CN114525250A

CN114525250A - Method for differentiating tumor stem cells towards neurons

Info

Publication number: CN114525250A
Application number: CN202210436760.1A
Authority: CN
Inventors: 肖海蓉; 刘庆喜; 魏卿; 房帅
Original assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Current assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Priority date: 2022-04-25
Filing date: 2022-04-25
Publication date: 2022-05-24
Anticipated expiration: 2042-04-25
Also published as: CN114525250B

Abstract

The invention provides a method for differentiating tumor stem cells towards neurons, which comprises the steps of obtaining tumor stem cells, inducing and differentiating the tumor stem cells towards the neurons, culturing the differentiated neurons and the like; the invention also carries out certain verification work on the differentiated neurons, verifies that the neurons from the tumor stem have the specific markers of the neurons, and verifies that the neurons from the tumor stem have sodium ion inflow signals through patch clamp.

Description

Method for differentiating tumor stem cells towards neurons

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a method for differentiating tumor stem cells towards neurons.

Background

Tumor stem cells (CSCs) are a subpopulation of cancer cells that have the ability to resemble somatic stem cells. CSCs play a key role in the development, metastasis, recurrence and resistance of tumors. The vast majority of tumors are considered to originate from CSCs, which may be one of the important reasons for primary and acquired drug resistance in clinical tumor treatment, so that the research on tumor stem cells has attracted extensive attention in the basic research of tumors at home and abroad in recent years. At present, a few human tumors are known, which spontaneously regress from an undifferentiated malignant tumor to a completely benign tumor, and differentiation of neuroblastoma cells into neuronal cells belongs to one of them.

With the continuous development of stem cells and regenerative medicine technology, the original cell population with unlimited self-renewal, replication, differentiation and regeneration capacity of stem cells has shown huge potential in the aspects of tissue repair and regeneration. Therefore, many researchers have been working on the repair of the degenerative nervous system using stem cells. Through years of exploration of researchers, until now, the field of intervention of stem cells in neurodegenerative diseases has been broken through successively, and a new hope is brought to patients with neurodegenerative diseases. At present, stem cells for repairing neurons mainly originate from neural stem cells, and the neural stem cells are difficult to source and obtain, so that the research progress of nerve regeneration is restricted. Therefore, the directed differentiation of the CSCs is not only a potential cancer treatment strategy, but also provides a new approach for the regeneration and repair of nerves.

At present, the tumor stem is mainly obtained from tumor tissues and tumor cell lines, and because the tumor tissues obtained from clinical sources are limited, and part of the tumor tissues need to be subjected to clinical pathological analysis, most of the tumor tissues for separating the tumor stem are mainly cancer-adjacent tissues, which also restricts the obtaining of the tumor stem cells. For the above reasons, more and more researchers are focusing on the isolation of tumor stem cells from tumor cell lines.

In the research of the differentiation of the tumor stem cells into the nerve cells, most researches mainly refer to a method for inducing the nerve stem cells into the nerve cells, wherein the method mainly comprises the steps of removing serum and adding neurotrophic factors and ATRA (atom transfer radical amplification) inducers in the culture process, the capacity and the efficiency of promoting the differentiation of the tumor stem cells into the nerve cells are low, and the functions of the differentiated nerve cells are required to be further improved.

Disclosure of Invention

In view of the above, the present invention is directed to a method for differentiating tumor stem cells into neurons, which comprises inducing the tumor stem cells into neurons rapidly, wherein the differentiated neurons have neuron-specific markers and sodium current signals.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a method for differentiating tumor stem cells towards neurons comprises the following steps:

s1, obtaining tumor stem cells;

s2, inducing and differentiating the tumor stem cells to neurons;

s21, inoculating the tumor stem cells in a differentiation medium and culturing for 3-7 days, and changing the medium every 2 days for 1 time, wherein the differentiation medium is a DMEM/F12 complete medium containing FBS, MEM-100x, streptomycin 100x, all-trans retinoic acid ATRA and curcumin;

s22, introducing the differentiated cells into a neuron maintenance Medium, wherein the neuron maintenance Medium is Neurobasal Medium A complete Medium containing BDNF, NGF, MEM-100x, streptomycin qinghaosu 100x and B27, and continuing to culture for 7-14 days.

Further, the volume fraction of FBS in the differentiation medium is 1-8%, the volume fraction of MEM-100x is 0.5-2%, the volume fraction of streptomycin 100x is 0.1-0.4%, the dosage of ATRA is 1-10 μ M per liter, and the dosage of curcumin is 0.5-5 μ M per liter.

Further, the dosage of FBS in each 100mL of differentiation medium is 5mL, the dosage of MEM-100x is 1mL, the dosage of streptomycin 100x is 200 muL, the dosage of ATRA is 1 muM, and the dosage of curcumin is 0.5 muM.

Further, the concentration of BDNF in the neuron maintenance medium is 10ng/mL-80ng/mL, the concentration of NGF is 5ng/mL-40ng/mL, the volume fraction of MEM-100x is 0.5-2%, the volume fraction of penicillin 100x is 0.1-0.4%, and the volume fraction of B27-50x is 0.5-4%.

Further, the concentration of BDNF in each 100mL of neuron maintenance culture medium is 50ng/mL, the concentration of NGF is 10ng/mL, the dosage of MEM-100x is 1mL, the dosage of streptomycin is 200 mu L, and the dosage of B27-50x is 2 mL.

Further, the obtained tumor stem cells were digested with trypsin to obtain single cells before the step of S2.

Further, the cultivation in step S21The conditions are as follows: 37 ℃ and 5% CO₂Culturing in an incubator.

Further, step S1 specifically includes the following steps:

1) dry culturing glioma cell line U-118 MG;

inoculating U-118MG cells in tumor stem cell culture medium containing 1-40ng/ml bFGF, 1-20ng/ml EGF, 0.5-4% B27-50x, 0.1-0.4% streptomycin 100x serum-free DMEM/F12 complete medium, and culturing for 3-5 days;

2) sorting immunomagnetic beads;

3) differential adherent purification after sorting;

4) tumor stem is rapidly amplified.

The present invention also provides a neural stem cell obtained by the differentiation method according to any one of the above-mentioned methods.

Compared with the prior art, the method for differentiating the tumor stem cells to the neuron direction has the following advantages:

the method for differentiating the tumor stem cells towards the neurons combines all-trans-retinoic acid (ATRA) and curcumin (curcumin) in the process of differentiating towards the neurons, the curcumin cooperates with the ATRA to inhibit proliferation of the tumor stem cells, the differentiation of the tumor stem cells towards the neurons is promoted by inhibiting a Notch1 signal channel, the strong differentiation capability towards the neurons is shown, and the differentiated neurons have markers of mature neurons and sodium ion signals.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a diagram of tumor stem cells in this example;

FIG. 2 is a diagram illustrating flow identification of tumor stem cells in this example;

FIG. 3 is a graph showing the differentiation of tumor stem cells into tumor cells according to this example;

FIG. 4 is a transmission photoplethysm of the tumor stem cells after differentiation into neurons according to the present embodiment;

FIG. 5 is a fluorescent plot of β III Tubulin following neuronal differentiation of tumor stem cells according to this example;

FIG. 6 is a graph of the sodium current I-V after differentiation of tumor stem cells into neurons according to this example;

FIG. 7 is a photograph of light transmitted after differentiation of comparative example tumor stem cells into neurons.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

Examples

The method for differentiating the tumor stem cells towards the neuron direction comprises the following steps:

1) and (3) recovering U-118MG cells: the cancer cell culture medium and DMEM/F12 basal medium were preheated to 37 ℃ in a water bath and 6mL of preheated basal DMEM/F12 medium was added to a 15mL centrifuge tube. Taking out frozen U-118MG cells from liquid nitrogen tank, rapidly thawing in 37 deg.C water bath pan (thawing degree is suitable for small amount of ice crystal left in freezing tube), sucking cell suspension into a pre-placed centrifuge tube containing 6mL of basic culture medium, centrifuging at room temperature of 1000 r/min for 5 min, discarding supernatant, adding appropriate amount of culture solution with cell concentration of 1 × 10⁵cells/mL were grown in culture flasks at 37 ℃ with 5% CO₂Culturing in an incubator, and performing liquid change treatment every 2 d.

2) Passage of U-118MG cells: and the growth density of the U-118MG cells reaches more than 95% of the bottom area of the culture flask for cell passage.

The method comprises the following specific steps:

(1) the medium and PBS were pre-warmed at 37 ℃ in advance.

(2) The cell culture fluid was aspirated off and a small amount of PBS was added to rinse the cells.

(3) Removing PBS by suction, adding appropriate amount of pancreatin to cover cells, adding 5% CO at 37 deg.C₂Digesting in incubator for about 3 min, adding culture medium with 2 times of pancreatin volume to stop digestion when cells tend to be round, intercellular space becomes large and a small amount of cells float, and using gun to stop digestionThe cells were gently tapped with a head to completely suspend the cells.

(4) Collecting cell suspension, centrifuging at room temperature 1000 r/min for 5 min, sucking off supernatant, and blowing suspending with culture medium to make cell concentration 1 × 10⁵The individual cells/mL were planted in culture flasks at 37 ℃ with 5% CO₂Culturing in an incubator.

3) Dry culture of tumor cells: the cells were cultured at 1X 10⁵The cells/mL are inoculated in a tumor stem cell culture medium for 5 days, each 100mL of the culture medium comprises 10ng/mL of bFGF, 20ng/mL of EGF, 2mL of B27-50x, and 200 mu L of DMEM/F12 serum-free complete medium of streptomycin 100x, and the cells are cultured for 3 times and 15 minutes each time in an environment at 4 ℃ during the culture process.

4) And (3) sorting immunomagnetic beads: the sorting operation was performed exactly according to the Miltenyi Biotec magnetic bead sorting instructions, with the following specific steps:

(1) the U-118MG adherent cells were washed with PBS to remove dead cells, and digested with pancreatin.

(2) The cells were resuspended in a basal medium, centrifuged at 1000 rpm for 5 min and the cell pellet collected.

(3) At 10⁷60 mu L of buffer solution is added to each total cell to resuspend the cell pellet.

(4) Every 10 th ⁷20 μ L of FcR blocking reagent was added to each total cell.

(5) Every 10 th ⁷20 mu L of CD133 magnetic beads are added into each total cell, and after fully and uniformly mixing, the cells are incubated in a refrigerator (2-8 ℃) for 15 min. The incubation was continued with slow rotation using a MACSmix rotator to prevent cell sedimentation.

(6) The incubated cells were washed, 1-2 mL of buffer was added to wash the cells, and then centrifuged at 300 g/min for 10min, and the supernatant was discarded.

(7) At 10⁷The cells were suspended by blowing 500 μ L buffer solution for each cell (10)⁷Individual cells).

(8) The separation column (500 μ L) was placed in a separator, and the separation column was rinsed with a rinse solution.

(9) Slowly adding the incubated cells into the separation column, collecting the unlabeled cells, and adding the unlabeled cells into the separation column again.

(10) Washing the column with appropriate amount of buffer, repeating 3 times, and washing to remove unlabeled cells (CD 133) to obtain positive cells (CD 133)⁺) Left in the separation column.

(11) And taking the separation column off the separation frame, and putting the separation column on the collecting pipe for elution.

5) And (3) carrying out ball-forming culture on the sorted cells: the cells sorted by the magnetic beads were treated at 1X 10⁵Inoculating each cell/mL in a tumor stem cell culture medium, attaching more first-generation cells in spherulitic culture to the wall, collecting cells growing in spherulitic growth, carrying out pancreatin digestion, repeatedly collecting the spherulitic cells to remove the attached cells, facilitating the purification of the tumor stem cells, and obtaining the dry cells completely suspended in spherulitic growth after repeated passage.

6) And (3) identifying the tumor stem cells:

(1) collecting the dry U-118MG cells, digesting with 0.05% pancreatin, terminating the culture medium, centrifuging at 1000 r/min for 5 min, washing with PBS, and counting.

(2) The cell suspension was centrifuged at 1000 r/min for 5 min and the supernatant was discarded.

(3) Adding 4% paraformaldehyde stationary liquid, fixing for 15 min, washing with PBS, centrifuging at 1000 r/min for 5 min, and blowing and suspending with PBS to obtain suspension with density of 1 × 10⁶Individual cells/mL of cell suspension.

(4) FITC-CD133, APC-OCT4, PE-CD44 antibodies were added at 2 μ L each (the antibodies were diluted 1:50 according to the recommended proportion in the antibody instruction of America and whirlpool), and incubated for 1 h at room temperature in the dark.

(5) PBS was washed 3 times to remove unbound CD133, OCT4, CD44 antibody.

(6) The cells were resuspended in 500 μ L PBS and flow cytometric assay was performed, the results are shown in figure 2.

7) Differentiation of tumor stem cells into U-118MG cells: collecting the suspension-grown U-118MG stem cells, centrifuging the cell suspension at 1000 r/min for 5 min, removing the supernatant, and washing with appropriate amount of PBS for 1 time. The PBS was removed by suction and 0.05% pancreatin was added and the mixture was left at 37 ℃ with 5% CO₂Culture boxDigesting for 5 min. Gently blowing to single cell, adding sufficient PBS to dilute pancreatin, centrifuging at room temperature of 1000 r/min for 5 min, removing supernatant, adding appropriate amount of tumor cell culture solution containing serum (DMEM/F12 +10% FBS +1% streptomycin 100 x), and adjusting cell density to 1 × 10⁵The cells/mL were inoculated into 24-well plates and cultured for 7 d (1 change every 2 d), observed under a microscope, and recorded by photographing, and the results are shown in FIG. 3.

8) Differentiation of tumor stem cells into neurons:

(1) collecting the suspension-grown U-118MG stem cells, centrifuging the cell suspension at 1000 r/min for 5 min, removing supernatant, and washing with appropriate amount of PBS for 1 time. Then adding 0.05% pancreatin, and standing at 37 deg.C and 5% CO₂Digesting in incubator for 5 min. Slightly blowing and beating to a single cell, adding sufficient PBS to dilute pancreatin, centrifuging for 5 min at the speed of 1000 r/min, removing supernatant, adding tumor stem cells into a neuron differentiation culture medium, wherein the dosage of FBS in each 100mL of differentiation culture medium is 5mL, the dosage of MEM-100x is 1mL, the dosage of streptomycin cyaneus 100x is 200 mu L, the dosage of ATRA is 1 mu M, and the dosage of curcumin is 0.5 mu M; then placing at 37 ℃ and 5% CO₂Culturing in incubator for 5d, and changing liquid every 2 d for 1 time.

(2) After differentiation, the differentiation medium is sucked and discarded, PBS is used for cleaning for 2 times, the neuron maintenance medium is added for continuous culture for 10 days, the BDNF concentration in each 100mL of neuron maintenance medium is 50ng/mL, the NGF concentration is 10ng/mL, the MEM-100x dosage is 1mL, the streptomycin 100x dosage is 200 mu L, and the B27-50x dosage is 2 mL; changing the culture medium for neuron maintenance 1 time every 2 days, and identifying the nerve cells after the cell synapses are stretched.

And (4) analyzing results:

1. tumor stem cells

Tumor stem cells fig. 1 shows the obtained tumor stem cells, which grow in suspension and aggregate to grow as spheres.

As is clear from FIG. 2, the obtained tumor stem cells had CD133⁺CD44⁺OCT4⁺Three positive properties.

2. Tumor stem cell differentiation

FIG. 3 is a graph showing the differentiation of tumor stem cells into U-118MG cells, from which it can be seen that the tumor stem cells can be rapidly differentiated into tumor cells in a tumor cell culture environment, indicating that the obtained tumor stem cells have a multi-differentiation capability.

FIG. 4 is a diagram of transmitted light after differentiation of tumor stem cells into neurons, which illustrates that the neural cells derived from the tumor stem cells have the basic characteristics of neurons, have obvious synapse generation, and can be rapidly differentiated into the neurons under the environment of differentiation into the neurons, and thus the multi-directional differentiation characteristics of the tumor stem are verified.

3. Fluorescent identification

Identifying a neuron marker beta III-Tubulin by adopting an immunofluorescence method, comprising the following steps:

(1) discarding the supernatant, gently washing the residual culture solution with PBS, fixing 4% paraformaldehyde at room temperature for 20 min, adding excessive PBS, gently mixing, centrifuging, and washing with PBS for 3 min 2 times. The cell samples were then incubated with 0.25% Triton-X100 in PBS for 30 minutes to increase permeability.

(2) Blocking with 5% sheep serum for 30 min to block non-specific staining.

(3) The blocking solution was aspirated, and the prepared murine primary anti-. beta.III Tubulin incubation solution (1. mu.g/mL) was added thereto and incubated at 4 ℃ for 12 hours.

(4) PBS was washed 3 times, goat anti-mouse FITC labeled secondary antibody was added and incubated for 2 h at room temperature in the dark.

(5) PBS was washed 3 times, DAPI dilution (1: 200) was added, and incubation was carried out at room temperature for 3-5 min in the absence of light.

(6) PBS is washed for 3 times, a proper amount of PBS is added (the PBS needs to cover the cells), and the cells are observed under a fluorescence microscope and photographed and recorded.

The result is shown in FIG. 5, which shows that the differentiated nerve cells have positive characteristics of the neuron-specific marker β III Tubulin.

4. Performance testing of differentiated neural stem cells

The electrophysiological experiment method comprises the following steps:

the patch clamp electrophysiology technique is a method of using micro-glass tube electrode (patch suction tube or patch electrode) to contact cell membrane, using impedance above giga ohm to make it seal-connect, making the cell membrane small region (patch) connected with electrode tip opening be electrically separated from its periphery, fixing point position on the basis of said electric separation, and monitoring and recording ion current (pA grade) of ion channel on said patch.

The recording method comprises the following steps: the I-V converter formed by the field effect tube operational amplifier is the core part of the measuring loop. When the positive and negative input terminals of the FET operational amplifier are equipotential and a command potential is applied to the positive input terminal, the purpose of clamping is achieved equipotential by both the short-circuit negative terminal and the diaphragm, when a 10G omega or more seal is formed between the diaphragm microelectrode tip and the diaphragm, the shunt current therebetween is minimized, and 100% of the current across the diaphragm can be measured as the recording current (l p) from the diaphragm electrode.

Preparing a microelectrode used for electrophysiological detection: the electrode drawing can be directly carried out by purchasing a clean glass blank, or the electrode drawing can be carried out by purchasing an unclean glass blank after cleaning. In order to reduce the noise that occurs in single-pass recordings, the glass blank is preferably cleaned again. The glass blank is cleaned by soaking in absolute ethyl alcohol, ultrasonic treating for 10min, cleaning with deionized water for 3 times, and oven drying at 200 deg.C. In order to prevent the generation of surface tension in the electrode, cleaning is not performed using a cleaning agent. Since the microelectrode used in patch clamp electrophysiological experiments does not need to penetrate into the cell, the electrode tip is not required to be very sharp. The electrodes are drawn by a second draw, which is generally required because the first draw softens the glass and pulls it apart a distance, which pulls the glass electrodes apart to form a lumen, i.e., a neck, for an electrode. The second draw is to use a lower temperature to facilitate the breaking of the tubules to form two identical electrodes. The diameter of the drawn electrode tip is typically 1-5 μ M, while the resistance of Ringer's fluid after filling is 1-5M Ω. The resistance can be detected using a patch-clamp amplifier. When the glass electrode is used, the glass electrode needs to be pulled at present so as to avoid dust from adhering to the electrode. The electrode box, the electrode can and the glass vessel for storing the buffer solution can not be stored at night, and the buffer solution can only be temporarily stored.

The instruments for drawing the electrodes are of various kinds: there are horizontal, vertical and some equipment that are also equipped with polishing systems. The manufacturers of glass microelectrodes mainly include Narishige company in Japan and Sutter company in the United states (products are classified as P-200, P-100, P-97). The apparatus for drawing the electrode using a platinum sheet was as follows: p-100 and P-97 from Sutter, USA. Such devices require attention to the following details:

(1) the drawing apparatus was preheated for 30 min before drawing the glass electrode.

(2) Note that the platinum sheet cannot be touched by hand when the glass electrode is drawn, so as not to influence various parameters during drawing and even replace the platinum sheet.

(3) The platinum sheet needs to be replaced in time because the platinum sheet is aged after long-time use, and the resistance of the electrode is influenced.

(4) The platinum sheet cannot be replaced by pliers, otherwise the service life of the platinum sheet is reduced.

(5) When the electrodes are drawn, it is not easy to draw a plurality of electrodes at one time so as to avoid large difference between the electrodes.

(6) The cooling air outlet of the drawing instrument needs to be provided with a drying agent, and when the drying agent becomes powder after being moistened, the drying agent needs to be replaced by a new drying agent in time.

(7) The parameters of the drawing instrument are not changed at will, and the original parameters must be backed up or other channel settings must be selected when the experiment requires the parameters to be changed.

The microelectrode used in the experiment is obtained by drawing twice, the glass is softened after the drawing speed, the pressure, the tension and the temperature are adjusted, the microelectrode is drawn into a thin rod-shaped structure, then the microelectrode is drawn into an electrode with two sharp ends from the center of the rod again, and the resistance is kept between 2 and 4M omega after the microelectrode is filled with electrode liquid. To prevent dust contamination, the electrodes are drawn prior to use. Because the compensation requirements of the film capacitance and the series resistance are high, the micro-electrode resistance is about 2-4M omega during the experiment, so that the accuracy of the experiment result is ensured.

Electrophysiological recording: after the cells are sealed, the electrode instrument is operated under a microscope, the recording electrode is connected to the detected cells, then negative pressure is pumped to complete G omega sealing, capacitance compensation is carried out, negative pressure is applied to rupture the membrane, and a whole cell recording mode is formed. Experimental data signals were collected using an EPC-10 amplifier and the collected data was stored in the PatchMaster software.

FIG. 6 is a graph of sodium current I-V, from which it can be seen that differentiated neurons have significant sodium current signals.

Comparative example 1

On the basis of the above examples, no curcumin was added.

After the induction culture by the same method, the differentiation condition of the neurons without adding curcumin is shown in fig. 7, and synapses of the differentiated neurons are shorter than those of the embodiment with curcumin, which indicates that the addition of curcumin can promote the differentiation of the neurons, enhance the differentiation capability to the neurons, and the differentiated neurons have markers of mature neurons and sodium ion signals.

TABLE 1 list of reagent names

Name of reagent	Chinese name and explanation
		FBS	Fetal bovine serum
MEM-100x	Non-essential amino acid solution
		Penicillin streptomycin 100x	Penicillin streptomycin
BDNF	Brain-derived neurotrophic factor
		NGF	Nerve growth factor
B27-50x	B27 additive
		DMEM/F12	DMEM/F12 medium
Neurobasal Medium A complete Medium	Basic culture medium for neuron growth and maintenance
		bFGF	Recombinant human fibroblast growth factor
EGF	Recombinant human epithelial growth factor

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for differentiating tumor stem cells towards neurons is characterized in that: the method comprises the following steps:

s1, obtaining tumor stem cells;

s2, inducing and differentiating the tumor stem cells to neurons;

s22, introducing the differentiated cells into a neuron maintenance Medium, and continuing to culture for 7-14 days, wherein the neuron maintenance Medium is a Neurobasal Medium A complete Medium containing BDNF, NGF, MEM-100x, streptomycin qinghaosu 100x and B27-50 x.

2. The method for differentiating tumor stem cells in a neuronal direction according to claim 1, wherein: the volume fraction of FBS in the differentiation medium is 1-8%, the volume fraction of MEM-100x is 0.5-2%, the volume fraction of streptomycin 100x is 0.1-0.4%, the dosage of ATRA is 1-10 μ M per liter, and the dosage of curcumin is 0.5-5 μ M per liter.

3. The method for differentiating tumor stem cells in a neuronal direction according to claim 2, wherein: the dosage of FBS in each 100mL of differentiation medium is 5mL, the dosage of MEM-100x is 1mL, the dosage of streptomycin 100x is 200 muL, the dosage of ATRA is 1 muM, and the dosage of curcumin is 0.5 muM.

4. The method for differentiating tumor stem cells in a neuronal direction according to claim 1, wherein: the concentration of BDNF in the neuron maintenance medium is 10ng/mL-80ng/mL, the concentration of NGF is 5ng/mL-40ng/mL, the volume fraction of MEM-100x is 0.5-2%, the volume fraction of streptomycin 100x is 0.1-0.4%, and the volume fraction of B27-50x is 0.5-4%.

5. The method for differentiating tumor stem cells in a neuronal direction according to claim 4, wherein: the BDNF concentration in each 100mL of neuron maintenance medium is 50ng/mL, the NGF concentration is 10ng/mL, the MEM-100x dosage is 1mL, the penicillin streptomycin 100x dosage is 200 mu L, and the B27-50x dosage is 2 mL.

6. The method for differentiating tumor stem cells in a neuronal direction according to claim 1, wherein: the obtained tumor stem cells were digested with trypsin to obtain single cells before the step of S2.

7. The method for differentiating tumor stem cells in a neuronal direction according to claim 1,the method is characterized in that: the culture conditions of step S21 were: 37 ℃ and 5% CO₂Culturing in an incubator.

8. The method for differentiating tumor stem cells in a neuronal direction according to claim 1, wherein: step S1 specifically includes the following steps:

1) dry culturing glioma cell line U-118 MG;

2) sorting immunomagnetic beads;

3) differential adherent purification after sorting;

4) tumor stem is rapidly amplified.

9. A neural stem cell obtained by the differentiation method according to any one of claims 1 to 8.