CN1621515A - Method for adjusting the multiplication of human nerve stem cell cultured in vitro - Google Patents
Method for adjusting the multiplication of human nerve stem cell cultured in vitro Download PDFInfo
- Publication number
- CN1621515A CN1621515A CNA2003101168139A CN200310116813A CN1621515A CN 1621515 A CN1621515 A CN 1621515A CN A2003101168139 A CNA2003101168139 A CN A2003101168139A CN 200310116813 A CN200310116813 A CN 200310116813A CN 1621515 A CN1621515 A CN 1621515A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- oxygen
- cell
- low
- human nerve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses one method of regulating the proliferation of extracorporeal cultured human nerve stem cell. By means of proper oxygen concentration, the present invention regulates the proliferation of extracorporeal cultured human nerve stem cell to make the cells maintain the proliferation state, and the nerve stem cell proliferated under low oxygen environment can differentiate into neure with function. The said physical measure may be used in small amount sampling, extracorporeal proliferation and obtaining in great amount, and this makes it possible to apply human nerve stem cell in treating various nerve damage diseases and retrogressive diseases clinically.
Description
Technical field
The present invention relates to a kind of method of regulating cell proliferation, relate in particular to a kind of method of regulating the human nerve stem cell propagation of vitro culture.
Background technology
Central nervous system just can not be repaired after causing a large amount of neuronal damages owing to reasons such as wound, ischemic and degenerative diseases.Utilized neural stem cells transplantation trial multiple nerve of treatment and degenerative disease to become the focus of research in recent years, and be that functional rehabilitation has brought hope behind the central nervous system injury.Neural stem cell is a kind of initiating cell with many differentiation potentials, ability with self and propagation, and under the specific factor influence or inducing, neuralward unit or neurogliocyte differentiation, because neural stem cell has the potential differentiation capability, promptly under certain condition can directed differentiation, therefore, after somatic nerves tissue damaged or sex change, can utilize particular environment factor induced nerve stem cells to corresponding nervous tissue or cytodifferentiation, and then with its alternative damaged tissue or cell, so, no matter neural stem cell is the donor as brain tissue graft, still as the target cell of inducing differentiation in the body, for central nervous system function rebuild and neurotization provide one new by way of.
The importance of research human nerve stem cell mainly is its potential therapeutic value, and at present, the human nerve stem cell that is used for clinical treatment is mainly derived from people embryo or adult cerebral tissue.The adult neural stem cell is difficult for obtaining in the individual brain, and content is less; There is ethnics Problem in the neural stem cell that derives from the embryo, particularly neural stem cell is under external serum-free culture condition, propagation slowly, and be in negative vegetative state along with the increase of passage number, therefore, acquisition abundance, safe and qualified neural stem cell are research and use the precondition of neural stem cell treatment.Yet, also do not have a practicable method to promote the in-vitro multiplication of neural stem cell at present.
We know that oxygen is the prerequisite that life exists, and also are a kind of important regulatory factors of cell physiological function.And hypoxemia is the basic environment that life is grown, and is development growth in low-oxygen environment as mammiferous embryo; The in-house local oxygen levels of adult mammal brain has only 1%-5%, also is a kind of physiological low-oxygen environment.Yet the standard conditions of mammalian cells in vitro cultivation at present are under 37 ℃, 5% carbonic acid gas and 95% Air mixing gas (oxygen level is 20%).About hypoxemia the propagation of stem cell and the regulating effect of differentiation had only odd report in recent years, Sean J.Morrison andStuder Lorenz studied report respectively in 2000: hypoxemia (3%) can promote to derive from peripheral nerve ridge stem cell (the Culture in reduced levels of oxygen promotes clonogenic sympathoadrenaldifferentiation by isolated neural crest stem cells of tire mouse, JNeurosciencee, 2000,20:7370-7376) with nervus centralis precursor cell (Enhancedproliferation, survival, and dopaminergic differentiation of CNS precursors inlowered oxygen, J Neurosciencce, 2000, propagation 20:7377-7383), and induced nerve stem cells breaks up to the dopaminergic neuron direction.Yet, do not see the report of relevant hypoxemia so far to the influence of vitro culture human nerve stem cell proliferation and differentiation.
Summary of the invention
The invention discloses a kind of method of regulating the human nerve stem cell propagation of vitro culture.
The present inventor is unexpected in experiment to find that suitable low oxygen concentration has its proliferation function of promotion to the human nerve stem cell of vitro culture, makes these cells continue to keep vegetative state, and can be divided into the neurone of function under suitable condition.
Method of the present invention comprises following content:
At first carry out the separation and Culture and the evaluation of human nerve stem cell
In the fetal brain of miscarriage, to take out hippocampal tissue and be cut into fragment, after adding pancreatin and the DNA enzyme I digestion, the centrifugal supernatant of abandoning adds serum-free DMEM/F12 substratum, is inoculated in culturing bottle behind the counting and cultivates.Former neurocyte ball of being commissioned to train foster after trysinization, is made single cell suspension with complete culture solution, and going down to posterity behind the counting is inoculated in the plate that scribbles poly-lysine, puts incubator and cultivates.Marker Nestin with neural stem cell identifies the neural ball of separation and Culture and the neural stem cell that goes down to posterity.
Hypoxemia is to the short proliferation function of human nerve stem cell
It is that 20% normal oxygen and oxygen level are respectively in 3% and 10% the low-oxygen environment and cultivate that former generation or the neural stem cell that goes down to posterity are placed on oxygen level respectively, and microscopically is observed the neurocyte ball of cultivating and increased in low-oxygen environment.Analyze by statistics, under 10% low-oxygen environment, the formation number of neural ball is apparently higher than normal oxygen group, and the neural nodule number order that forms in 3% hypoxia group also increases to some extent equally.With the analogue of uracil, be incorporated in the dna double chain in the synthesis phase of cell, as the index of cell proliferation, observe the influence of hypoxemia to the cell proliferation of nerve cord of adherent growth.Before hypoxemia, BrdU is joined in the substratum, then neural stem cell is placed on respectively under the different environment cultivate after, the BrdU coloration result shows: the positive cell number of BrdU mark is all apparently higher than normal oxygen group in 3% and 10% low-oxygen environment.
The human nerve stem cell of cultivating in the low-oxygen environment can be divided into the neurone with function
With the neural stem cell ball of cultivating in the varying environment, blow and beat in the liquid unicellularly in differentiation, be seeded in the gummed plate, in containing the differentiation liquid of foetal calf serum, the equal adherent growth of visible cell after 24 hours, and grow projection, generally needed 9-12 days just can become sophisticated neurone.The result shows: at 3%O
2Good with the neure growth state that forms after the cell differentiation of nerve cord of cultivating in 10% low-oxygen environment, neuron number is more than normal oxygen group.The differentiation capability that shows the neural stem cell of breeding in the low-oxygen environment is not subjected to tangible influence, and more helps its neuralward unit direction differentiation.
Hypoxemia is to the influence of the neurocyte electrophysiological characteristics after breaking up
Known sophisticated neurone has distinctive sodium-potassium current, and the existence of sodium-potassium current also is the prerequisite that neurone has function, and the maturity of the big I reacting cells of membrane potential.The contriver has detected the influence of hypoxemia to sodium-potassium current on the neuron membrane after breaking up, the result is presented at the neural stem cell of breeding in 3% low-oxygen environment, differentiation back neuron membrane sodium-potassium current is big, apparently higher than the neurone of in 20% and 10% low-oxygen environment, inducing differentiation, and the neural stem cell of in 10% low-oxygen environment, breeding, differentiation back neuron membrane sodium-potassium current is less than the neurone that breaks up in 20% and 3% the low-oxygen environment (as shown in Figure 6).This results suggest 3% low-oxygen environment more helps the maturation of neurocyte.
In sum, hypoxemia can promote the in-vitro multiplication of human nerve stem cell, make these cells continue to keep vegetative state, the neural stem cell of breeding in low-oxygen environment can be divided into the neurone with function, and more helps the maturation of neural stem cell at 3% low-oxygen environment.
The propagation of utilizing the hypoxemia in the environment to regulate the human nerve stem cell of vitro culture, it is simple to have an experiment flow, and repeatability is strong, reliable results is at external easy-regulating, to neural stem cell not damaged and toxic side effect, practical characteristics, and the ethics that can avoid using fetal brain tissue to cause limits.
Description of drawings
Fig. 1. for the human nerve stem cell of separation and Culture is identified figure.A wherein: the people hippocampus Culture of neural stem cells of former pickup kind is after 3 days, and formed neural ball is the Nestin stained positive.B: former being commissioned to train supported formed neural ball after 3 days, and 3 days cell of adherent growth is Nestin stained positive (200 *) after going down to posterity.
Fig. 2. be the proliferation function figure of hypoxemia to neural ball formation.Wherein the A:10% low-oxygen environment is cultivated formed neural ball after 3 days down.B: normal oxygen environment is cultivated the neural ball (100 *) that forms after 3 days down.
Fig. 3. be the action diagram of hypoxemia to the cell proliferation of nerve cord of adherent growth.A: the neural stem cell of cultivating under 10% low-oxygen environment is mixed the BrdU positive cells.B: the neural stem cell of cultivating under normal oxygen environment is mixed the BrdU positive cells.Black is represented BrdU positive cells (200 *).
Fig. 4. hypoxemia is to the statistical graph of cell proliferation of nerve cord influence.Length axis is illustrated in the neural stem cell of cultivating under the oxygen environment of different concns and mixes BrdU positive cells number, mix BrdU positive cells number apparently higher than normal oxygen control group in the neural stem cell that low-oxygen environment (3% and 10%) is cultivated down, P＜0.01 is compared with control group).
Fig. 5. the neural stem cell of cultivating in the low-oxygen environment can be divided into sophisticated neurone.A:10%O
2B:3%O
2C:20%O
2The nuclear of black is NeuN-male neurone (200 *).
Fig. 6. the sodium of representational neuron membrane-potassium current figure.A:20%O wherein
2B:3%O
2C:10%O
2
Embodiment
The separation and the cultivation of embodiment one human nerve stem cell
1. the separation and Culture of human nerve stem cell
Cultural method (Transplantation of expandedmesencephalic precursors leads to recovery in parkinsonian rats with reference to the neural stem cell of Studer L., Nat Neurosci1:290-295), get the tire brain of the aborted fetus in pregnant 9-12 week, under anatomical lens, peel off meninx, separate the fragment that hippocampal tissue is cut into the 1mm size, add and contain 0.125% trysinization liquid.Place 37 ℃ CO
2Hatched in the incubator 30 minutes, and added 37 ℃ of digestion of DNA enzyme I 10 minutes again, stop digestion with the nutrient solution that contains serum.After blowing and beating gently with suction pipe, 1000 rev/mins 5 minutes, abandon supernatant and add serum-free bFGF 20ng/ml and 1%N
2And B
27), blow and beat into single cell suspension gently, the counting back is by 1 * 10
6/ ml is inoculated in culturing bottle, puts incubator, changes half liquid in every 3-4 days.Former neurocyte ball of being commissioned to train foster after trysinization, is made single cell suspension with complete culture solution, behind the counting, by 1 * 10
5/ ml goes down to posterity and is inoculated in the 35mm plastics plate that scribbles poly-lysine, puts incubator and cultivates.
2. the evaluation of human nerve stem cell
The former foster neural stem cell of being commissioned to train is cultivated after 3 days in the substratum of serum-free, or the former foster neural ball of being commissioned to train, and is seeded in after going down to posterity in the culture dish of gummed 35mm, cultivates after 3 days, after the Paraformaldehyde 96 with 4% is fixing, carries out the Nestin immunohistochemical staining.Mouse Nestin antibody (1: 1000), 4 ℃ are spent the night, biotin labeled two anti-(Vector, 1: 1000), 4 ℃ are spent the night, and 60 fens kinds of ABC mixture (1: 1000) normal temperature with the colour developing of DAB method, are brown endochylema throw out.Identify that through neural stem cell marker Nestin dyeing the former neural stem cell ball Nestin that is commissioned to train foster is positive, and neural stem cell Nestin as the Fig. 1 and the back adherent growth that goes down to posterity is positive in Fig. 2.The experiment of back all is that the human nerve stem cell with the 2nd and 3 generations carries out.
Embodiment two low-oxygen environments are to the short proliferation function of human nerve stem cell
1. experiment grouping and hypoxia condition determines
Experiment divides 3 groups, is respectively normal oxygen control group (20%O
2) 3% hypoxia group (3%O
2) and 10% hypoxia group (10%O
2).Self-control cell hypoxemia incubator feeds respectively in closed cabinet and contains 3% or 10%O
2, 5%CO
2With the mixed gas of nitrogen balance, beginning air flow 1L/min, continue ventilation 30min after, be adjusted to maintenance dose 0.1L/min and continue ventilation, keep oxygen level in the encloses container to be respectively 3% or 10%O
2
2. the detection method of cell proliferation of nerve cord
1) neural ball count method
Isolating neural stem cell was placed on respectively in the environment of different oxygen concentrations and cultivated after 3 days former generation, and double-blind method is counted the formation number of neural ball, and its mean value, is calculated in 50 visuals field of every bottle of counting by 4 bottles every group under 200 power microscopes.Under 200 power microscopes, the number of double-blind method numeration BrdU stained positive cell, 50 visuals field of every ware counting, every group of 5-6 ware calculates its average, relatively uses non-matching Student ' sT-test between group.
2) BrdU mixes and immunohistochemical staining
After going down to posterity, neural ball warp is seeded in the culture dish of gummed 35mm, every ware adds BrdU 20ul (final concentration is 10uM), be placed on respectively then contain 3%, 10% and normal oxygen environment in cultivated 3 days, after Paraformaldehyde 96 with 4% is fixing, carry out the BrdU immunohistochemical staining.After 30 minutes denaturing treatment of 2N HCl normal temperature, add mouse BrdU-antibody (MolecularProbe, 1: 1000) 4 ℃ 48 hours, biotin labeled two anti-(Vector, 1: 1000), 4 ℃ are spent the night, ABC mixture (1: 1000) normal temperature 60 minutes, with single nickel salt amine-DAB staining colour developing, BrdU immune response positive cell is the nucleus throw out of black.Under 200 power microscopes, the number of double-blind method numeration BrdU stained positive cell, 50 visuals field of every ware counting, every group of 5-6 ware calculates its average, relatively uses non-matching Student ' sT-test between group.
3. low-oxygen environment is to the short proliferation function of human nerve stem cell
1) with former generation cultured cells be placed on respectively in normal oxygen (oxygen level is 20%) and hypoxemia (oxygen level the is respectively 3% and 10%) environment and cultivated 3 days, microscopically is observed the neurocyte ball of cultivating and is increased in low-oxygen environment.Analyze by statistics: under 10% low-oxygen environment, the formation number of neural ball is apparently higher than normal oxygen group, and the neural nodule number order of propagation is normal oxygen group 2.4 times (as Fig. 3), and is same, the neural nodule number order that forms in 3% low-oxygen environment also increases to some extent, is 1.5 times of normal oxygen group.
2) hypoxemia is to the short proliferation function of the neural stem cell of adherent growth.5-bromouracil deoxyribose (BrdU) is the analogue of uracil, is incorporated in the dna double chain in the synthesis phase of cell, can be used as the index of cell proliferation.Before hypoxemia, BrdU is joined in the substratum, then neural precursor is placed on respectively and cultivates after 72 hours under the different environment, the BrdU coloration result shows: the positive cell number of (3% and 10%) BrdU mark has increased by 63% and 88% than normal oxygen group respectively in low-oxygen environment, apparently higher than normal oxygen group (as Fig. 3).BrdU mixes experiment and shows: the positive cell number of BrdU mark is apparently higher than control group in low-oxygen environment.
The differentiation capability of the human nerve stem cell of cultivating in embodiment three low-oxygen environments
1. the method for cell differentiation of nerve cord
The neural stem cell in the 2nd or 3 generations is blown and beaten into single cell suspension, by 1 * 10
5/ ml goes down to posterity and is inoculated in 25cm
2Culturing bottle in, be placed on normal oxygen control group (20%O respectively
2), 3% hypoxia group (3%O
2) and 10% hypoxia group (10%O
2) propagation is long to 50-100 cell arranged to neural ball in the environment, takes out the back and removes growth media, blows and beats into unicellularly in containing the DMEM/F12 differentiation liquid of 1%FBS and 1%N3, presses 1 * 10 behind the counting
5/ ml is seeded in 35mm respectively
2In the gummed plate, under the normal oxygen condition, cultivated 9-12 days, changed liquid once in every 3-4 days, observe influence its differentiation.
2. be divided into the detection method of mature neuron: the NeuN immunohistochemical staining
Cell after the differentiation is fixed with 4% Paraformaldehyde 96, add mouse NeuN antibody (MolecularProbe, 1: 1000) 4 ℃ 48 hours, biotin labeled two anti-(Vector, 1: 1000), 4 ℃ are spent the night, ABC mixture (1: 1000) normal temperature 60 minutes, with benzidine (DAB) colour developing, NeuN immune response positive cell is brown nucleus throw out.
3. the human nerve stem cell of cultivating in the low-oxygen environment can detect by differentiation capability
Above result shows that hypoxemia (oxygen level is respectively 3% and 10%) can promote the amplification in vitro of neural stem cell, so, does its differentiation capability of the neural stem cell of breeding in low-oxygen environment change? for this reason, the contriver observes the differentiation capability of the neural stem cell of breeding in the low-oxygen environment.With the neural stem cell ball of cultivating in the varying environment, in differentiation liquid, blow and beat into unicellular, be seeded in the gummed plate, in containing the differentiation liquid of 1% foetal calf serum, the equal adherent growth of visible cell after 24 hours, and grow projection, generally needed 9-12 days just can become sophisticated neurone, and the result show: at 3%O
2Good with the neure growth state that forms after the cell differentiation of nerve cord of cultivating in 10% low-oxygen environment, number is more than normal oxygen group (as shown in Figure 5).This result shows: the differentiation capability of the neural stem cell of breeding in the low-oxygen environment is not subjected to tangible influence, helps its neuralward unit direction differentiation all the better.
The function of neurons of cultivating in embodiment four low-oxygen environments is identified
1. neuronic electrophysiological characteristics detection method
With the 3rd generation the neural stem cell ball be seeded in the 35cm gummed plate and break up, differentiation liquid is the DMEM/F12 (1%N3) that contains 1% foetal calf serum, changed liquid once in every 3-4 days, cultivated 9-12 days, and treated that neurosome became big, the elongated one-tenth net of projection, method with the outer record of cell born of the same parents, sodium-the potassium current of record cell is surveyed 6-8 cell for every group at random, calculates the mean of sodium-potassium current and carries out statistical study.
2. hypoxemia is to the influence of the neurocyte electrophysiological characteristics after breaking up
Known sophisticated neurone has distinctive sodium-potassium current, and the existence of sodium-potassium current also is the prerequisite that neurone has function, and therefore, the contriver has further detected the influence of hypoxemia to sodium-potassium current on the neuron membrane after breaking up again.The result shows: the neural stem cell of breeding in hypoxemia (3%) environment, differentiation back neuron membrane sodium-potassium current is big, apparently higher than the neurone of in 20% and 10% low-oxygen environment, inducing differentiation, and the neural stem cell of in hypoxemia (10%) environment, breeding, differentiation back neuron membrane sodium-potassium current is less than the neurone that breaks up in 20% and 3% the low-oxygen environment (as shown in Figure 6).This results suggest 3% low-oxygen environment helps the maturation of neurocyte.
*This research has obtained the subsidy of state natural sciences fund major project.
Claims (5)
1, a kind of method of regulating the human nerve stem cell propagation of vitro culture is characterized in that adopting low-oxygen environment, promotes the propagation of the human nerve stem cell of vitro culture.
2, method according to claim 1 is characterized in that said low-oxygen environment is that oxygen concn is lower than 20% environment.
3, method according to claim 1 and 2, the concentration that it is characterized in that oxygen in the low-oxygen environment is 3% to 10%.
4, method according to claim 3 is characterized in that the oxygen concn in the low-oxygen environment is 3%.
5, method according to claim 3 is characterized in that the oxygen concn in the low-oxygen environment is 5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101168139A CN1621515A (en) | 2003-11-28 | 2003-11-28 | Method for adjusting the multiplication of human nerve stem cell cultured in vitro |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101168139A CN1621515A (en) | 2003-11-28 | 2003-11-28 | Method for adjusting the multiplication of human nerve stem cell cultured in vitro |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1621515A true CN1621515A (en) | 2005-06-01 |
Family
ID=34760777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003101168139A Pending CN1621515A (en) | 2003-11-28 | 2003-11-28 | Method for adjusting the multiplication of human nerve stem cell cultured in vitro |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1621515A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811410A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | External extremely low oxygen inducing function nerve unit forming method based on micro-fluidic chip |
| CN111454905A (en) * | 2020-03-03 | 2020-07-28 | 中国人民解放军军事科学院军事医学研究院 | Application of FG-4592 or salt thereof in expansion of neural stem cells |
| EP3766506A4 (en) * | 2018-03-16 | 2022-03-09 | Medinno Inc. | Angiogenesis-inducing method using neural stem cell |
-
2003
- 2003-11-28 CN CNA2003101168139A patent/CN1621515A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811410A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | External extremely low oxygen inducing function nerve unit forming method based on micro-fluidic chip |
| EP3766506A4 (en) * | 2018-03-16 | 2022-03-09 | Medinno Inc. | Angiogenesis-inducing method using neural stem cell |
| CN111454905A (en) * | 2020-03-03 | 2020-07-28 | 中国人民解放军军事科学院军事医学研究院 | Application of FG-4592 or salt thereof in expansion of neural stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230115507A1 (en) | Agent for accelerating growth of pluripotent stem cells | |
| CN103031275A (en) | Inducing method for differentiating umbilical cord mesenchymal stem cells into neural stem cells | |
| Sellés et al. | Effect of sucrose on growth and galanthamine production in shoot-clump cultures of Narcissus confusus in liquid-shake medium | |
| US20020142463A1 (en) | Method for the mass propagation of adventitious roots of ginseng, camphor ginseng and wild ginseng by tissue culture and the improvement of their saponin content | |
| Yong et al. | In vitro micropropagation of Eucheuma seaweeds | |
| CN103789268B (en) | A kind of method of separation and Culture hippocampal neurons and special culture solution thereof | |
| CN108753736A (en) | Produce the method and its application of varicella virus stoste | |
| CN103805565B (en) | Hippocampal neuron is separated and primary culture method and reagent | |
| CN1221660C (en) | Method for exosomatic separation and amplification of filled stem cells between human marrow and funic blood and directional induction toward nerve cells | |
| CN1621515A (en) | Method for adjusting the multiplication of human nerve stem cell cultured in vitro | |
| CN100344756C (en) | A method for in vitro derivativing nerve stem cell directional differentiation to cholinergic neuron | |
| CN108998410A (en) | Kinases inhibitor is inhibiting the purposes in haploid cell diplodization | |
| CN104789531A (en) | Method for inducing umbilical cord mesenchymal stem cells to be differentiated into dopaminergic neurons | |
| CN103074300A (en) | Co-culture system establishment of mesenchymal stem cells and tumor cells as well as mesenchymal stem cells heredity stability change characteristic in tumor microenvironment | |
| CN102600465A (en) | Newcastle disease (ND) vaccine, and its production method | |
| CN107164325B (en) | The preparation method and kit of the oligodendroglia in the source MSCs | |
| CN111676194A (en) | Method for improving differentiation maturity of induced neural stem cells to TH positive neurons by using ginkgolides | |
| CN102250841A (en) | Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof | |
| CN103013919A (en) | Method for separating and subculturing primary embryo-derived neural stem cell | |
| CN114703126A (en) | Muscle stem cell separation and extraction, novel hydrolysate culture system and application thereof | |
| CN104726407A (en) | Method for increasing yield of neural stem cells in adult nerve tissues by utilizing organotypic culture | |
| CN105567569B (en) | A kind of cultural method of dendrobium candidum brown patch germ | |
| CN105420117B (en) | For cultivating the culture medium containing special sugar source of dendrobium candidum brown patch germ | |
| CN1342461A (en) | Process for preparing embryo nutrient | |
| Willoughby | The fruiting behaviour and nutrition of Cladochytrium replicatum Karling |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |