CN102363767A - Neuron-like cells obtained by processing neural stem cells by use of saikoside alpha, development of kit thereof and composition for treating nervous system diseases - Google Patents
Neuron-like cells obtained by processing neural stem cells by use of saikoside alpha, development of kit thereof and composition for treating nervous system diseases Download PDFInfo
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Abstract
The invention provides neuron-like cells (NLC) obtained by processing human neural stem cells by the use of saikoside alpha and a proliferous differentiation medium thereof. The selective medium comprises a liquid basic medium. The volume of the liquid basic medium is reference. The selective medium also contains 1-500 ug/mL of saikoside alpha, 1-20% of fetal calf serum by volume, 5-200ng/mL of human epidermal growth factor and 5-200ng/mL of basic fibroblast growth factor. The invention also provides a method for preparing a saikoside alpha differentiation kit by using the above mentioned differentiation medium, and also provides a pharmaceutical composition for treating nervous system diseases by the use of the neuron-like cells obtained by the above mentioned method. The method by using the selective medium can be adopted to efficiently differentiate neural stem cells so as to obtain the neuron-like cells. The pharmaceutical composition for treating nervous system diseases can be used to repair damaged neural cells in vivo and eliminate neuropathy.
Description
Technical field
The present invention relates to a kind of saikoside α handler NSC and obtain neuron cell (neuron-like cells; NLC) and propagation division culture medium; And the method for test kit development, and the pharmaceutical composition that is used to treat nervous system disorders that comprises the neuron cell that preceding method obtains.More specifically; The present invention relates to the division culture medium that a kind of saikoside α differentiation of human NSC obtains neuron cell; Use this substratum optionally to obtain the method for neuron cell, and the pharmaceutical composition that is used to treat nervous system disorders that comprises the neuron cell that preceding method obtains.
Background technology
The self-replacation that stem cell has and the characteristic of many differentiation potentials make it to become very ideal cell resource.But there are a lot of problem puzzlement clinical treatments in simple neural stem cells transplantation; Multiple complicated factor affecting stem cell transplantation effect; Wherein stem cell survival rate is low in vivo; The cell that the real differentiation of stem cells that arrives the focus position becomes termination function seldom is difficult to form effective result of treatment.Before stem cell transplantation, earlier stem cell is induced into the neural like cell of functional one-tenth, after be transplanted in the body and treat, possibly reach than independent stem cell transplantation better therapeutic.
Neural system has a large amount of neurones.Neurone is the staple of brain, and neuron pool is realized the analytic function of brain through each neuronic message exchange, and then realizes the exchange output of sample; The sample of output is lighted mound feel generation consciousness through connecting the path.The pathology of neuronal cell, sex change or damage etc. must cause various nervous system disorderss, like apoplexy, cerebral plasy, brain injury, hematencephalon, marrow lateral sclerosis disease and Parkinson's disease etc.
For example; CN 1923195A is disclosed to be broken up by the coumarin kind compound inducing nerve stem cell orienting; Can be divided into oligodendrocyte precursor cells, it shows the medicine that can be used as inducing nerve stem cell orienting differentiation, but and unexposed its can directed differentiation be neuron cell.
Existing bibliographical information Chinese medicine can be divided into neuron cell by inducing mesenchymal stem cell, for example Radix Notoginseng total arasaponins, TANSHINONES, thioglycerin and Radix Salviae Miltiorrhizae Injection etc.These reports show that all Chinese medicine can the inducing mesenchymal stem cell commentaries on classics be divided into neuron cell, and differentiation efficiency and the propagation ability that goes down to posterity is not confirmed.
To sum up, need badly a kind of simple to operate, cost is low, differentiation purity is high, proliferate efficiency is high and the method for the acquisition neuron cell of the safety of passage cell stable in properties.
Summary of the invention
The object of the invention breaks up NSC efficiently through Chinese medicine and becomes neuron cell; And propagation is not broken up; The method operation that overcomes existing acquisition and propagation neuron cell is complicated, cost is high; The neuron cell purity that differentiation obtains is low, proliferate efficiency is low, break up sex change easily; And in the substratum of differentiation and breeding use, contain a lot of harmful medicines; Make the shortcoming of differentiation and neuron cell poor stability when being prepared into pharmaceutical composition of obtaining of propagation, a kind of division culture medium of neuron cell is provided, use the method for the differentiation of this substratum and the neuron cell that propagation obtains simple to operate, cost is low, differentiation purity is high, proliferate efficiency is high and the passage cell stable in properties.
The invention provides the division culture medium that a kind of cell differentiation of nerve cord is neuron cell and propagation thereof; Said division culture medium comprises the liquid base substratum; Wherein, Volume with said liquid base substratum is a benchmark, and said division culture medium also contains the saikoside α of 1-500ug/mL, the foetal calf serum of 1-20 volume %, the Urogastrone of 5-200ng/mL and the Prostatropin of 5-200ng/mL.
The present invention also provides a kind of saikoside α differentiation NSC to become the method for neuron cell and propagation thereof, and wherein, said method uses division culture medium of the present invention to cultivate said NSC.
The present invention also provides a kind of saikoside α differentiation NSC to become the method for the test kit development of neuron cell and propagation thereof, and wherein, described test kit comprises:
1) basic medium;
2) saikoside α;
3) cytokine (Urogastrone and Prostatropin);
4) enzyme of peptic cell and;
5) working instructions;
The present invention also provides a kind of pharmaceutical composition that is used to treat nervous system disorders; Wherein, Said pharmaceutical composition comprises the neuron cell that becomes the method for neuron cell and propagation thereof to obtain from cell differentiation of nerve cord according to the invention, and and pharmaceutically acceptable carrier or vehicle.
Use the method that becomes neuron cell and propagation thereof from cell differentiation of nerve cord of the present invention of selection substratum of the present invention; Can efficiently break up and a large amount of propagation neuron cells, and still keep and original proterties through the neuron cell of the propagation that goes down to posterity.Because selection substratum selectivity of the present invention is strong, the neuron cell purity that the cultivation of going down to posterity obtains can reach more than 85%.Therefore; Method of the present invention need not to use the medicine of expensive poor stability such as magnetic bead just can reach more highly purified separating effect; Avoided complicated operation; Thereby reduced cost, and the security of the neuron cell that is obtained by this method improves greatly, can the preparation cost invention be used to treat the pharmaceutical composition of nervous system disorders.The present invention is used to treat the pharmaceutical composition of nervous system disorders, and not only security is good, and can repair the neuronal cell that damages in the nervous system disorders in vivo, thus for the treatment nervous system disorders provides maybe.
Description of drawings
Fig. 1 is the fluorescence immunoassay photo of the neuron cell expressing protein of embodiment 1 and 3 acquisitions; Wherein, Figure 1A is two anti-colour developing fluorescence immunoassay photos behind mouse-anti people microtubule associating albumen-2IgG mark, and Figure 1B is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people nidogen IgG mark;
Fig. 2 is the PCR product gel electrophoresis detection picture of the neuron cell expressing gene of embodiment 1 and 3 acquisitions;
Fig. 3 is the neuron cell transplantation treatment cerebral infarction rat model that uses embodiment 2 and 3 to obtain, and transplants the back and improves curve along with the time variation improves the neurological deficit scoring;
The neuron cell transplantation treatment cerebral infarction rat model of Fig. 4 for using embodiment 2 and 3 to obtain transplanted the back and carried out the brain injury area and improve curve along with the time variation;
The neuron cell transplantation treatment cerebral infarction rat model of Fig. 5 for using embodiment 2 and 3 to obtain transplanted section BrdU positive staining NLC photo (40 times of immunofluorescence microscopy magnifications) in the Ipsilateral striatum of back.
Embodiment
The invention provides the division culture medium that a kind of cell differentiation of nerve cord is neuron cell and propagation thereof; Said division culture medium comprises the liquid base substratum; Wherein, Volume with said liquid base substratum is a benchmark; Said selection substratum also contain the foetal calf serum of saikoside α, the 1-20 volume % of 1-500ug/mL, the Urogastrone of 5-200ng/mL (epidermal growth factor, EGF) with the Prostatropin of 5-200ng/mL (basic fibroblast growth factor, bFGF).
Preferably; Volume with said liquid base substratum is a benchmark, and said selection substratum also contains the saikoside α of 5-300ug/mL, the foetal calf serum of 5-15 volume %, the Urogastrone of 10-100ng/mL and the Prostatropin of 10-100ng/mL.Further preferably; Volume with said liquid base substratum is a benchmark, and said selection substratum also contains the saikoside α of 50-200ug/mL, the foetal calf serum of 5-10 volume %, the Urogastrone of 20-50ng/mL and the Prostatropin of 20-50ng/mL.
Cell differentiation of nerve cord of the present invention is that the division culture medium of neuron cell and propagation thereof can comprise the various minimum mediums that this area is commonly used; For example; Be selected from neuronal cell substratum, BME cell culture medium, Dole's Becquerel improvement Iger (Dulbecco ' s modified Eagle ' s medium, DMEM) one or more in cell culture medium, DMEM/F12 cell culture medium, Fischer ' s cell culture medium, IMDM cell culture medium, 199 cell culture mediums, MEM cell culture medium, F10 cell culture medium, F12 cell culture medium and 1640 cell culture mediums.The said minimum medium that preferably includes is 1: 1 F12 cell culture medium of weight ratio and DMEM cell culture medium.The composition of said minimum medium is known in this field, and the above-mentioned minimum medium name of enumerating is called their trade(brand)name, the commercial confession.
It is the method for neuron cell and propagation thereof from Hu saponin(e α differentiation NSC that the present invention also provides a kind of, and wherein, said method uses division culture medium of the present invention to cultivate said NSC.
Provided by the invention is that the method for neuron cell and propagation thereof goes for various NSCs from saponin(e α differentiation NSC.NSC belongs to the adult multipotential stem cell with certain differentiation degree.Theoretically; The NSC in any source can be used method differentiation of the present invention and propagation obtains neuron cell, and for example marrow, Cord blood, placenta, umbilical cord, miscarriage embryo (the for example embryo in the 12-14 of miscarriage week) and aborted fetus etc. are originated.But consider to run counter to ethics, social custom custom even legal provisions, therefore generally do not utilize miscarriage embryo and aborted fetus, and the source of embryo and aborted fetus is limited.Preferred said in vitro tissue is selected from one or more in marrow, placenta, Cord blood and the umbilical cord.Wherein, marrow and Cord blood can be from public umbilical cord blood bank (for example, Chinese umbilical cord blood bank etc.) and marrow storehouse (for example, Chinese Marrow Donor Program data banks etc.).Abundance considers that preferred said in vitro tissue is a umbilical cord from the source, is processed as Biohazard Waste as the one of which.
Neuron cell differentiation culture system according to the invention comprises 5 * 10
5To 5 * 10
6Individual/ml, preferred 1 * 10
6The neuron cell division culture medium of individual/ml neuron cell and 1mL-100mL, preferred 5-10mL is at 37 ℃, 5%CO
2Cultivate in the incubator, obtain neuron cell by cell differentiation of nerve cord.Use the method for the present invention of division culture medium of the present invention, the neuron cell of breeding still keeps and identical proterties through going down to posterity.For example; No matter be that the neuron cell or their neurofilament protein of neuron cell (NF-M), nidogen (nestin), the microtubule associating albumen-2 (MAP-2) that repeatedly obtains that goes down to posterity that cell differentiation of nerve cord obtains is expressed the positive, and glial fibrillary acidic protein (GFAP) is negative.Detect the standard whether neuron cell break up sex change according to this and judge, the neuron cell that method of the present invention obtains can guarantee not break up sex change at passage number repeatedly.The said neuron cell that repeatedly goes down to posterity and obtain can be as the raw material of preparation pharmaceutical composition of the present invention.In addition, for the ease of transportation and preservation, said neuron cell also can carry out freezing preservation, and before closing on use, recovering gets final product.
The present invention also provides a kind of pharmaceutical composition that is used to treat nervous system disorders; Wherein, Said pharmaceutical composition comprise according to the invention from from Hu saponin(e α differentiation NSC be neuron cell and propagation thereof the neuron cell that obtains of method, and with pharmaceutically acceptable carrier or vehicle.
Said pharmaceutically acceptable carrier or vehicle refer to nontoxic solid-state, semi-solid state or liquid weighting agent, thinner, coating material or other pharmaceutical adjuncts, for example, include but not limited to salt solution, buffering saline solution, water, glycerine, ethanol and composition thereof.Administrations such as said pharmaceutical composition is suitable for that intravenously, waist are worn, canalis spinalis, intervention, intramuscular, subcutaneous, intradermal injection and infusion.
The pharmaceutical composition that is suitable for administration comprises sterilized water body lotion or non-aqueous solution, dispersion liquid, suspension-s or emulsion, and is used for before closing on use in sterile injectable solution or dispersion liquid powder formulated.Suitable water-based or non-aqueous carrier, thinner, solvent or vehicle comprise water, ethanol, Gan Bo, Ucar 35, polyoxyethylene glycol, complete methylcellulose gum, vegetables oil and injectable organic enzyme as mooring sour second vinegar.
These compsns can also contain adjuvants such as sanitas, wetting agent, emulsifying agent and dispersion agent.It possibly be favourable adding isotonic agent such as carbohydrate, sodium-chlor etc.
Preferred said pharmaceutical composition is an injection liquid, and said injection liquid comprises pharmaceutically acceptable carrier, and is benchmark with said injection liquid volume, 1 * 10
5To 1 * 10
8The neuron cell that the method for the present invention of individual/milliliter obtains.Preferred said injection liquid comprises that with said injection liquid volume be benchmark, 5 * 10
5To 1 * 10
7The neuron cell that the method for the present invention of individual/milliliter obtains.Preferred said pharmaceutically acceptable carrier is a saline water.Saline water described here is meant the solution that Physiology Experiment or osmotic pressure commonly used clinically equate with the osmotic pressure of animal or human's body blood plasma.It when preferably, being used for mammal and human body the sodium chloride solution of 0.85-0.9%.More preferably 0.9% sodium chloride solution.
Contriver of the present invention is surprised to find that neuron cell can be repaired (referring to embodiment 5) with losing to neuronal damage.Therefore the present invention is used for treating nervous system disorders that the pharmaceutical composition of nervous system disorders mentions and is selected from apoplexy, cerebral plasy, brain injury, hematencephalon, marrow lateral sclerosis disease and the Parkinson's disease etc. one or more.
The neuron cell that use is obtained by method of the present invention; Be in the division culture medium of the present invention; Therefore before preparation is used to treat the pharmaceutical composition of nervous system disorders, can wash and resuspended said neuron cell with saline water through removing division culture medium of the present invention.The washing times of said saline water can residual nutrient media components decides in the elutant through measuring.The pharmaceutical composition residual nutrient media components when the patient is given in injection that is used to treat nervous system disorders of the present invention can not caused that the patient is uncomfortable to get final product.Said compsn comprises 1 * 10
5To 1 * 10
8Individual/mL new neuron cell of the present invention; Be preferably 1 * 10
6To 5 * 10
7Individual/mL.The application of neuron cell in preparation treatment nervous system disorders medicine that the present invention also provides method of the present invention to obtain preferably, the invention provides the application of neuron cell in preparation treatment apoplexy medicine that method of the present invention obtains.
The present invention also provides a kind of method of treating nervous system disorders, and it comprises to the patient uses the pharmaceutical composition that the present invention is used to treat nervous system disorders.
Use mode that the present invention is used for treating the pharmaceutical composition of nervous system disorders can be selected from get involved that injection, waist are worn, intravenous injection, subcutaneous injection and intradermal injection one or more; The more preferably said mode of using gets involved injection for the brain stereotaxis.The pharmaceutical composition that is used to treat nervous system disorders according to the invention preferably contains 1 * 10
5To 1 * 10
8The neuron cell of cell/mL, its per injection dosage are that 0.1mL-5mL/ is individual; More preferably, the pharmaceutical composition that is used to treat nervous system disorders according to the invention comprises 1 * 10
6To 5 * 10
7The neuron cell of individual/mL, its per injection dosage are that 0.1mL-2mL/ is individual.
Provided the dosage that the pharmaceutical composition that is used to treat nervous system disorders according to the invention is injected each time with last, three injections course of treatment, every double injection is a week at interval.The generally onset in the 3rd day to the 7th day after having injected for the second time of pharmaceutical composition that is used to treat nervous system disorders according to the invention.Said effective standard is: (modified Neurological Severity Scores mNSS) reduces the scoring of improvement neurological deficit gradually, and the brain injury area improves, and is target to reduce or to stop the brain injury area.After finish a course of treatment, can carry out the treatment of second course of treatment at once continuously, the treatment that also can carry out second course of treatment after one to three months at interval is to consolidate curative effect, and general treatment can be eliminated the nervous system disorders illness two to four courses of treatment; Be six months or 1 year pitch time, and the patient is necessary as thinking, also can consolidate the treatment of curative effect.
The preferred in vitro tissue (for example marrow) that uses method of the present invention to separate patient with nervous system disease; The gained neuron cell is prepared into pharmaceutical composition administration patient with nervous system disease of the present invention from body, the spinoff that can avoid immunological rejection to cause to greatest extent like this.
Below, specific embodiment of the present invention is described, but technical scope of the present invention is not limited to these examples.
Embodiment 1:
Present embodiment is used for explaining and uses division culture medium of the present invention from the method for the isolating cell differentiation of nerve cord of people's marrow as neuron cell.
Marrow has been signed Informed Consent Form from the patients with cerebral hemorrhage of donation in 45 years old with this patient and hospital side, when it is performed the operation, gathers its marrow.
Marrow is diluted with 1 * PBS equal-volume; Obtain the marrow diluent; With the marrow diluent: the volume ratio of the Ficoll-Hypaque of 1.077 kilograms per cubic meter (Ficoll-Hypaque) is to join in 50ml centrifuge tube at 2: 1, carries out density gradient centrifugation 20 minutes down at 2000rpm, 25 degree, centrifugal back collection intermediate layer cell; And used 1 * PBS 1000rpm centrifugal 10 minutes; Washing gained cell three times adds perfect medium, contains the DMEM/F12 substratum (1: 1) of 10% foetal calf serum, 20ng/mL Urogastrone and 20ng/mL Prostatropin (U.S. R&D company).After the cell counting, by 3 * 10
5Cell/cm
2Density be inoculated in 75cm
2Culturing bottle in, every bottle adds above-mentioned substratum again to make final volume is 10ml.At 37 ℃, 5%CO
2Incubator in cultivate after the 3rd day, half amount is changed liquid.With the slow sucking-off 5ml of old substratum, add fresh above-mentioned substratum when changing liquid, every bottle adds 5ml, continues at 37 ℃, 5%CO
2Cultivate.When cell attachment growth reach the culturing bottle bottom surface 85% the time; Substratum is inclined; And with cell through use trysinization, 800rpm collected the cultivation of then in containing the DMEM/F12 substratum (1: 1) of 10% foetal calf serum, 20ng/mL Urogastrone and 20ng/mL Prostatropin, going down to posterity in centrifugal 5 minutes.Same when the cell attachment growth reach the culturing bottle bottom surface 85% the time, carry out the had digestive transfer culture cultivation.1 bottle of primary culture can go down to posterity three bottles, and every bottle of initial cultured cells density is 2 * 10
5Cell/mL.Three bottles of 75cm
2Culturing bottle is at 37 ℃, 5%CO
2Cultivate in the incubator after 6 days, obtain 90% the cell that adherent and growth reaches the culturing bottle bottom surface.The NSC that obtains is got 1 bottle and carries out immunofluorescence detection (following detailed description method); Use mouse-anti people nidogen (nestin) IgG and mouse-anti people glial fibrillary acidic protein (DFAP) IgG to dye; Detected result shows: nestin and DFAP are positive, meet the NSC phenotypic characteristic.
Get above-mentioned cultured NSC; The substratum that inclines, every bottle of neuronal cell substratum 10ml that adds 200ug/mL saikoside α, 10% foetal calf serum, 20ng/mL Urogastrone (EGF) and 50ng/mL Prostatropin (bFGF) carries out differentiation culture (division culture medium), and later half amount was changed liquid in 48 hours; When changing liquid with the slow sucking-off 5ml of old substratum; Add fresh division culture medium, every bottle adds 5ml, continues at 37 ℃, 5%CO
2Cultivate.Neural stem cell form changed in the 5th day, and carried out half amount and change liquid, continued to cultivate the 7th day mesenchyme and was divided into neuron cell basically.
Get wherein 1 bottle of cell and carry out immunofluorescence and detect, discard substratum, through trysinization, 800rpm is after centrifugal 5 minutes by aforementioned condition, and the collecting cell deposition is resuspended with 1mL 1 * PBS (phosphoric acid buffer of pH 7.4), counts.(every part contains 1 * 10 to get 6 part of 50 μ L
6Individual cell) above-mentioned going down to posterity obtained in six holes that cell suspension is seeded in six well culture plates respectively adherent growth 4 hours; Wash once with 1 * PBS behind the sucking-off substratum; Added 2% formaldehyde fixed 3 minutes; With 0.5% triton 100 (Triton-100), 1 * PBS washing three times, mouse-anti people neurofilament protein (NF-M) IgG, mouse-anti people nidogen (nestin) IgG, the anti-people MAP-2IgG of rabbit and mouse-anti people glial fibrillary acidic protein (DFAP) IgG that in six holes of six orifice plates, add 1 * PBS more respectively and dilute with 1 * PBS.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times, in each hole, add two anti-IgG-PE or IgG-FITC, under 37 degree, hatch 1h with 1 * PBS dilution; Hatch the back and wash gently 3 times, seal with 90% glycerine after having washed with 1 * PBS.Fluorescent microscope is observation down; The result shows; Cell more than 85% presents neurofilament protein (NF-M), nidogen (nestin), microtubule associating albumen-2 (MAP-2) the expression positive; And glial fibrillary acidic protein (GFAP) is negative, meets the specific characteristics of neuron cell, thereby proof uses division culture medium of the present invention can differentiate highly purified neuron cell from people's derived from bone marrow NSC.
Table 1
Different group cell differentiation of nerve cord are NF-M stained positive neuron cell percentage
Can learn that from table 1 NSC is under 200ug/mL saikoside α+50ng/mL bFGF component cultivation, the neuron cell NF-M stained positive percentage of acquisition is apparently higher than other groups, and T detects the P value much smaller than 0.01.
Embodiment 2:
Present embodiment is used for explaining and uses division culture medium of the present invention from the method for the isolating cell differentiation of nerve cord of human cord blood as neuron cell.
Cord blood divides the Cord blood of puerperium collection from 28 years old healthy pregnant women.Signed Informed Consent Form with this pregnant woman and hospital side, gathered its Cord blood in the puerperium its minute.
Cord blood is diluted with 1 * PBS equal-volume; Obtain the Cord blood diluent; With the Cord blood diluent: the volume ratio of the Ficoll-Hypaque of 1.077 kilograms per cubic meter (Ficoll-Hypaque) is to join in 50ml centrifuge tube at 2: 1, carries out density gradient centrifugation 20 minutes down at 2000rpm, 25 degree, centrifugal back collection intermediate layer cell; And used 1 * PBS 1000rpm centrifugal 10 minutes; Washing gained cell three times adds perfect medium, contains the DMEM/F12 substratum (1: 1) of 10% foetal calf serum, 20ng/mL Urogastrone and 20ng/mL Prostatropin.After the cell counting, by 3 * 10
5Cell/cm
2Density be inoculated in 75cm
2Culturing bottle in, every bottle adds above-mentioned substratum again to make final volume is 10ml.At 37 ℃, 5%CO
2Incubator in cultivate after the 3rd day, half amount is changed liquid.With the slow sucking-off 5ml of old substratum, add fresh above-mentioned substratum when changing liquid, every bottle adds 5ml, continues at 37 ℃, 5%CO
2Cultivate.When cell attachment growth reach the culturing bottle bottom surface 85% the time; Substratum is inclined; And with cell through use trysinization, 800rpm collected the cultivation of then in containing the DMEM/F12 substratum (1: 1) of 10% foetal calf serum, 20ng/mL Urogastrone and 20ng/mL Prostatropin, going down to posterity in centrifugal 5 minutes.Same when the cell attachment growth reach the culturing bottle bottom surface 85% the time, carry out the had digestive transfer culture cultivation.1 bottle of primary culture can go down to posterity three bottles, and every bottle of initial cultured cells density is 2 * 10
5Cell/mL.Three bottles of 75cm
2Culturing bottle is at 37 ℃, 5%CO
2Cultivate in the incubator after 6 days, obtain 90% the cell that adherent and growth reaches the culturing bottle bottom surface.The NSC of above-mentioned acquisition can continue the 10th generation of increasing; This NSC still has identical proterties; The NSC that obtains is got 1 bottle and carries out immunofluorescence detection (seeing embodiment 1 detailed description method); Use mouse-anti people nidogen (nestin) IgG and mouse-anti people glial fibrillary acidic protein (DFAP) IgG to dye, detected result shows: nestin and DFAP are positive, meet the NSC phenotypic characteristic.
Get above-mentioned cultured NSC; The substratum that inclines, every bottle of neuronal cell substratum 10ml that adds 200ug/mL saikoside α, 10% foetal calf serum, 20ng/mL Urogastrone (EGF) and 50ng/mL Prostatropin (bFGF) carries out differentiation culture (division culture medium), and later half amount was changed liquid in 48 hours; When changing liquid with the slow sucking-off 5ml of old substratum; Add fresh division culture medium, every bottle adds 5ml, continues at 37 ℃, 5%CO
2Cultivate.Neural stem cell form changed in the 5th day, and carried out half amount and change liquid, continued to cultivate the 7th day mesenchyme and was divided into neuron cell basically.
Get wherein 1 bottle of cell, discard substratum, through trysinization, 800rpm is after centrifugal 5 minutes by aforementioned condition, and the collecting cell deposition is resuspended with 1mL 1 * PBS (phosphoric acid buffer of pH 7.4), counting.(every part contains 1 * 10 to get 6 part of 50 μ L
6Individual cell) above-mentioned going down to posterity obtained in six holes that cell suspension is seeded in six well culture plates respectively adherent growth 4 hours; Wash once with 1 * PBS behind the sucking-off substratum; Added 2% formaldehyde fixed 3 minutes; With 0.5% triton 100 (Triton-100), 1 * PBS washing three times, the mouse-anti people neurofilament protein IgG, mouse-anti people nidogen IgG, the anti-people MAP-2IgG of rabbit and the mouse-anti people glial fibrillary acidic protein IgG that in six holes of six orifice plates, add 1 * PBS more respectively and dilute with 1 * PBS.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times, in each hole, add two anti-IgG-PE or IgG-FITC, under 37 degree, hatch 1h with 1 * PBS dilution; Hatch the back and wash gently 3 times, seal with 90% glycerine after having washed with 1 * PBS.Fluorescent microscope is observation down; The result shows; Cell more than 85% presents neurofilament protein (NF-M), nidogen (nestin), microtubule associating albumen-2 (MAP-2) the expression positive; And glial fibrillary acidic protein (GFAP) is negative, meets the specific characteristics of neuron cell, thereby proof is used division culture medium of the present invention, and NSC can differentiate highly purified neuron cell from the human cord blood source.
Embodiment 3
Present embodiment is used to explain the method for use division culture medium of the present invention for neuron cell subculture in vitro separately multiplication culture, and the method for the freezing preservation of gained neuron cell.
Get the neuron cell that obtains in embodiment 1 or 2; When its adherent growth reach the culturing bottle bottom surface reach 90% the time; With cell through use trysinization, contain after the 800rpm collection in centrifugal 5 minutes 200ug/mL saikoside α, 10% foetal calf serum, 20ng/mL Urogastrone and 50ng/mL basic fibroblast growth because of neuronal cell culture medium culturing base in the cultivation of going down to posterity.Same neuron cell adherent growth reaches 90% o'clock of culturing bottle bottom surface, repeats the above-mentioned culturing process that goes down to posterity.When increasing to, neuron cell cultivates algebraically during the 5th generation; 1 bottle of neuron cell is used trysinization; 800rpm collected and obtains the neuron cell deposition in centrifugal 5 minutes; With resuspended this neuron cell deposition of foetal calf serum, the microscopically counting makes the concentration of neuron cell suspension reach 5 * 10
6/ ml.(DMSO Sigma) is mixed with the foetal calf serum that contains 20%DMSO earlier with foetal calf serum, and the back adds makes in the above-mentioned resuspended liquid that DMSO concentration is 10% and to adjust neuron cell concentration be 5 * 10 to use DMSO 99.8MIN.
6/ ml.Every 1ml gained neuron cell suspension branch is installed in the frozen pipe of 2ml, according to working instructions, after-80 degree refrigerators are preserved 2 days, go in the liquid nitrogen frozen earlier in the placement programmed cooling box (U.S. Nalgene company).
Getting the 1 bottle of algebraically that goes down to posterity is the neuron cell in the 4th generation, through trysinization, 800rpm after centrifugal 5 minutes cell precipitation resuspended with 1mL 1 * PBS, the counting.(every part contains 1 * 10 to get 6 part of 50 μ L
6Individual cell) above-mentioned going down to posterity obtained in six holes that cell suspension is seeded in six well culture plates respectively adherent growth 4 hours; Wash once with 1 * PBS behind the sucking-off substratum; Added 2% formaldehyde fixed 3 minutes; With 0.5% triton 100 (Triton-100), 1 * PBS washing three times, in six holes of six orifice plates, add 1 * PBS more respectively and unite albumen-2IgG and mouse-anti people glial fibrillary acidic protein IgG with the anti-people's microtubule of mouse-anti people neurofilament protein IgG, mouse-anti people nidogen IgG, rabbit of 1 * PBS dilution.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times; In each hole, add two anti-IgG-PE and two anti-IgG-FITC with 1 * PBS dilution; Under 37 degree, hatch 1h, hatch the back and wash gently 3 times, seal with 90% glycerine after having washed with 1 * PBS.Fluorescent microscope is observation down; The result shows; Cell more than 90% presents neurofilament protein, nidogen, the microtubule associating albumen-2 expression positive, and glial fibrillary acidic protein is negative, meets the specific characteristics of neuron cell; Thereby the proof neuron cell that uses division culture medium of the present invention to go down to posterity to cultivate phenomenon that sex change do not occur breaking up, kept the cell proterties of neuron cell.
In addition; In order more to clearly illustrate effect of the present invention; The applicant also will breed to the neuron cell in the 4th generation and cultivate in six well culture plates according to the method for embodiment 1 and 3, and Fig. 1 has shown the fluorescence immunoassay photo of this neuron cell expressing protein, wherein; Figure 1A is two anti-colour developing fluorescence immunoassay photos behind mouse-anti people microtubule associating albumen-2IgG mark, and Figure 1B is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people nidogen IgG mark.
The neuron cell that embodiment 1 and 3 obtains extracts mRNA; Gene through PCR method amplification expression; Be neurofilament protein, nidogen, microtubule associating albumen-2 and glial fibrillary acidic protein; Fig. 2 is a gel electrophoresis figure behind the pcr amplification, and swimming lane 1 is a DNA standard molecule sign, and swimming lane 2 extracts PCR product (blank) behind the mRNA for inoblast; Swimming lane 3 extracts PCR product (GFAP positive control) behind the mRNA for astroglia cell; Swimming lane 4 extracts PCR product (differentiation contrast) behind the mRNA for NSC, and swimming lane 5 be that neuron cell that embodiment 1 obtains extracts PCR product behind the mRNA, and swimming lane 6 be a PCR product behind the neuron cell extraction mRNA of embodiment 3 acquisitions.Obviously learning neuron cell expression neurofilament protein, nidogen and microtubule associating albumen-2 gene that embodiment 1 and 3 obtains among Fig. 2, and do not express glial fibrillary acidic protein, is the notable feature of neuron cell.
The test kit preparation that present embodiment explanation the inventive method obtains.
Human nerve stem cell is induced the division culture medium test kit of differentiated neuron like cell and propagation thereof, and its component is prepared as follows:
1) basic medium is NM, DMEM/F12 (1: 1) or DMEM substratum, and 500ml uses the aseptic reagent bottle packing of 500ml;
2) saikoside α, 100mg uses 2ml sterile seal cillin bottle or reagent tube packaging;
3) cytokine (Urogastrone and Prostatropin);
The Urogastrone is that 10ug and Prostatropin are 25ug, uses 2ml sterile seal cillin bottle or reagent tube packaging;
4) enzyme of peptic cell and;
Pancreatin is 100ml, uses the aseptic reagent bottle packing of 100ml;
5) working instructions; Introducing cell differentiation of nerve cord in detail is the use step and the precaution of neuron cell and propagation thereof.
Embodiment 5:
The pharmaceutical composition that is used for treating apoplexy that present embodiment explanation contains neuron cell that the inventive method obtains is used at the cerebral infarction animal model.
The algebraically that goes down to posterity of getting embodiment 2 and 3 gained is that the neuron cell culture in the 5th generation contains 1 * 10
6Neuron cell is collected neuron cell born of the same parents' cell precipitation behind the centrifugal 5min under the 800rpm, the sodium chloride solution washing with 0.9% 3 times uses this sodium chloride solution of 0.9% resuspended to 2 * 10 then
6The concentration of individual/mL, preparation 2 * 10
6The neuron cell injection liquid of individual/mL concentration is preserved subsequent use down in 4 ℃.
Will be available from the cerebral infarction rat model of the Capital University of Medical Sciences; Be divided into A, B and three groups of groups of C at random; Every group 10; The A group is the neuron cell injection for treating group of Cord blood source cell differentiation of nerve cord, the B group Cord blood source cell differentiation of nerve cord and the neuron cell injection for treating group in 5 generations of going down to posterity, and the C group is the sodium chloride solution blank control group with volume 0.9%.A and B group the 0th day rat model with microsyringe with the 5uL/min speed injection in the Ipsilateral striatum, inject 1 * 10
6Individual/the mL neuron cell, promptly 0.5mL neuron cell injection liquid was injected with same dose in the 7th day and the 14th day once more; C organizes in identical time point and position injection 0.5mL saline water; The 1st, 2,3,5,7 weeks all rats were improved neurological deficit scoring (modified Neurological Severity Scores respectively at transplant time point with after transplanting; MNSS), the result is as shown in table 2 below.
Table 2
Explain: (modified Neurological Severity Scores is mNSS) from 0～18 minute (normally, 0 minute for the scoring of improvement neurological deficit; Maximum neurological deficit, 18 minutes), 1 fen not damaged; 2～6 fens minor injuries; 7～12 fens Moderate lesion; 13～18 fens severe injuries.
Can find out from table 2; The C group is compared P much smaller than 0.01 with A group or B group; And as can be seen from Figure 3 the mNSS scoring of A and B group is organized apparently higher than C; The pharmaceutical composition that promptly is used to treat cerebral infarction has excellent result of treatment for the cerebral infarction rat model, and the cell differentiation of nerve cord in Cord blood source is that the immunological rejection that produces of the neuron cell of neuron cell and propagation thereof is very little, even the xenogenesis administration still has the obvious treatment effect.
Transplant each time point at neuron cell, put to death rat, get brain after the perfusion of 4% Paraformaldehyde 96 is fixing with vetanarcol anesthesia back.Crown 4 parts that are cut into equalization of big capsules of brain, paraffin embedding, section (5um).HE dyeing is with NIH image analysis software (Image-Pro plus
TM, Media cybernetics) and analyze the area of brain injury.Method according to Swanson: (normal side cerebral tissue area-Ipsilateral injured brain tissue area)/normal side cerebral tissue area * 100%, calculate the brain injury area.Fig. 4 transplants the back for the neuron cell that uses embodiment 2 and 3 to obtain and carries out the brain injury area and improve curve along with the time variation; The C group is compared P much smaller than 0.01 with A group or B group; And the brain injury area that can find out A and B group improves to be organized apparently higher than C; A and B group neuron cell are transplanted the hindbrain damaged area and are significantly reduced, and do not have between the two than big difference (P＞0.05).
Use the neuron cell transplantation treatment cerebral infarction rat model of the BrdU mark of embodiment 2 and 3 acquisitions, Fig. 5 is the BrdU positive staining NLC photo that neuron cell is transplanted section in the rat Ipsilateral striatum of back.Show in the photo to show that neuron cell repaired the cerebral tissue position of damage in the cerebral infarction rat model that obviously growth is closely for tissue site.
Claims (12)
1. a human nerve stem cell is divided into the division culture medium of neuron cell and propagation thereof; Said division culture medium comprises the liquid base substratum; It is characterized in that; Volume with said liquid base substratum is a benchmark, and said selection substratum also contains the saikoside α of 1-500ug/mL, the foetal calf serum of 1-20 volume %, the Urogastrone of 5-200ng/mL and the Prostatropin of 5-200ng/mL.
2. division culture medium according to claim 1; Wherein, Volume with said liquid base substratum is a benchmark, and said division culture medium also contains the saikoside α of 5-300ug/mL, the foetal calf serum of 5-15 volume %, the Urogastrone of 10-100ng/mL and the Prostatropin of 10-100ng/mL.
3. division culture medium according to claim 2; Wherein, Volume with said liquid base substratum is a benchmark, and said selection substratum also contains the saikoside α of 50-200ug/mL, the foetal calf serum of 5-10 volume %, the Urogastrone of 20-50ng/mL and the Prostatropin of 20-50ng/mL.
4. division culture medium according to claim 1; Wherein, Said liquid base substratum be selected from the neuronal cell substratum (Neuronal Medium, NM), in BME cell culture medium, Dole's Becquerel improvement Iger cell culture medium, DMEM/F12 cell culture medium, Fischer ' s cell culture medium, IMDM cell culture medium, 199 cell culture mediums, MEM cell culture medium, F10 cell culture medium, F12 cell culture medium and 1640 cell culture mediums one or more.
5. one kind becomes the method for neuron cell with cell differentiation of nerve cord, it is characterized in that, said method uses among the claim 1-5 any described division culture medium to cultivate described human nerve stem cell.
6. the neuron cell that obtains by the described method of claim 1-5.
7. one kind prepares the described saikoside α of claim 6 differentiation agents box, it is characterized in that said test kit comprises:
1) NSC basic medium;
2) saikoside α;
3) cytokine (Urogastrone and Prostatropin);
4) enzyme of peptic cell and;
5) working instructions.
8. a pharmaceutical composition that is used to treat nervous system disorders is characterized in that, said pharmaceutical composition comprises any neuron cell that described method obtains among the claim 6-7, and and pharmaceutically acceptable carrier or vehicle.
9. pharmaceutical composition according to claim 8, wherein, said pharmaceutical composition is an injection liquid, said injection liquid comprises pharmaceutically acceptable carrier, and is benchmark with said injection liquid volume, 1 * 10
5To 1 * 10
8Any neuron cell that described method obtains among the claim 6-7 of individual/milliliter.
10. pharmaceutical composition according to claim 9, wherein, said injection liquid comprises that with said injection liquid volume be benchmark, 5 * 10
5To 1 * 10
7Any neuron cell that described method obtains among the claim 6-7 of individual/milliliter.
11. pharmaceutical composition according to claim 9, wherein, said pharmaceutically acceptable carrier is a saline water.
12. pharmaceutical composition according to claim 8, wherein, said nervous system disorders is selected from one or more in apoplexy, cerebral plasy, brain injury, hematencephalon, marrow lateral sclerosis disease and the Parkinson's disease etc.
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