CN104020292A - Method for researching NSCS differentiation regulation and control of HBO by using functional proteome technology - Google Patents
Method for researching NSCS differentiation regulation and control of HBO by using functional proteome technology Download PDFInfo
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Abstract
A method for researching the regulation and control of HBO to NSCS differentiation by functional proteome technology includes in-vivo research and in-vitro research, preparing cerebral ischemia and anoxia rat model, differentiating the double-label neural stem cells into neuron, oligodendrocyte and astrocyte by immunofluorescence staining, obtaining differentiated neural cells and undifferentiated neural stem cells by laser capture microdissection, extracting the total protein of each component of each cell by protein sequential fractional extraction, searching the attribution of determined protein by Internet, and performing protein identification and function analysis by bioinformatics technology and MS-Fit software method.
Description
Technical field
The present invention relates to a kind of test method, particularly relate to a kind of by the function protein technique research HBO method that differentiation regulates and controls to NSCS.
Background technology
Neural stem cell (NSC) is that a class is present in the cell in nervous system with self-renewal capacity and multi-lineage potential, they can be divided into neuron, astroglia and oligodendroglia under certain condition, and performance has corresponding form and Electrophysiological characteristics.Through the research of more than 10 years, the neural stem cell that it is found that grow up vertebrate and adult concentrates on three specific regions in brain: one is to be positioned at ventricles of the brain district and ventricles of the brain inferior segment (SVZ), the mixed cellularity group that the ependymocyte in these two regions is made up of neuroblast, they can move to olfactory bulb and produce precursor, astrocyte; Another is connecting the region of telocoele and olfactory bulb; The 3rd is hippocampus.The discovery of neural stem cell makes growth, the neural regeneration etc. of people to central nervous system have new understanding, for the treatment of cranial vascular disease has brought new hope.There are some researches show when in brain, there is some pathological change or under some Cytokine these NSC be activated, breed, migrate and break up.Cerebral ischemia not only can be impelled the nerve stem cell proliferation of dentate fascia (DG), also can affect the neural stem cell of ventricles of the brain inferior segment, and the propagation of ischemic side cerebral hemisphere NSC does not have increase yet, and wherein ischemic side cerebral hemisphere NSC propagation is the most obvious.Research also finds that cerebral ischemia can activate NSC differentiation, and the neuron that substitutes and repair damage of the neuron of differentiation energy part.More than studies confirm that in cerebral ischemia situation, NSC can be activated, bred, move and break up, illustrated that NSC can participate in the pathophysiological process of cranial vascular disease.Thereby point out us, inquire into propagation to NSC of the molecular mechanism of nerve stem cell proliferation and differentiation and intervention means (as hyperbaric oxygen, rehabilitation etc.) and the facilitation of differentiation and neuron regeneration and be of great significance for the research tool of cranial vascular disease.
Along with finishing successfully of the Human Genome Project (HGP), make life science enter the genome times afterwards comprehensively, study hotspot is transferred to proteomics, studies have found that between the existence of gene and the existence of diversity and protein and diversity be unbalanced.Therefore, utilizing genomic achievement in research to carry out large-scale proteome research has become inevitable.First Australian scholar Wilkins in 1994 and Williams propose " protein group " concept (PROTEOME), proteomics is exactly from overall angle, protein composition, expression and the decorating state of dynamic change in analysis of cells, understand the interaction between protein and contact, disclosing a new research field of protein function and cell activities rule.Can find the protein relevant with pathological change and disease specific protein, these protein both can be given a clue for understanding disease pathogenesis, also can be used as the molecular labeling of medical diagnosis on disease, can also be as the target spot for the treatment of and drug development.
Proteomics refers to expression pattern and the functional mode of all protein of genomic expression, its content comprises the expression of identification of protein, existing way (modified forms), structure, function and interaction etc., and in view of the difficulty of proteomics research, many scholars have proposed the Research Thinking of funcational protenome, study at special time, the active protein of expressing of genome under specific environment and experiment condition, funcational protenome is a part for gross protein group, by the research to funcational protenome, can not only illustrate the function of a certain colony protein, also can study a certain physiology, the situation of protein expression under pathological state, enrich gross protein group database, and finally depict the proteomics close to " all protein ".In the great vital movements such as Cell Differentiation, protein not only has the difference of expression time, expression, and there is environmental stimulus to external world, comprise the stimulations such as physiological signal, pathological signals and extraneous physics, chemical factor, actively aitiogenic ability, this is that biological phenomena is different from one of essential characteristic of non-biological phenomena.
Hyperbaric oxygen (HBO) refers to that body is breathed in hyperbaric environment and the pure oxygen of environment equipressure, and utilizes the method that sucks hyperbaric oxygentherapy disease to be called hyperbaric oxygen ation.A large amount of Clinical and experimental studies confirm that HBO can improve oxygen content and the oxygen reserve of brain tissue, reduce brain cell because of anoxic degeneration necrosis; Improve aerobic metabolism, reduce anerobic glycolysis, reduce lactate concentration in brain, thereby correct acidosis, improve environment in brain; The collapsible cerebrovascular, reduces the volume of blood flow of brain tissue, thereby alleviates encephaledema, reduces intracranial pressure; Can improve the content of superoxide dismutase (SOD), strengthen removing free radical and oxidation resistance, reduce the damage of pouring into again brain tissue; " ischemic penumbra " function be can recover, recovery and the regeneration of neurocyte promoted.At a specified future date and recent clinical and experiment all shows, and the acute symptom of hyperbaric oxygen to cranial vascular disease and motion, sensation, language, intelligence and the memory disorders of rear something lost have good curative effect.Research recently finds that HBO can mobilize the stem cell in marrow, makes the CD34+ cell in blood peripheral circulation increase by 8 times, and stem cell factor increases by 50%, and height ratio VEGF expression 2 and SDGF acceptor; Can raise the expression of neurotrophic factor-3, Basic Fibroblast Growth Factor, and the change of the concentration of these factors can affect cell peripheral microenvironment and activate NSC propagation and differentiation; Can increase the expression of cerebral hypoxia ischemia neural stem cells in rats Nestin albumen, Brdu immunostaining positive cell is rolled up; Our preliminary experiment has also shown that hyperbaric oxygen can raise brain source property Differentiation of Neural Stem Cells.Therefore, we think that HBO must cause molecule, organelle, the structure of integral multi-layered and the change of functional status in realizing from hereditary information to allomeric function in the effect in NSCs intervention, and determine that the structure of these aspects and the basis of function are genes, and directly determinative is mainly protein.Therefore taking protein expression as index, taking protein regulation change and functionalized modification as research direction, carry out the research of hyperbaric oxygen too many levels, the corrective action of many target spots, be expected the Study on mechanism of hyperbaric oxygentherapy cranial vascular disease to make a breakthrough.
The morbidity rate of China's cranial vascular disease increases year by year at present, and hyperbaric oxygentherapy has obtained important progress to the improvement of acute neuro-protective and sequal of cerebrovascular diseases, but the molecular mechanism of neural stem cell differentiation regulation and control is understood really seldom.This project adopts the methods such as functional proteomics technology and bioinformatics technique, from in vitro and in the molecular mechanism of research hyperbaric oxygen to neural stem cell differentiation regulation and control aspect two of body experiments, set up the technology platform such as functional proteomics and bioinformatics, utilize this platform that many target spots of hyperbaric oxygen, multipath effect feature and protein expression are associated, more different differential expression spectrum, corresponding protein expression target spot, illustrates the mechanism of action of hyperbaric oxygentherapy.For hyperbaric oxygentherapy cranial vascular disease is opened up new research and application, simultaneously for solid theoretical foundation is established in its clinical practice.
Summary of the invention
Cerebral hypoxia ischemia animal model is made: except Normal group, sham-operation group, all the other respectively organize equal modeling, and cerebral hypoxia ischemia model adopts classical Rice model; Hyperbaric oxygen processing: rat model is made in rear 3h, 6h, 12h, 24h and put into hyperbaric oxygen chamber, and pressure is 0.2MPa, and in cabin, oxygen concentration is 80%, each 1h, 1 time/d, 7d continuously; The two marks of immunofluorescence dyeing: Qu Gezu rat cerebral tissue, fix with paraformaldehyde, brain tissue after fixing brain hat is carried out to routine dehydration, the transparent rear paraffin embedding of dimethylbenzene, section, adds respectively primary antibodie Brdu, Nestin, GFAP, Tuji, Galc, and 4 DEG C are spent the night, use again PBS rinsing, two anti-what add Cy3/FITC mark, 37 DEG C of incubation 2h, observe the differentiation situation of neural stem cell with immunofluorescence microscopy; Adopt laser capture microdissection technology free each group of fluorescently-labeled various cells respectively, adopt protein ordinal ranking extraction process to extract respectively the gross protein of the each component of cell, language function protein technique and bioinformatics technique research hyperbaric oxygen affect the expression of neural stem cell differentiation associated protein;
Vitro study comprises the following steps:
Culture of neural stem cells: separate newborn rat brain tissue under aseptic condition, machinery is divided into 1mm
3size tissue, adds nutrient solution to blow and beat into single cell suspension, and the cell suspension inoculation that takes a morsel, in culture flask, is added with the nutrient solution of the DMEM/F12 of bFGF and B27, is placed in 5%CO
2in incubator, after neural ball forms, mechanical separating clone is made single cell suspension again, part cell suspension inoculation is cultivated in new culture flask, after 7d, mechanical separating clone goes down to posterity 1 time, method is the same, by cultivate the 3rd generation neural stem cell make single cell suspension, be divided in different 25ml culture flasks, be divided into Normal group, model control group, high concentration oxygen group, pressure-air group, hyperbaric oxygen group, 1 bottle/group, manufacture stem cell hypoxic-ischemic model: except Normal group, the equal modeling of all the other each groups, in the time that the neural stem cell of cultivating reaches 80% fusion, be changed to fresh complete medium, discard nutrient culture media to replace fresh serum-free DMEM/F12 nutrient culture media next day, and cell is placed in to 93%N
2, 5%CO
2, 2%O
237 DEG C of cultivations of incubator, neural stem cell acts on 3h, 6h, 12h, 24h in this environment, then Brdu is used to related experiment after hatching altogether 48h, the mensuration of neural stem cell vigor is got 1ml cell suspension from every group and is moved into 96 hole ELISA Plate, cell density is 103～104, every hole cell, every hole adds the MTT solution 20ul of 5mg/ml, hatch 4h for 37 DEG C, stop cultivating, suck culture supernatant, every hole adds 150ulDMSO, concussion 10min fully dissolves crystal, select 490nm wavelength, on enzyme-linked immunosorbent assay instrument, measure each hole optical density value, induced nerve stem cells differentiation under hyperbaric oxygen: by above-mentioned hyperbaric oxygen group hypoxic-ischemic cell suspension inoculation in scribbling 24 well culture plates of poly-D-lysine, a part of cell is carried out after adherent 2h to Nestin immunofluorescence dyeing, another part adds the serum DMEM/F12 nutrient culture media of removing growth factor to continue to cultivate 7d, and it is placed under hyperbaric oxygen environment simultaneously, and pressure is 0.2MPa, each 1h, 1 time/d, 7d continuously.All the other each group be simultaneously as under each group of environment by each group; Neurocyte specific immune fluorescent dye: by the above-mentioned each group of abundant rinsing of cell PBS, fix with paraformaldehyde solution.Add respectively primary antibodie Brdu, Nestin, GFAP, Tuji, Galc, 4 DEG C are spent the night, and use PBS rinsing, then add two of Cy3/FITC mark to resist, and 37 DEG C of incubation 2h, by the differentiation situation of immunofluorescence microscopy observation neural stem cell; Application flow cytometer obtains respectively each group of fluorescently-labeled various cells, adopt protein ordinal ranking extraction process to extract respectively the gross protein of the each component of cell, language function protein technique and bioinformatics technique research hyperbaric oxygen break up relevant protein expression impact to neural stem cell.
Embodiment
The present invention is described in further detail below.
Embodiment 1
Cerebral hypoxia ischemia animal model is made: except Normal group, sham-operation group, all the other respectively organize equal modeling, and cerebral hypoxia ischemia model adopts classical Rice model; Hyperbaric oxygen processing: rat model is made in rear 3h, 6h, 12h, 24h and put into hyperbaric oxygen chamber, and pressure is 0.2MPa, and in cabin, oxygen concentration is 80%, each 1h, 1 time/d, 7d continuously; The two marks of immunofluorescence dyeing: Qu Gezu rat cerebral tissue, fix with paraformaldehyde, brain tissue after fixing brain hat is carried out to routine dehydration, the transparent rear paraffin embedding of dimethylbenzene, section, adds respectively primary antibodie Brdu, Nestin, GFAP, Tuji, Galc, and 4 DEG C are spent the night, use again PBS rinsing, two anti-what add Cy3/FITC mark, 37 DEG C of incubation 2h, observe the differentiation situation of neural stem cell with immunofluorescence microscopy; Adopt laser capture microdissection technology free each group of fluorescently-labeled various cells respectively, adopt protein ordinal ranking extraction process to extract respectively gross protein, language function protein technique and the expression impact of bioinformatics technique research hyperbaric oxygen on neural stem cell differentiation associated protein of the each component of cell.
Vitro study comprises the following steps:
Culture of neural stem cells: separate newborn rat brain tissue under aseptic condition, machinery is divided into 1mm
3size tissue, adds nutrient solution to blow and beat into single cell suspension, and the cell suspension inoculation that takes a morsel, in culture flask, is added with the nutrient solution of the DMEM/F12 of bFGF and B27, is placed in 5%CO
2in incubator, after neural ball forms, mechanical separating clone is made single cell suspension again, part cell suspension inoculation is cultivated in new culture flask, after 7d, mechanical separating clone goes down to posterity 1 time, method is the same, by cultivate the 3rd generation neural stem cell make single cell suspension, be divided in different 25ml culture flasks, be divided into Normal group, model control group, high concentration oxygen group, pressure-air group, hyperbaric oxygen group, 1 bottle/group, manufacture stem cell hypoxic-ischemic model: except Normal group, the equal modeling of all the other each groups, in the time that the neural stem cell of cultivating reaches 80% fusion, be changed to fresh complete medium, discard nutrient culture media to replace fresh serum-free DMEM/F12 nutrient culture media next day, and cell is placed in to 93%N
2, 5%CO
2, 2%O
237 DEG C of cultivations of incubator, neural stem cell acts on 3h, 6h, 12h, 24h in this environment, then Brdu is used to related experiment after hatching altogether 48h, the mensuration of neural stem cell vigor is got 1ml cell suspension from every group and is moved into 96 hole ELISA Plate, cell density is 103～104, every hole cell, every hole adds the MTT solution 20ul of 5mg/ml, hatch 4h for 37 DEG C, stop cultivating, suck culture supernatant, every hole adds 150ulDMSO, concussion 10min fully dissolves crystal, select 490nm wavelength, on enzyme-linked immunosorbent assay instrument, measure each hole optical density value, induced nerve stem cells differentiation under hyperbaric oxygen: by above-mentioned hyperbaric oxygen group hypoxic-ischemic cell suspension inoculation in scribbling 24 well culture plates of poly-D-lysine, a part of cell is carried out after adherent 2h to Nestin immunofluorescence dyeing, another part adds the serum DMEM/F12 nutrient culture media of removing growth factor to continue to cultivate 7d, and it is placed under hyperbaric oxygen environment simultaneously, and pressure is 0.2MPa, each 1h, 1 time/d, 7d continuously.All the other each group be simultaneously as under each group of environment by each group; Neurocyte specific immune fluorescent dye: by the above-mentioned each group of abundant rinsing of cell PBS, fix with paraformaldehyde solution.Add respectively primary antibodie Brdu, Nestin, GFAP, Tuji, Galc, 4 DEG C are spent the night, and use PBS rinsing, then add two of Cy3/FITC mark to resist, and 37 DEG C of incubation 2h, by the differentiation situation of immunofluorescence microscopy observation neural stem cell; Application flow cytometer obtains respectively each group of fluorescently-labeled various cells, adopt protein ordinal ranking extraction process to extract respectively the gross protein of the each component of cell, language function protein technique and bioinformatics technique research hyperbaric oxygen break up relevant protein expression impact to neural stem cell.
The extraction of gross protein with quantitatively: the gross protein in body and the in vitro each component of various cells extracts with reference to " proteomics " operation, and the BradFord method that quantification of protein press described in Ausubel operates.
Two dimensional gel electrophoresis (2-DE technology): gross protein and the Chong Pao liquid that rises is mixed, add IPG to hold in glue groove, be placed on aquation on IPGphor electrophoresis apparatus, isoelectric focusing; Adhesive tape is moved to sds gel upper end, and continuous current electrophoresis, adds 2D standard protein as interior mark in sample.
Gel-colored and image acquisition analysis: the silver ammino solution decoration method of pressing Hochstrasser etc.Molecular imaging instrument transmission scan for gel after dyeing, the digitized image obtaining for file PDQuest (Bio-Rad) carry out software analysis, in utilizing, the mark standard molecular weight of 2D-Marher and isoelectric point are determined molecular weight and the isoelectric point of glue internal protein point.
Protein in situ enzymolysis: the method with reference to Bergman etc. is carried out, getting a device will be put in EP pipe under interested protein spots cutting; Add decolouring working fluid, acetonitrile dehydration repeatedly, adds TPCK-trypsase 10uL to be placed in ice bath, and after enzymolysis, supernatant is transferred in EP pipe, carries out mass spectrophotometry after extract freeze-drying.
MADLI-TOF peptide mass fingerprinting map analysis: by " proteomics " operation, select matrix, sample dissolution; With the ZipTipTMC18 device mouth desalination of Millipore company of the U.S.; The sample preparing can use the ProFlexTM III MADLI-TOF mass spectrometer of Bruker company of the U.S. to analyze, and carries out internal data correction simultaneously.
HPLC-Edman edman degradation Edman is measured the amino acid sequence of polypeptide: by " proteomics " operation.Enzymolysis mixture solution, after centrifugal, go supernatant ABI173AMiroBlotter kapillary HPLC system to separate, sample peak uses ABI173AMiroBlotter to collect on pvdf membrane, blade cuts the pvdf membrane corresponding with going out peak position, the sample on pvdf membrane is carried out to sequencing with ABI475 gas phase protein sequence instrument.
The function of differential protein particle in bioinformatics identification of protein collection of illustrative plates: by " proteomics " operation.Search to determine the ownership of protein by Internet net, use bioinformatics technique and MS-Fit software approach, carry out identification of proteins and functional analysis.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.Above-described embodiment is only explanation technical conceive of the present invention and feature; its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented; can not limit the scope of the invention with this; all equivalences that Spirit Essence is done according to the present invention change or modify; effect is similar, all should be encompassed in protection scope of the present invention.
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