diazol sulfanilamide generation diazo reaction, and generate diazonium compound, the latter further with naphthyl vinyl diamidogen generation coupled reaction, production concentration and NO that this reaction generates
2-Concentration has linear relationship, at the 540nm place, maximum absorption band is arranged.Adopt the Griess method to measure NO/NO
2-, the supernatant of getting cell culture adds equal-volume Griess reagent (1.0% anhydrous p-anilinesulfonic acid., 0.10%N-1-naphthodiamide hydrochlorate, 2.5% phosphoric acid), surveys the absorbance at 540nm place.
Result: NO is that microglia participates in immunoreactive important cytokine, and after microglia was activated, the expression of NO can significantly increase.LPS can effectively induce microglia induced expression type nitricoxide synthase (iNOS), only is induced to activate under the stimulation of inflammatory factor, can induce to generate a large amount of NO, produces obvious neurotoxicity.Use the NO content in Giress method detection cell culture supernatant.As shown in Figure 3, the microglia that Carrageenan sugar can suppress to induce through LPS is expressed NO, thereby reduces cell viability.
2.2 kappa-carrageenin oligose discharges the ELISA method testing result of TNF-α, IL-10 amount impact on microglia
Use ELISA test kit (R﹠amp; D Systems, Minneapolis, MN) detect the burst size of TNF-α, IL-10.Strict by specification operation, and use microplate reader to measure absorbance at 450nm.In sample, TNF-α and IL-10 concentration can be read by the standard curve that standard substance are made.
Result: the microglia after being activated by LPS can discharge inflammatory factor, and as NO, TNF-α, IL-10 etc., these inflammatory factors can further be strengthened the carrying out of inflammatory reaction.If protect in advance 12h with Carrageenan sugar, re-use LPS and activate microglia, can see that the burst size of TNF-α and IL-10 is suppressed, and this depression effect has dosage correlation (as accompanying drawing 4,5).The reagent of inducing microglial activation that adopts in the present invention is LPS, it does not have obvious cytotoxic effect to neurocyte, but can cause immunoreation by activating microglia, the microglia of activation can discharge inflammatory factor, the damage of mediation neuronal cell.LPS itself is with negative charge, thereby can produce the inflammation factor with the Cell binding inducing cell.As with Carrageenan sugar, microglia being protected in advance, can effectively stop LPS and Cell binding, and Carrageenan sugar desulfurization acyloxy derivatives, due to its with melanoma cells, so protective effect weakens relatively.This phenomenon also can be explained the microglia that activates with LPS simultaneously, then adds the Carrageenan sugar effect, can not play the effect of obvious inhibition cell viability.Illustrate the immunoregulation effect of Carrageenan sugar and its with sulfate group in close relations.
Embodiment 3: kappa-carrageenin oligose acts on the mode of microglia
3.1 laser co-focusing detects the kappa-carrageenin oligose of FITC labelling to the effect of microglia
Fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) sterling is yellow or orange-yellow crystalline powder, soluble in water and spirit solvent.Molecular weight is 389.4, and the absorption maximum optical wavelength is 490～495nm, and the emission maximum optical wavelength is 520～530nm, presents bright yellow-green fluorescence, is present most widely used fluorescein.Because kappa-carrageenin oligose itself has negative charge, therefore can mutually combine with charge effect with FITC, can be used for indicating the interaction of oligosaccharide and cell under fluorescence microscope.
3.1.1FITC labelling kappa-carrageenin oligose:
With kappa-carrageenin oligose with etc. quality FITC mixing, be dissolved in a certain amount of distilled water, use magnetic stirring apparatus at room temperature to stir 12h, with the bag filter dialysis of solution in 500 molecular cut offs, extracellular fluid dialysis is single water that steams, be placed in 4 ℃, every 8h changes extracellular fluid dialysis one time, detects extracellular fluid dialysis under uviol lamp, when can't detect fluorescence, stop dialysis, with gained dialysis solution lyophilisation, 4 ℃ keep in Dark Place.
3.1.2 laser confocal microscope detects:
Collection is in the N9 cell of exponential phase, and adjusting its density is 5 * 10
4Individual/ml is connected in 24 well culture plates, and every hole adds 1ml cell suspension, in 37 ℃, hatches 12h in the incubator of 5%CO2, carefully draws culture fluid, and PBS cleans 2 times, and creep plate is hatched the fixed time with FITC-KCOS/LPS respectively under 4 ℃.PBS with pre-cooling washs creep plate 3 times.The 4% paraformaldehyde fixative that creep plate is placed in pre-cooling is 30min fixedly.Observe under laser confocal microscope and take pictures.The excitation wavelength of FITC is 495nm, and emission wavelength is 520nm.Overall process is carried out under the lucifuge condition.
Result: laser co-focusing detects the kappa-carrageenin oligose of FITC labelling to the effect of microglia
FITC is a kind of green fluorescence dyestuff, can be combined with KCOS by charge effect, indicates its behavior in microglia.Laser co-focusing experimental results show that the interaction of fluorescently-labeled kappa-carrageenin oligose (FITC-KCOS) and microglia, as shown in Figure 6, FITC-KCOS 4 ℃ acting on cell 10min before with it in conjunction with less, as effect 30min, on most of oligosaccharide gathering and cell membrane, small part enters cell, and during effect 60min, the oligosaccharide major part has entered cell.For whether the effect of further verifying LPS and KCOS has competitive relation, we act on the microglia that activates through LPS with FITC-KCOS.As shown in Figure 7, activate microglia 30min through LPS, then act on respectively microglia 5-60min with FITC-KCOS, oligosaccharide enters the process of microglia and obviously slows down.During 30min, FITC-KCOS obviously assembles and cell surface, can clearly observe the appearance of circulus, and after 60min, oligosaccharide has begun to enter cell.
Results suggest, As time goes on, KCOS can enter cell by cell membrane, if still surface of cell membrane is in conjunction with LPS, entering of KCOS will be obstructed.The research report is arranged, after LPS induces, having specific binding receptor TRL4 at cell surface expresses, may there be competitive relation in KCOS and LPS, can infer that it is to enter cell interior in conjunction with protein mediated its that the microglia surface may exist KCOS specific, regulates the immunoregulatory activity of cell.
In order further to verify the motor process of FITC-KCOS in microglia, microglia is removed FITC-KCOS after FITC-KCOS effect and microglia 30min, continue to cultivate respectively 30min, 60min.PI is a kind of red fluorescence dyestuff, position that can clear showed cell core, as shown in Figure 8, begin to enter in cell after FITC-KCOS effect 30min, after continuing cultivation, beginning is assembled in core, infer according to experimental result, KCOS can enter the nucleus performance and regulate immunoreactive effect.
3.2 the effect of the kappa-carrageenin oligose of Flow cytometry FITC labelling to microglia
Collection is in the N9 cell of exponential phase, and adjusting its density is 5 * 10
4Individual/ml is connected in Tissue Culture Flask, and every hole adds 8ml cell suspension, in 37 ℃, and 5%CO
2Incubator in hatch 12h, carefully draw culture fluid, PBS cleans 2 times, collecting cell makes 1 * 10 with cell
6The cell suspension of individual/ml is hatched the fixed time with FITC-KCOS/LPS under 4 ℃.With the PBS of pre-cooling washing 3 times, the 4% paraformaldehyde fixative that is placed in pre-cooling is 30min fixedly, 300 purpose screen filtrations, and flow cytometer detects.Overall process is carried out under the lucifuge condition.
Result: the laser confocal microscope detection validation KCOS can be combined with microglia, and can enter cell interior assembles to nucleus, in order to verify further whether KCOS and LPS have competitive relation, adopt flow cytometry detect respectively LPS and FITC-KCOS co-cultivation and act on cell with FITC-KCOS separately.As shown in Figure 9, the existence of LPS can affect cell to the combination of KCOS, than removing the LPS group, KCOS and LPS co-cultivation group fluorescence intensity a little less than, hint that there are competitive relation in LPS and KCOS.
Claims (5)
1. application of regulating the kappa-carrageenin oligose of microglial activation state, it is characterized in that: kappa-carrageenin oligose can act on the microglia of quiescent condition, can promote the propagation of cell, improves cell viability; Simultaneously, also can effectively suppress activity through LPS excessive activation microglia.
2. a kind of application of regulating the kappa-carrageenin oligose of microglial activation state according to claim 1, it is characterized in that it can suppress the hyper-proliferative of microglia, reduce the ability that it discharges inflammatory factor, weaken the damage of the peripheral nerve-cell that the release due to the hyper-proliferative of microglia and inflammatory factor causes, can be used for preparing treatment and prevention by the medicine of the nervous system disease of microglia mediation.
3. the disease that participates in 2 described nervous system by the microglia mediation according to claim 1, comprises: Alzheimer, parkinsonism, epilepsy, nervous system infection and ischemical reperfusion injury class disease.
4. kappa-carrageenin oligose can promote the propagation of the immunocytes such as microglia according to claim 1, is used for improving immunity of organisms.
5. according to claim 1-5 is described, and kappa-carrageenin oligose can add the prevention nervous system disease to and improve in the health food and medicine of immunity of organisms, also can add in the middle of various liquid and solid food.
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| CN108210504A (en) * | 2017-12-27 | 2018-06-29 | 中国科学院海洋研究所 | The application of sulphation galactooligosacchari(es and pharmaceutical composition |
| CN111303310A (en) * | 2020-03-12 | 2020-06-19 | 大连大学 | Preparation method of marine sulfuric acid galactohexaose and application of marine sulfuric acid galactohexaose in preparation of medicines for resisting Alzheimer disease |
| CN114209645A (en) * | 2021-12-17 | 2022-03-22 | 大连大学 | Preparation method of CS-KOS temperature-sensitive hydrogel for slowly releasing k-carrageenan oligosaccharides |
| CN114209645B (en) * | 2021-12-17 | 2023-11-07 | 大连大学 | Preparation method of CS-KOS temperature-sensitive hydrogel for slowly releasing k-carrageenan oligosaccharides |
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