CN102337246A - Development of oligodendrocyte progenitor cells prepared by differentiating mesenchymal stem cells through eleutheroside and kit, and medicine for nerve system disease - Google Patents
Development of oligodendrocyte progenitor cells prepared by differentiating mesenchymal stem cells through eleutheroside and kit, and medicine for nerve system disease Download PDFInfo
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- CN102337246A CN102337246A CN201110307969XA CN201110307969A CN102337246A CN 102337246 A CN102337246 A CN 102337246A CN 201110307969X A CN201110307969X A CN 201110307969XA CN 201110307969 A CN201110307969 A CN 201110307969A CN 102337246 A CN102337246 A CN 102337246A
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Abstract
The invention provides a differential medium for obtaining oligodendrocyte progenitor cells and proliferation thereof by processing mesenchymal stem cells through eleutheroside. The differential medium comprises a liquid basal medium, and also comprises 1-100ug/mL eleutheroside, 1 to 20 percent of fetal calf serum, 5 to 300ng/mL human epidermal growth factor (EGF) and 5 to 300ng/mL basic fibroblast growth factor (bFGF) based on the volume of the liquid basal medium. The invention also provides a method for preparing an eleutheroside processing kit by using the differential medium, and also provides a medicinal composition comprising the oligodendrocyte progenitor cells prepared by the method and used for treating nerve system disease. By using the differential medium, the method has the advantages that: the mesenchymal stem cells can be efficiently differentiated and the oligodendrocyte progenitor cells are obtained. The medicinal composition for treating diseases such as traumatic spinal cord injury, multiple sclerosis and the like can repair injured nerve cells in vivo and eliminate neurologic disorder.
Description
Technical field
The present invention relates to a kind of Radix Et Caulis Acanthopanacis Senticosi glucoside handler mescenchymal stem cell and obtain oligodendrocyte precursor cells (Oligodendrocyte Progenitor cells; OPC) and propagation division culture medium; And the method for test kit development, and the pharmaceutical composition that is used to treat diseases such as traumatic Spinal injury and multiple sclerosis that comprises the precursor cell that preceding method obtains.More specifically; The present invention relates to the division culture medium that a kind of Radix Et Caulis Acanthopanacis Senticosi glucoside handler mescenchymal stem cell obtains oligodendrocyte precursor cells and propagation thereof; Use this substratum optionally to obtain the method for oligodendrocyte precursor cells, and the pharmaceutical composition that is used to treat diseases such as traumatic Spinal injury and multiple sclerosis that comprises the oligodendrocyte precursor cells that preceding method obtains.
Background technology
Oligodendrocyte is a kind of macroglia in the cns.Oligodendrocyte is a myelin founder cell unique in the cns, also secretes multiple neurotrophic factor simultaneously and keeps normal physiological effect mutual between oligodendrocyte and other neurocyte.Major function is to produce myelin to hold neural axon, keeps nerve impulse and conducts along the aixs cylinder jumping characteristic.Myelin (myelin) is made up of the cytoplasmic membrane that ripe oligodendrocyte extends.Spinal injury causes oligodendrocyte death, is the immediate cause that the aixs cylinder demyelination changes.Multiple sclerosis is a cns chronic inflammation demyelinating disease, extensively the losing of oligodendrocyte in the demyelination process, and the damage of primary oligodendrocyte also causes the Secondary cases demyelination.
The stem cell that is used for nervous system disease at present comprises human mesenchymal stem cell (mesenchymal stem cells; MSC); Their external neuralward cytodifferentiation; The more use of foreign study 2-thioglycol, methyl-sulphoxide, thioglycerin etc. are as inductor, but these chemicals all have certain toxicity.It can not be applied in the human body, makes the cell poor stability when being prepared into pharmaceutical composition that obtains.
For example; CN200480019375.7 is disclosed to obtain Oligodendrocyte precursor cells by separation and Culture in the tissues such as the mammiferous hippocampus that comprises the people, cerebellum, spinal cord; Through the ciliary nerves cytotrophy factor and 3; The oligodendrocyte that 3 ', 5 '-triiodothyronine can obtain to break up, its ethnics Problem possibly limit its application.
CN200710195844.6 discloses the method for human marrow mesenchymal stem cell to the oligodendrocyte differentiation; Adopt adhesion molecule F3/Contactin to induce differentiation; It induces differentiation cost possibility expensive, obtains oligodendrocyte terminal differentiation, does not have stem cell potential property.
To sum up, need badly a kind of simple to operate, cost is low, differentiation purity is high, the method for the acquisition oligodendrocyte precursor cells of the safety of propagation stable in properties.
Summary of the invention
The method operation that the objective of the invention is to overcome existing inducing differentiation of human mescenchymal stem cell oligodendroblast is complicated, cost is high; The oligodendrocyte purity that differentiation obtains is low; And in the inductor of separation and amplification procedure use, contain harmful medicine; Make and induce the shortcoming of breaking up the oligodendrocyte poor stability when being prepared into pharmaceutical composition that obtains; A kind of division culture medium of inducing differentiation of human mescenchymal stem cell oligodendroblast is provided, uses the method for separation and amplification oligodendrocyte of this substratum simple to operate, cost is low, high, the stable in properties of differentiation purity.
Utilization induces differentiation agent to have inexpensive, safe advantage through the traditional Chinese medicine conduct of clinical verification, is expected to become clinical from now on nerve cells transplantation and induces differentiation agent safely and effectively.More verified Chinese medicines have the effect in external evoked MSC neuralward like cell differentiation like Radix Salviae Miltiorrhizae Injection, rhizoma Gastrodiae, baicalin etc., are mainly neuron cell.
The invention provides the division culture medium that a kind of Radix Et Caulis Acanthopanacis Senticosi glucoside handler mescenchymal stem cell obtains oligodendrocyte precursor cells 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 Radix Et Caulis Acanthopanacis Senticosi glucoside of 1-500ug/mL, foetal calf serum, 5-300ng/mL Urogastrone (EGF) and the 5-300ng/mL Prostatropin (bFGF) of 1-20 volume %.
The method that the test kit that the present invention also provides a kind of Radix Et Caulis Acanthopanacis Senticosi glucoside handler mescenchymal stem cell to obtain oligodendrocyte precursor cells and propagation thereof is developed, wherein, described test kit comprises:
1) mescenchymal stem cell basic medium;
2) Radix Et Caulis Acanthopanacis Senticosi glucoside;
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 diseases such as traumatic Spinal injury and multiple sclerosis; Wherein, Said pharmaceutical composition comprises the oligodendrocyte precursor cells that obtains from the human mesenchymal stem cell differentiation method according to the invention, and and pharmaceutically acceptable carrier or vehicle.
Use the of the present invention of division culture medium of the present invention, can efficiently break up and a large amount of oligodendrocyte precursor cells that obtain from the oligodendrocyte precursor cells of human mesenchymal stem cell differentiation acquisition and the method for propagation thereof.Because differentiation culture based selective of the present invention is strong, obtaining oligodendrocyte precursor cells purity can reach more than 70%.Therefore; Method of the present invention need not to use the medicine of expensive poor stability such as nutritional factor or chemical inducer just can reach more highly purified separating effect; Avoided complicated operation; Thereby reduced cost, and the security of the oligodendrocyte precursor cells that is obtained by this method improves greatly, can the preparation cost invention be used to treat the pharmaceutical composition of diseases such as traumatic Spinal injury and multiple sclerosis.The present invention is used to treat the pharmaceutical composition of diseases such as traumatic Spinal injury and multiple sclerosis, and not only security is good; And can repair the oligodendrocyte of extensively losing or damaging in the diseases such as traumatic Spinal injury and multiple sclerosis in vivo, and the myelinic formation of aixs cylinder is arranged.
Description of drawings
Fig. 1 is divided into the growthhabit photo (40 times of opticmicroscope magnifications) of oligodendrocyte for embodiment 1 mesenchymal stem cells MSCs;
Fig. 2 is the fluorescence immunoassay photo (40 times of fluorescent microscope magnifications) of the oligodendrocyte precursor cells expressing protein of embodiment 1 and 3; Wherein, Fig. 2 A is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people O4 IgM mark; Fig. 2 B is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people A2B5 IgM mark, and Fig. 2 C is the fluorescence immunoassay photo that Fig. 2 A and Fig. 2 B stack obtain;
Fig. 3 transplants the back along with Spinal injury BBB (Basso, Beatlie, Bresnahan) function score curve is carried out in the time variation for using embodiment 2 and the traumatic rats with spinal cord injury model of 3 oligodendrocyte precursor cells transplantation treatments;
Fig. 4 is for using embodiment 2 and the 3 oligodendrocyte precursor cells pharmaceutical compositions microstructure photograph (400 times of opticmicroscope magnifications) to effect comparison before and after the traumatic rats with spinal cord injury model treatment.
Embodiment
The invention provides the division culture medium that a kind of Radix Et Caulis Acanthopanacis Senticosi glucoside handler mescenchymal stem cell obtains oligodendrocyte precursor cells 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 Radix Et Caulis Acanthopanacis Senticosi glucoside of 1-500ug/mL, foetal calf serum, 5-300ng/mL Urogastrone (EGF) and the 5-300ng/mL Prostatropin (bFGF) of 1-20 volume %.
Preferably; Volume with said liquid base substratum is a benchmark, and said division culture medium also contains the Radix Et Caulis Acanthopanacis Senticosi glucoside of 5-300ug/mL, foetal calf serum, 10-200ng/mL Urogastrone (EGF) and the 10-200ng/mL Prostatropin (bFGF) of 5-15 volume %.Further preferably; Volume with said liquid base substratum is a benchmark, and said division culture medium also contains the Radix Et Caulis Acanthopanacis Senticosi glucoside of 10-200ug/mL, foetal calf serum, 10-100ng/mL Urogastrone (EGF) and the 10-100ng/mL Prostatropin (bFGF) of 5-10 volume %.
People's mesenchyme of the present invention is that the division culture medium of oligodendrocyte precursor cells and propagation thereof can comprise the various minimum mediums that this area is commonly used in cytodifferentiation; For example; Be selected from 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 the MEM 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.
Human mesenchymal stem cell provided by the invention goes for the various in vitro tissues that contain mescenchymal stem cell, for example marrow, Cord blood, umbilical cord, miscarriage embryo (the for example embryo in the 12-14 of miscarriage week) and aborted fetus etc.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, Cord blood and the umbilical cord.Wherein, marrow and Cord blood can be from public umbilical cord blood bank (for example, Beijing 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.In addition, the marrow of traumatic Spinal injury and multiple sclerosis patients can be used as from the source of body mescenchymal stem cell, can avoid immunological rejection.
The human mesenchymal stem cell that is used to be divided into oligodendrocyte precursor cells of the present invention is from safe conservative consideration; Preferably using the algebraically that goes down to posterity is the mescenchymal stem cell in 10 generations of the 1st generation to the; Wherein, the go down to posterity mescenchymal stem cell in algebraically 4 generations of the 1st generation to the can be used as the seed mescenchymal stem cell and uses.From the consideration of mescenchymal stem cell quantity with stability, the algebraically that preferably goes down to posterity is that the mescenchymal stem cell in the 4th generation uses as the seed mescenchymal stem cell.Said seed stem cell can be used as the initiator cell of large scale culturing, also can adopt the technology of the conventional freezing preservation cell in this area that it is preserved as the cell strain long-term frozen, uses in order to recovery.The article mescenchymal stem cell used in the middle of algebraically 10 generation of the 5th generation to the mescenchymal stem cell that goes down to posterity can be used as.The article stem cell is meant the mescenchymal stem cell that obtains after the large scale culturing in the middle of said, can be divided into the raw material of oligodendrocyte precursor cells or pharmaceutical composition as preparation the present invention.In addition, for the ease of transportation and preservation, said oligodendrocyte precursor cells 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 diseases such as traumatic Spinal injury and multiple sclerosis; Wherein, Said pharmaceutical composition comprises the oligodendrocyte precursor cells that the differentiation of stem cells method obtains of supplementing with money from the human world according to the invention, and and 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 oligodendrocyte precursor cells 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 oligodendrocyte precursor cells 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, oligodendrocyte precursor cells can be lost or damages and repair oligodendrocyte, and can useful effect in the myelinic formation of aixs cylinder (referring to embodiment 5).Therefore pharmaceutical composition of the present invention is used to treat diseases such as traumatic Spinal injury and multiple sclerosis.
The oligodendrocyte precursor cells 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 diseases such as traumatic Spinal injury and multiple sclerosis; Can be through removing division culture medium of the present invention, with saline water washing and resuspended said oligodendrocyte precursor cells.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 diseases such as traumatic Spinal injury and multiple sclerosis 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 oligodendrocyte precursor cells of the present invention; Be preferably 5 * 10
5To 1 * 10
7Individual/mL.The oligodendrocyte precursor cells that the present invention also provides method of the present invention to obtain, the application in disease medicaments such as preparation traumatic Spinal injury of treatment and multiple sclerosis.
The present invention also provides a kind of method of treating diseases such as traumatic Spinal injury and multiple sclerosis, and it comprises to the patient uses the pharmaceutical composition that the present invention is used to treat diseases such as traumatic Spinal injury and multiple sclerosis.
Use mode that the present invention is used for treating the pharmaceutical composition of diseases such as traumatic Spinal injury and multiple sclerosis can be selected from get involved that injection, intravenous injection, waist are worn, subcutaneous injection and intradermal injection one or more; The more preferably said mode of using is that waist is worn.The pharmaceutical composition that is used to treat diseases such as multiple sclerosis and traumatic Spinal injury according to the invention preferably contains 1 * 10
5To 1 * 10
8The oligodendrocyte precursor cells of cell/mL, its per injection dosage are that 0.1mL-5mL/ is individual; More preferably, the pharmaceutical composition that is used to treat diseases such as traumatic Spinal injury and multiple sclerosis according to the invention comprises 1 * 10
5To 5 * 10
7The oligodendrocyte precursor cells 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 diseases such as traumatic Spinal injury and multiple sclerosis 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 diseases such as multiple sclerosis and traumatic Spinal injury according to the invention.Said effective standard is: the scoring of Spinal injury BBB function obviously improves, and can impel medullated aixs cylinder to generate.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 Spinal injury 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 disease patients such as traumatic Spinal injury and multiple sclerosis; The gained oligodendrocyte precursor cells is prepared into disease patients such as traumatic Spinal injury of pharmaceutical composition administration of the present invention and multiple sclerosis 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 to explain the method for using division culture medium of the present invention to be divided into oligodendrocyte precursor cells from human marrow mesenchymal stem cell.
People's marrow has been signed Informed Consent Form with this donor and hospital side from 31 years old donor.People's marrow is diluted with 1 * PBS equal-volume; Obtain people's marrow diluent; With people's 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 10 minutes down at 1500rpm, 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 α-MEM substratum (U.S. Gibco company) of 10% foetal calf serum (U.S. Hyclone 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 mescenchymal stem cell 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 α of 10% foetal calf serum-MEM substratum, 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 mescenchymal stem cell that obtains is got 1 bottle and is carried out the fluidic cell detection, and through trysinization, centrifugal back cell precipitation is resuspended with 1mL 1 * PBS, counting.Get 3 part of 20 μ L (1 * 10
6Individual) above-mentioned going down to posterity obtain cell suspension and add to respectively 20 μ L FITC-CD34 (BD company) and 20 μ L PE-CD90 (BD company) are arranged; 20 μ L FITC-CD45 (BD company) and 20 μ L PE-CD44 (BD company); In the centrifuge tube of 20 μ LFITC-HLA-DR (BD company) and 20 μ L PE-CD105 (BD company).Place 4 ℃ of refrigerators dyeing 30 minutes, use 1 * phosphate buffered saline buffer (PBS) washing three times of 1mL then, use the cell after the resuspended washing of 1 * PBS of 0.5mL at last, the cell after the gained washing detects with FACSCalibur basic model flow cytometer.The result shows that CD44＞95.56%, CD90＞98.60%, CD105＞91.3%, CD34＜3.45%, CD45＜2.10%, HLA-DR＜1.33% meet the mescenchymal stem cell phenotypic characteristic.
Get above-mentioned cultured mescenchymal stem cell; The substratum that inclines, every bottle of α-MEM substratum 10ml that adds 50ug/mL Radix Et Caulis Acanthopanacis Senticosi glucoside, 10% foetal calf serum, 20ng/mL Urogastrone (EGF) and 20ng/mL Prostatropin (bFGF) (U.S. R&D company) 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.The mescenchymal 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 oligodendrocyte precursor cells basically.
Get wherein one 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, in six holes of six orifice plates, add the mouse-anti people A2B5IgM of 1 * PBS and the mouse-anti people O4IgM that dilutes with 1 * PBS, 1 * PBS dilution and the mouse-anti human myelin basic protein IgG of 1 * PBS dilution more respectively.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times; In the hole of mouse-anti human myelin basic protein IgG, add two anti-IgG-FITC, and in the hole of mouse-anti people O4IgM and mouse-anti people A2B5IgM, add two anti-IgM-PE and two anti-IgM-FITC respectively, under 37 degree, hatch 1h with 1 * PBS dilution 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 70% presents A2B5 and O4 is positive; Myelin basic protein is negative, meets the specific characteristics of oligodendrocyte precursor cells, thereby proof uses division culture medium of the present invention can differentiate highly purified oligodendrocyte precursor cells from human marrow mesenchymal stem cell.
Embodiment 2:
Present embodiment is used to explain and uses division culture medium of the present invention from the method for umbilical cord derived mesenchymal differentiation of stem cells as oligodendrocyte precursor cells.
Umbilical cord divides the umbilical cord of puerperium collection from 26 years old healthy pregnant women.Signed Informed Consent Form with this pregnant woman and hospital side, collected its umbilical cord in the puerperium its minute.
With the 1 * PBS washing that contains 2% pair anti-(penicillium mould and Streptomycin sulphate) three times, the back shreds with eye scissors becomes about 1mm with the in vitro tissue umbilical cord
3Fritter.The fritter of organizing that gained shreds restrains with 1 with 0.25% pancreatin solution: the weightmeasurement ratio digestion of 0.5mL is after 5 minutes; Adding is that the foetal calf serum of pancreatin liquor capacity 1/5th stops digestion; Collected the fritter of organizing that digests well in centrifugal 5 minutes with 1000rpm, the pancreatin solution supernatant that inclines, the fritter of organizing that gained shreds then washs three times with containing 1% couple of anti-α-MEM substratum; Collected the fritter of organizing that washs well in centrifugal 5 minutes with 1000rpm, the washing soln supernatant inclines.Collect the said fritter of organizing,, the fritter of not organizing just, place in the petridish, at 37 ℃, 5%CO to wherein adding the α-MEM substratum that contains 10% foetal calf serum
2Cultivate.Changed liquid on the 3rd day, and slowly drew substratum, the back adds fresh culture, and at 37 ℃, 5%CO
2Cultivate in the incubator.Every not good liquor that changed at a distance from three days; Cover with petridish until climbing out of mescenchymal stem cell, can use trysinization to carry out amplification cultivation, substratum is inclined; And with cell through use trysinization, 800rpm collected the cultivation of then in containing the α of 10% foetal calf serum-MEM substratum, 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 mescenchymal stem cell of above-mentioned acquisition can continue the 10th generation of increasing; This mescenchymal stem cell still has identical proterties, gets 1 flask culture to the, 10 generation mescenchymal stem cell and carries out the fluidic cell detection, through trysinization; Centrifugal back cell precipitation is resuspended with 1mL1 * PBS, counting.Get 3 part of 20 μ L (1 * 10
6Individual) above-mentioned going down to posterity obtain cell suspension and add to respectively 20 μ L FITC-CD34 (BD company) and 20 μ LPE-CD90 (BD company) are arranged; 20 μ L FITC-CD45 (BD company) and 20 μ L PE-CD44 (BD company); In the centrifuge tube of 20 μ L FITC-HLA-DR (BD company) and 20 μ L PE-CD105 (BD company).Place 4 ℃ of refrigerators dyeing 30 minutes, use 1 * phosphate buffered saline buffer (PBS) washing three times of 1mL then, use the cell after the resuspended washing of 1 * PBS of 0.5mL at last, the cell after the gained washing detects with FACSCalibur basic model flow cytometer.The result shows that CD44＞95.56%, CD90＞98.60%, CD105＞91.3%, CD34＜3.45%, CD45＜2.10%, HLA-DR＜1.33% meet the mescenchymal stem cell phenotypic characteristic.
Get above-mentioned cultured mescenchymal stem cell; The substratum that inclines, every bottle of α-MEM substratum 10ml that adds 50ug/mL Radix Et Caulis Acanthopanacis Senticosi glucoside, 10% foetal calf serum, 20ng/mL EGF and 20ng/mL bFGF carries out differentiation culture, 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.The mescenchymal 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 oligodendrocyte precursor cells basically.
Get wherein one 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, in six holes of six orifice plates, add the mouse-anti people A2B5IgM of 1 * PBS and the mouse-anti people O4IgM that dilutes with 1 * PBS, 1 * PBS dilution and the mouse-anti human myelin basic protein IgG of 1 * PBS dilution more respectively.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times; In the hole of mouse-anti human myelin basic protein IgG, add two anti-IgG-FITC, and in the hole of mouse-anti people O4IgM and mouse-anti people A2B5IgM, add two anti-IgM-PE and two anti-IgM-FITC respectively, under 37 degree, hatch 1h with 1 * PBS dilution 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 70% presents A2B5 and O4 is positive; Myelin basic protein is negative, meets the specific characteristics of oligodendrocyte precursor cells, thereby proof uses division culture medium of the present invention can differentiate highly purified oligodendrocyte precursor cells from the mescenchymal stem cell in umbilical cord source.
Embodiment 3
Present embodiment is used to explain the method for use division culture medium of the present invention for oligodendrocyte precursor cells subculture in vitro separately amplification cultivation, and the method for the freezing preservation of gained oligodendrocyte precursor cells.
Get the oligodendrocyte precursor cells that obtains in embodiment 1 and 2; When its adherent growth reach the culturing bottle bottom surface reach 90% the time; Cell is passed through to use trysinization, the cultivation of in the α that contains 50ug/mL Radix Et Caulis Acanthopanacis Senticosi glucoside, 10% foetal calf serum, 20ng/mL EGF and 20ng/mL bFGF-MEM culture medium culturing base, going down to posterity after 800rpm collected in centrifugal 5 minutes.Same oligodendrocyte precursor cells 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, oligodendrocyte precursor cells cultivates algebraically during the 5th generation; 1 bottle of oligodendrocyte precursor cells is used trysinization; 800rpm collected and obtains the oligodendrocyte precursor cells deposition in centrifugal 5 minutes; With resuspended this oligodendrocyte precursor cells deposition of foetal calf serum, the microscopically counting makes the concentration of oligodendrocyte precursor cells 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 oligodendrocyte precursor cells concentration be 5 * 10 to use DMSO 99.8MIN.
6/ ml.Every 1ml gained oligodendrocyte precursor cells 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 oligodendrocyte precursor cells 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 the mouse-anti people A2B5IgM of 1 * PBS and the mouse-anti people O4IgM that dilutes with 1 * PBS, 1 * PBS dilution and the mouse-anti human myelin basic protein IgG of 1 * PBS dilution more respectively.Dye/combine in 4 degree refrigerator overnight; Use 0.5%Triton-1001 * PBS to wash afterwards 3 times; In the hole of mouse-anti human myelin basic protein IgG, add two anti-IgG-FITC, and in the hole of mouse-anti people O4IgM and mouse-anti people A2B5IgM, add two anti-IgM-PE and two anti-IgM-FITC respectively, under 37 degree, hatch 1h with 1 * PBS dilution 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 70% presents A2B5 and O4 is positive, and myelin basic protein is negative, meets the specific characteristics of oligodendrocyte precursor cells; Thereby proof use division culture medium of the present invention to go down to posterity phenomenon that sex change do not appear breaking up in cultured oligodendrocyte precursor cells, kept the cell proterties of oligodendrocyte precursor cells.
In addition; In order more to clearly illustrate effect of the present invention, the applicant also will breed to the oligodendrocyte precursor cells in the 4th generation and cultivate in six well culture plates according to the method for embodiment 1 and 3, and Fig. 2 has shown the fluorescence immunoassay photo of this oligodendrocyte precursor cells expressing protein; Wherein, Fig. 2 A is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people O4IgM mark, and Fig. 2 B is two anti-colour developing fluorescence immunoassay photos behind the mouse-anti people A2B5IgM mark, and Fig. 2 C is the fluorescence immunoassay photo that Fig. 2 A and Fig. 2 B stack obtain.
Embodiment 4:
The test kit preparation that present embodiment explanation the inventive method obtains.
Human mesenchymal stem cell is induced the division culture medium test kit of differentiation oligodendrocyte precursor cells and propagation thereof, and its component is prepared as follows:
1) mescenchymal stem cell basic medium is α-MEM, DMEM/F12 (1: 1) or DMEM substratum, and 500ml uses the aseptic reagent bottle packing of 500ml;
2) Radix Et Caulis Acanthopanacis Senticosi glucoside, 25mg uses 2ml sterile seal cillin bottle or reagent tube packaging;
3) cytokine (Urogastrone and Prostatropin);
Urogastrone and Prostatropin all are 10ug, use 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; Introduce use step and precaution that mescenchymal stem cell is divided into oligodendrocyte precursor cells and propagation thereof in detail.
The pharmaceutical composition that is used for treating traumatic Spinal injury that present embodiment explanation contains oligodendrocyte precursor cells that the inventive method obtains is used at traumatic animal model with spinal cord damnification.
Get the umbilical cord derived mesenchymal differentiation of stem cells of embodiment 3 gained and the oligodendrocyte precursor cells culture in 5 generations of going down to posterity contains 2 * 10
6Oligodendrocyte precursor cells.Collect the cell precipitation of mescenchymal stem cell under the 800rpm behind the centrifugal 5min, 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 is preserved subsequent use down in 4 ℃.Prepare 2 * 10 of Cord blood derived mesenchymal differentiation of stem cells according to going down to posterity to increase with embodiment 1 and 3 same methods
6The oligodendrocyte precursor cells injection liquid of individual/mL concentration.
Will be available from the rats with spinal cord injury model of Department Of Medicine, Peking University; Be divided into A, B and three groups of groups of C at random; Every group 10; A group for the mescenchymal stem cell differentiation in umbilical cord source and 5 generations of going down to posterity oligodendrocyte precursor cells injection for treating group, the B group is the oligodendrocyte precursor cells injection liquid of the mescenchymal stem cell differentiation in Cord blood source, it is the sodium chloride solution blank control group with volume 0.9% that C organizes.Two groups of A and B were mouse model Spinal injury position injection 2 * 10 in the 0th day
6Individual/mL, promptly 1mL oligodendrocyte precursor cells injection liquid was injected with same dose in the 7th day and the 14th day once more; C organizes in identical time point and injects 0.5mL saline water at the Spinal injury position.Form respectively at the week of the 1st, 2,4,8,12,16 and the 24th after last injection monitoring mouse model myelin, and BBB function scoring before and after transplanting, the result is as shown in table 2 below.
Table 1
Explain: the scoring of BBB function is divided into 22 grades with the rat hindlimb motion.The hind leg pamplegia is 0 minute, fully normally is 21 minutes, is decided to be the 1-20 branch respectively according to function between the two, and its substance is: the number of joint motion and scope, heavy burden degree and front and back limb Harmony, the active situation of forward and backward pawl and afterbody.The BBB scoring observation period is 4min, and animal should remain on the central zone activity of scope of activity as far as possible.
Can find out that from table 1 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 BBB function scoring of A and B group is organized apparently higher than C; The pharmaceutical composition that promptly is used to treat Spinal injury has excellent result of treatment for the Spinal injury mouse model; And the immunological rejection that the mescenchymal stem cell in umbilical cord and Cord blood source is divided into the oligodendrocyte precursor cells generation is very little, even the xenogenesis administration still has the obvious treatment effect.
Fig. 4 has shown embodiment 2 and 3 oligodendrocyte precursor cells treatment situation in the rats with spinal cord injury model.Fig. 4 A is tissue slice (Toluidine blue staining) dyeing photo before the rat model treatment, is illustrated in rarefaction Remyelination very big between the aixs cylinder of normal medullated aixs cylinder and demyelination, has increased ECS; Fig. 4 B be oligodendrocyte precursor cells transplantation treatment Spinal injury mouse model after 6 months the tissue section strain photo.Show in the photo to show the aixs cylinder that tangible Remyelination is arranged in the Toluidine blue staining tissue slice, the stellate cell process that the is rich in midbody filament of increase surrounds aixs cylinder.
Claims (12)
1. a human mesenchymal stem cell is divided into the division culture medium of oligodendrocyte precursor cells 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 division culture medium also contains the Radix Et Caulis Acanthopanacis Senticosi glucoside of 1-500ug/mL, foetal calf serum, 5-300ng/mL Urogastrone (EGF) and the 5-300ng/mL Prostatropin (bFGF) of 1-20 volume %.
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 Radix Et Caulis Acanthopanacis Senticosi glucoside of 5-300ug/mL, foetal calf serum, 10-200ng/mL Urogastrone (EGF) and the 10-200ng/mL Prostatropin (bFGF) of 5-15 volume %.
3. division culture medium according to claim 2; Wherein, Volume with said liquid base substratum is a benchmark, and said division culture medium also contains the Radix Et Caulis Acanthopanacis Senticosi glucoside of 10-200ug/mL, foetal calf serum, 10-100ng/mL Urogastrone (EGF) and the 10-100ng/mL Prostatropin (bFGF) of 5-10 volume %.
4. division culture medium according to claim 1; Wherein, said liquid base substratum is selected from one or more 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.
5. the oligodendrocyte precursor cells of differentiation of stem cells acquisition and the method for propagation thereof are filled in a human world, it is characterized in that, any described division culture medium is cultivated said human mesenchymal stem cell among the said method use claim 1-5.
6. method according to claim 5, wherein, said mescenchymal stem cell is from people's marrow, Cord blood and the umbilical cord one or more.
7. the oligodendrocyte precursor cells that obtains by the described method of claim 1-6.
8. one kind prepares the described Radix Et Caulis Acanthopanacis Senticosi glucoside treatment kits of claim 7, it is characterized in that said test kit comprises:
1) mescenchymal stem cell basic medium;
2) Radix Et Caulis Acanthopanacis Senticosi glucoside;
3) cytokine (Urogastrone and Prostatropin);
4) enzyme of peptic cell and;
5) working instructions.
9. pharmaceutical composition that is used to treat diseases such as traumatic Spinal injury and multiple sclerosis; It is characterized in that; Said pharmaceutical composition comprises any oligodendrocyte precursor cells that described method obtains among the claim 7-8, and and pharmaceutically acceptable carrier or vehicle.
10. pharmaceutical composition according to claim 9, 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 oligodendrocyte precursor cells that described method obtains among the claim 7-8 of individual/milliliter.
11. pharmaceutical composition according to claim 10, wherein, said injection liquid comprises that with said injection liquid volume be benchmark, 5 * 10
5To 5 * 10
7Any oligodendrocyte precursor cells that described method obtains among the claim 7-8 of individual/milliliter.
12. pharmaceutical composition according to claim 11, wherein, said pharmaceutically acceptable carrier is a saline water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774807A (en) * | 2015-04-28 | 2015-07-15 | 安沂华 | Method for inductively differentiating umbilical cord mesenchymal stem cells into oligodendroglia cells |
| CN109362714A (en) * | 2018-12-18 | 2019-02-22 | 广州赛莱拉干细胞科技股份有限公司 | A kind of fat mesenchymal stem cell saves liquid and preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446907A (en) * | 2003-04-18 | 2003-10-08 | 中山大学中山医学院科技开发中心 | Method for proliferating and differentiating nerve stem cells under medication inducement and its application |
| CN101451123A (en) * | 2007-11-30 | 2009-06-10 | 中国人民解放军军事医学科学院基础医学研究所 | Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte |
| CN101914493A (en) * | 2010-07-16 | 2010-12-15 | 山东省齐鲁干细胞工程有限公司 | Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells |
-
2011
- 2011-10-12 CN CN201110307969XA patent/CN102337246A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446907A (en) * | 2003-04-18 | 2003-10-08 | 中山大学中山医学院科技开发中心 | Method for proliferating and differentiating nerve stem cells under medication inducement and its application |
| CN101451123A (en) * | 2007-11-30 | 2009-06-10 | 中国人民解放军军事医学科学院基础医学研究所 | Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte |
| CN101914493A (en) * | 2010-07-16 | 2010-12-15 | 山东省齐鲁干细胞工程有限公司 | Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells |
Non-Patent Citations (1)
| Title |
|---|
| 杨琴 等: ""刺五加注射液体外诱导骨髓基质细胞分化为神经元样细胞的机制研究"", 《中国中药杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774807A (en) * | 2015-04-28 | 2015-07-15 | 安沂华 | Method for inductively differentiating umbilical cord mesenchymal stem cells into oligodendroglia cells |
| CN104774807B (en) * | 2015-04-28 | 2018-05-08 | 安沂华 | The method that umbilical cord mesenchymal stem cells are induced differentiation into oligodendroglia |
| CN109362714A (en) * | 2018-12-18 | 2019-02-22 | 广州赛莱拉干细胞科技股份有限公司 | A kind of fat mesenchymal stem cell saves liquid and preparation method and application |
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Address after: 100085, B222, 5 Street, seven street, Beijing, Haidian District Applicant after: CELLONIS BIOTECHNOLOGIES CO., LTD. Address before: 100085 B110 room, Zhongguancun biological medicine Park, No. 5, Pioneer Road, seven street, Haidian District, Beijing Applicant before: Beijing Cellonis Biotechnologies Co., Ltd. |
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Application publication date: 20120201 |