WO2019100726A1

WO2019100726A1 - Application of d dna tetrahedron in promoting proliferation and differentiation of neural stem cells

Info

Publication number: WO2019100726A1
Application number: PCT/CN2018/094561
Authority: WO
Inventors: 林云锋; 马文娟; 蔡潇潇; 李谦顺; 赵丹
Original assignee: 四川大学
Priority date: 2017-11-22
Filing date: 2018-07-04
Publication date: 2019-05-31
Also published as: CN107881149A

Abstract

An application of a DNA tetrahedron in promoting proliferation and differentiation of neural stem cells. The sequences of four single strands of the DNA tetrahedron are as shown in SEQ ID NOs: 1-4. The application can effectively promote the proliferation and differentiation of neural stem cells.

Description

[Correct according to Rule 26 10.09.2018] Application of DNA tetrahedron in promoting proliferation and differentiation of neural stem cells

Technical field

The invention belongs to the technical field of cell proliferation and differentiation, and particularly relates to the application of a DNA tetrahedron in promoting the proliferation and differentiation of neural stem cells.

Background technique

Mouse neural stem cells (NE-4C), which are derived from the ATCC cell bank, are one of the better models for studying the nervous system in vitro. The NE-4C cell line can maintain the characteristics of neural stem cells under certain circumstances, but can differentiate into mature neurons or glial cells after specific induction treatment. At present, in the research on neural stem cells, the main direction is to study the effects of drugs or materials on the proliferation and differentiation of neural stem cells, such as the proliferation and differentiation of prostaglandin neural stem cells; and neural stem cells in some central degenerative diseases. Cellular therapeutic effects, such as the therapeutic role of neural stem cells in animal models of Alzheimer's disease.

DNA tetrahedral nanomaterials (TDNs) are a new type of DNA nanomaterials, which are currently widely studied and have great potential applications in biomedical fields. The current research found that the drugs or materials used in the process of proliferation and differentiation may have certain toxic effects on cells, that is, biocompatibility, biosafety, and poor biodegradability. TDNs are three-dimensional DNA nanomaterials formed by self-assembly of four ss DNA single strands under specific conditions. The base sequences of four ss DNAs (S1, S2, S3, S4) strictly follow the "base complementary pairing". The principle is then precisely and subtly designed. The synthesis method of TDNs is simple, the yield is high, and the tolerance to specific or non-specific nucleases is better than that of ordinary linear DNA, and it has good biocompatibility, biosafety and biodegradability. In addition, although there are many studies on TDNs, there are few studies on the effects of various physiological activities on cells, especially the research on promoting cell proliferation and differentiation.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the present invention provides a DNA tetrahedron for promoting proliferation and differentiation of neural neural stem cells by affecting Wnt/β-catenin and Notch signaling pathway-related genes and proteins. Expression changes, respectively, to promote the proliferation and differentiation of neural stem cells.

In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problem thereof is:

The application of DNA tetrahedron in promoting proliferation and differentiation of neural stem cells, D, wherein the four single-stranded sequences of DNA tetrahedra are as shown in SEQ ID NO: 1-4.

Further, DNA tetrahedron promotes proliferation of neural stem cells by activating Wnt/β-catenin signaling pathway; DNA tetrahedron promotes differentiation of neural stem cells by inhibiting Notch signaling pathway.

Further, the process of promoting neural stem cell proliferation includes promoting the expression of β-catenin, Lef-1, and Cyclin-D proteins.

Further, the process of promoting neural stem cell proliferation includes promoting the expression of the β-catenin, Lef-1, and Cyclin-D genes.

Further, the process of promoting differentiation of neural stem cells includes reducing the expression of Notch-1, Hes-1 and Hes-5 proteins.

Further, the process of promoting differentiation of neural stem cells comprises promoting expression of a β-III-Tubulin protein.

Further, the processes of promoting differentiation of neural stem cells include inhibition of expression of Notch-1, Hes-1 and Hes-5 genes, respectively, and promotion of expression of β-III-Tubulin gene.

Further, the concentration of the DNA tetrahedron in promoting proliferation and differentiation of the neural stem cells is 50 to 500 nM.

Further, the concentration of the DNA tetrahedron in promoting proliferation and differentiation of the neural stem cells is 100 to 300 nM.

Further, the concentration of the DNA tetrahedron in promoting proliferation and differentiation of neural stem cells was 250 nM.

The beneficial effects of the invention are:

1. Because TDNs have good biocompatibility, biosafety and biodegradability, they can effectively solve the problem of poor biological performance of traditional drugs or materials. DNA tetrahedron (TDNs) nanomaterials promote the proliferation and differentiation of neural stem cells, and at the same time solve the problems of slower proliferation of neural stem cells and slower differentiation and maturation, which lays a certain foundation for subsequent in vivo neural stem cell therapy experiments. Research basis.

2. DNA tetrahedrons (TDNs) can promote the proliferation and differentiation process by regulating Wnt/β-catenin and Notch signaling pathways, respectively, by promoting the expression of β-catenin, Lef-1 and Cyclin-D genes and proteins. Promote the proliferation of mouse neural stem cells; by reducing the expression of Notch-1, Hes-1 and Hes-5 proteins, and promoting the expression of β-III-Tubulin genes and proteins, the purpose of promoting differentiation of mouse neural stem cells can be achieved.

DRAWINGS

Figure 1 is a schematic representation of four single-stranded synthetic TDNs.

Figure 2 is a schematic diagram showing the results of TDNs polyacrylamide gel electrophoresis.

Figure 3 is a schematic illustration of the results of transmission electron microscopy identification.

Fig. 4 is a diagram showing the results of identifying the undifferentiated state of mouse neural stem cells by immunofluorescence technique.

Figure 5 is a graphical representation of the results of detection of TDNS uptake by mouse neural stem cells using fluorescent tracing techniques.

Figure 6 is a graphical representation of the results of the effect of TDNs concentration on the proliferation of mouse neural stem cells.

Figure 7 is a graph showing the results of cell cycle changes of mouse neural stem cells under the action of TDNs by flow cytometry.

Fig. 8 (a), (b) and (c) are schematic diagrams showing the detection of the expression levels of β-catenin, Lef-1 and Cyclin-D genes and proteins in mouse neural stem cells under the action of TDNs.

Fig. 9 (a), (b) and (c) are schematic diagrams showing the detection of β-III-Tubulin gene and protein expression in mouse neural stem cells under the action of TDNs.

Figures 10 and 11 are diagrams showing the results of detecting the expression level of β-III-Tubulin protein by immunofluorescence technique.

Fig. 12 (a), (b) and (c) are schematic diagrams showing the detection of the expression levels of Notch-1, Hes-1, Hes-5 genes and proteins in mouse neural stem cells under the action of TDNs.

Detailed ways

The embodiments of the present invention are described below in order to enable those skilled in the art to understand the present invention. It should be understood that the present invention is not limited to the scope of the specific embodiments. These variations are obvious in the spirit and scope of the invention as defined and claimed in the appended claims, and all inventions that are in the nature of the invention are in the protection.

Example 1 Synthesis and Identification of DNA Tetrahedrons (TDNs)

1. Synthesis of TDNs

Four equal concentrations of 4 ss DNA single strands (S1, S2, S3, S4) were added to a 200 μl EP tube containing 100 μl of TM buffer (10 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0), and the reaction solution was heated to 95. °C was maintained for 10 min, then rapidly cooled to 4 °C and kept for 20 min to obtain TDNs. The synthesis process is shown in Figure 1. The specific sequence of the four DNA single strands is as follows:

S1:

S2:

S3:

S4:

2. Identification of TDNs

(1) Identification by polyacrylamide gel electrophoresis

Polyacrylamide gel was prepared by using 40% acrylamide, 10×TAE, 10% APS solution, distilled water and TEMED;

Then, 1 μL of 6×loading buffer and 5 μL of the prepared TDNs were uniformly mixed, and respectively added to the corresponding electrophoresis tank, ice bath, constant pressure 100V, electrophoresis for 60 min; then GelRed and distilled water with a concentration ratio of 1:50 were used. In the dark, the shaker is treated for 15 to 25 minutes, then exposed, and then tested. The results are shown in Fig. 2.

As shown in Fig. 2, the four single-stranded S1, S2, S3 and S4 sizes of TDNs are about 60 bp, 50 bp, 50 bp and 50 bp, respectively, and the size of TDNs is about 210 bp.

(2) Transmission electron microscopy identification

The TDNs were identified by transmission electron microscopy. The results are shown in Fig. 3. As shown in Fig. 3, the shape of TDNs (triangular) is approximately triangular in shape under transmission electron microscopy, and the particle size is about 10-15 nM. The circle is labeled as a polymer.

Example 2 Identification of mouse neural stem cells by immunofluorescence technique

Identification of mouse neural stem cells by immunofluorescence technique, including the following steps at one time:

The cell suspension was inoculated into a confocal dish and placed in an incubator for 24 hours. The medium containing DMEM + 10% serum + 1% double antibody was aspirated, and washed three times with PBS for 5 minutes each time;

After fixing with 4% paraformaldehyde for 25 minutes, the paraformaldehyde was aspirated and washed three times with PBS for 5 minutes each time;

0.5% Triton-100 treatment for 20-25 minutes, aspirate Triton-100, wash 3 times with PBS for 5 minutes each time;

The sheep serum was treated for 1 hour, the sheep serum was aspirated, and the PBS was washed 3 times for 5 minutes each time;

Primary antibody (anti-nestin antibody) was treated at 4 ° C overnight. On the next day, the temperature was rewarmed at 37 ° C for 0.5 hours, and the primary antibody was recovered and washed three times with PBS for 5 minutes each time. Fluorescent secondary antibody treatment, protected from light, 37 ° C, 1 hour, the secondary antibody was aspirated, washed three times with PBS for 5 minutes each time;

Phalloidin treatment, protected from light, 10-30 minutes, aspirate phalloidin, wash 3 times in PBS for 5 minutes each time;

DAPI treatment, protected from light, 10 minutes, aspirate DAPI, wash 3 times with PBS for 5 minutes each time. 10% glycerol seal, protected from light, stored at 4 ° C. Check on the machine.

The detection results are shown in Fig. 4. As shown in Fig. 4, the mouse neural stem cells showed that the nestin antibody was positive, indicating that the cells were still in an undifferentiated state, and could be used for subsequent proliferation and differentiation experiments.

Example 3 Detection of TDNs uptake by mouse neural stem cells using fluorescent tracer technique

(1) Inoculate a cell suspension (100 μl/well) in a confocal dish, pre-culture the plate for 24 hours in an incubator (37 ° C, 5% CO ₂ ), then the fraction is DMEM + 10% serum +1 The serum concentration in the medium of % double antibody was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), and the serum concentration in the medium was reduced from 6% to 0. Incubate in an incubator for 1 hour (37 ° C, 5% CO ₂ ).

(2) The cultured cell suspension was divided into a control group and an experimental group. The concentration of 250 nM was added to the control group, and the Cy-5-modified DNA single-stranded S1 was added. The experimental group was added at a concentration of 250 nM and modified with Cy-5. The TDNs were cultured in an incubator for 6 hours (37 ° C, 5% CO ₂ ).

(3) The culture medium of the experimental group and the control group were respectively aspirated, and washed three times with PBS for 5 minutes each time. After fixing with 4% paraformaldehyde for 25 minutes, the paraformaldehyde was aspirated, washed three times with PBS for 5 minutes each time, and then treated with phalloidin, protected from light for 10-30 minutes, and the phalloidin was taken up. Wash PBS 3 times for 5 minutes each time; use DAPI treatment, avoid light, 10 minutes, aspirate DAPI, wash PBS 3 times for 5 minutes each time; finally seal with 10% glycerol, protect from light, and store at 4 °C. On the machine test, the test results are shown in Figure 5.

As shown in Figure 5, single-stranded S1 is less taken up by neural stem cells; neural stem cells take up more TDNs, and most of the TDNs that enter the cells accumulate in the cytoplasm of the cells and enter the nucleus less.

Example 4 TDNs promote proliferation and detection of mouse neural stem cells

1. Proliferation

(1) Inoculate a cell suspension (100 μl/well) in a 96-well plate, place the plate in an incubator for 24 hours (37 ° C, 5% CO ₂ ), and then mix the components with DMEM + 10% serum. The serum concentration in the +1% double-antibody medium was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), and then the serum concentration in the medium was reduced from 6% to 0, cultured in an incubator for 1 hour (37 ° C, 5% CO ₂ ).

(2) The cultured cell suspension was divided into a control group and an experimental group, and TDNs were added to the experimental group, and an equal amount of PBS was added to the control group, followed by incubation in an incubator for 24 hours (37 ° C, 5% CO _{2 ).} ).

(3) Add CCK-8 solution (10μl/well) to the experimental group and the control group, then incubate in the incubator for 1~4h (37°C, 5% CO ₂ ), and then measure the absorbance of each well at 450nm. The result is shown in Figure 6.

As shown in Fig. 6, when the concentration of TDNs was 62.5 nM, 125 nM, and 250 nM, the proliferation process of mouse neural stem cells in the experimental group was promoted to a certain extent by TDNs, and 250 nM was the most. Good concentration indicates that TDNs have the effect of promoting the proliferation of mouse neural stem cells.

2, using flow cytometry to detect cell proliferation

(1) Inoculate the cell suspension in a 25 ml culture flask, place the culture flask in an incubator for 24 hours (37 ° C, 5% CO ₂ ), and then divide the component into DMEM + 10% serum + 1% double antibody. The serum concentration in the medium was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), then the serum concentration in the medium was reduced from 6% to 0, in the incubator The medium was cultured for 1 hour (37 ° C, 5% CO ₂ ).

(3) The control cells and the experimental group cells were separately digested with 0.25% trypsin, placed in a 15 ml centrifuge tube (2000 rpm, 5 minutes), the supernatant was discarded, washed with PBS, centrifuged (2000 rpm, 5 minutes), and then added with ice. 500 μl of ethanol was fixed, and the cells were fixed at 4 ° C overnight. The next day, PBS was added to centrifuge, the supernatant was discarded, washed with PBS, centrifuged, and the supernatant was discarded. Then, 100 μl of RNase was added, and a 37 ° C water bath was added for 30 minutes. 400 μl of PI was added and mixed. Protected from light at 4 ° C for 30 minutes. The cells were transferred to a flow tube, detected by the machine, and analyzed by data. The results are shown in Fig. 7.

As shown in Fig. 7, compared with the control group, the number of cells in the S phase (DNA synthesis phase) in the experimental group increased significantly, indicating that TDNs changed the cell cycle of neural stem cells and promoted its proliferation.

Example 5 Mechanism of TDNs Promoting Proliferation of Mouse Neural Stem Cells

1. The mechanism of TDNs promoting the proliferation of mouse neural stem cells by Western blotting

(1) Inoculate a cell suspension (100 μl/well) in a 6-well plate, place the plate in an incubator for 24 hours (37 ° C, 5% CO ₂ ), and then mix the components with DMEM + 10% serum. The serum concentration in the +1% double-antibody medium was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), and then the serum concentration in the medium was reduced from 6% to 0, cultured in an incubator for 1 hour (37 ° C, 5% CO ₂ ).

(2) culturing the cell suspension obtained in the step (1) with a medium containing DMEM+1% double antibody, dividing the cell suspension into a control group and an experimental group, and simultaneously changing the medium at the same time; TDNs at a concentration of 250 nM were added to the group, and an equal amount of PBS was added to the control group, and then, after 24 hours of culture, the control group and the experimental histone protein were separately extracted using the whole protein extraction kit.

(3) SDS-PAGE electrophoresis was performed on the control group and the experimental histone protein obtained in the step (2), and the specific process was as follows: filling→loading→electrophoresis→transfer membrane→blocking liquid shaking for 1 hour→primary antibody 4°C overnight →Recover primary antibody, TBST wash 3 times, each time 5-10 minutes → secondary antibody, 1 hour → discard secondary antibody, TBST wash 3 times, each time 5-10 minutes → exposure, detection and data processing, the results see Figure 8 (a) and Figure 8 (b).

As shown in Fig. 8(a) and Fig. 8(b), the three proteins in the Wnt/β-catenin signaling pathway associated with the neural stem stem cell proliferation process in the experimental group were β-catenin, respectively. , Lef-1 and Cyclin-D, the expression levels of three proteins and corresponding control genes were increased by β-catenin, Lef-1 and Cyclin-D, further indicating that TDNs promoted the proliferation of neural stem cells.

2. The mechanism of TDNs promoting the proliferation of mouse neural stem cells by real-time PCR (Q-PCR)

(2) The suspension cells were divided into control group and experimental group, and TDNs at a concentration of 250 nM were added to the experimental group. The same amount of PBS was added to the control group, and then cultured in an incubator for 24 hours (37 ° C, 5%). CO ₂ ), then the gene extraction kit was used to extract the control and experimental group genes, respectively, and then the stable cDNA was obtained by the high purity total RNA rapid extraction kit and the reverse transcription kit.

(3) Q-PCR: 20 μl of reaction system (2 μl cDNA, 10 μl SYBR, 0.8 μl primer Forward, 0.8 μl primer Reserve, 6.4 μl ddH ₂ O) was added to each well, and the data was processed and the results were observed. Figure 8 (c).

As shown in Figure 8(c), the three proteins in the Wnt/β-catenin signaling pathway associated with neural stem cell proliferation in the experimental group were β-catenin, Lef-1, and Cyclin-, respectively. D, the expression levels of the three proteins and the corresponding control genes β-catenin, Lef-1 and Cyclin-D were increased, further indicating that TDNs promoted the proliferation of neural stem cells.

Example 6 Detection of TDNs Promoting Differentiation and Differentiation Mechanism of Mouse Neural Stem Cells

1. Western blotting method to detect the phenomenon of TDNs promoting differentiation of mouse neural stem cells and its mechanism

(2) culturing the cell suspension obtained in the step (1) with a medium containing DMEM/F-12+1% double-antibody + 1% B27, and dividing the cell suspension into a control group and an experimental group, and The medium was changed at the same time every day; TDNs at a concentration of 250 nM were added to the experimental group, and an equal amount of PBS was added to the control group, and then the whole protein extraction kit was used to extract the control group after 1 day, 3 days, and 7 days, respectively. And experimental histones.

(3) SDS-PAGE electrophoresis was performed on the control group and the experimental histone protein obtained in the step (2), and the specific process was as follows: filling→loading→electrophoresis→transfer membrane→blocking liquid shaking for 1 hour→primary antibody 4°C overnight →Recover primary antibody, TBST wash 3 times, each time 5-10 minutes → secondary antibody, 1 hour → discard secondary antibody, TBST wash 3 times, each time 5-10 minutes → exposure, detection and data processing, the results see Figure 9 (a), Figure 9 (b), Figure 12 (a) and Figure 12 (b).

As shown in Fig. 9(a), Fig. 9(b), Fig. 12(a) and Fig. 12(b), the expression levels of the differentiation-related protein (β-III-Tubulin) in the experimental group were higher than those in the control group. Compared with the control group, the expression levels of Notch-1, Hes-1 and Hes-5 were decreased in the three proteins related to the Notch signaling pathway in the experimental group, indicating that TDNs can promote the differentiation of mouse neural stem cells. mature.

2. The phenomenon and mechanism of TDNs promoting differentiation of mouse neural stem cells by real-time PCR (Q-PCR)

(1) (1) Inoculate the cell suspension (100 μl/well) in a 6-well plate, place the plate in an incubator for 24 hours (37 ° C, 5% CO ₂ ), and then the component is DMEM+ The serum concentration in the medium of 10% serum + 1% double antibody was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), and then the serum concentration in the medium was 6 % was lowered to 0 and incubated for 1 hour in the incubator (37 ° C, 5% CO ₂ ).

(2) culturing the cell suspension obtained in the step (1) with a medium containing DMEM/F-12+1% double-antibody + 1% B27, and dividing the cell suspension into a control group and an experimental group, and The medium was changed at the same time every day; TDNs at a concentration of 250 nM were added to the experimental group, and an equal amount of PBS was added to the control group, and then the cells were extracted for 1 day, 3 days, and 7 days, respectively, using a gene extraction kit. And the experimental group genes, and then obtain stable cDNA by high-purity total RNA rapid extraction kit and reverse transcription kit.

(3) Q-PCR: 20 μl of reaction system (2 μl cDNA, 10 μl SYBR, 0.8 μl primer Forward, 0.8 μl primer Reserve, 6.4 μl ddH ₂ O) was added to each well, and the data was processed and the results were observed. Figure 9 (c) and Figure 12 (c).

As shown in Fig. 9(c) and Fig. 12(c), compared with the control group, the expression level of the differentiation-related gene (β-III-Tubulin) in the experimental group was higher, and compared with the control group, The three proteins on the Notch signaling pathway in the differentiation group were Notch-1, Hes-1 and Hes-5, and the expression levels of the corresponding genes Notch-1, Hes-1 and Hes-5 were decreased. TDNs can promote the differentiation and maturation of mouse neural stem cells.

3. Immunofluorescence technology

(1) Inoculate the cell suspension (100 μl/well) in a confocal dish, place the plate in an incubator for 24 hours (37 ° C, 5% CO ₂ ), and then divide the component into DMEM + 10% serum. The serum concentration in the +1% double-antibody medium was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO ₂ ), and then the serum concentration in the medium was reduced from 6% to 0, cultured in an incubator for 1 hour (37 ° C, 5% CO ₂ ).

(2) The cell suspension obtained in the step (1) is cultured in a medium containing DMEM/F-12+1% double antibody + 1% B27, and the cell suspension is divided into a control group and an experimental group, and each day. At the same time, the medium was changed; TDNs at a concentration of 250 nM were added to the experimental group, and an equal amount of PBS was added to the control group for 1 day and 7 days.

(3) The culture medium of the experimental group and the control group were respectively aspirated, and washed three times with PBS for 5 minutes each time. After fixing with 4% paraformaldehyde for 25 minutes, the paraformaldehyde was aspirated, washed three times with PBS for 5 minutes, treated with 0.5% Triton-100 for 20-25 minutes, and then washed with Triton-100 and washed with PBS three times. 5 minutes each time. The sheep serum was treated for 1 hour, and the sheep serum was aspirated, and washed three times with PBS for 5 minutes each time. Monoclonal anti-beta-III-Tubulin antibody treatment, 4 ° C, overnight. On the next day, the temperature was rewarmed at 37 ° C for 0.5 hours, and the primary antibody was recovered and washed three times with PBS for 5 minutes each time. Fluorescent secondary antibody treatment, protected from light, 37 ° C, 1 hour, the secondary antibody was aspirated, washed three times with PBS for 5 minutes each time. DAPI treatment, protected from light, 10 minutes, aspirate DAPI, wash 3 times with PBS for 5 minutes each time. 10% glycerol seal, protected from light, stored at 4 ° C. Check on the machine. The results are shown in Figures 10 and 11.

As shown in Fig. 10 and Fig. 11, after 1 day and 7 days, the fluorescence intensity (β-III-Tubulin) was higher in the experimental group than in the control group, and the morphology of the cells was better.

Claims

The application of the DNA tetrahedron in promoting the proliferation and differentiation of neural stem cells, wherein the four single-stranded sequences of the DNA tetrahedron are shown in SEQ ID NOs: 1-4.
The use of the DNA tetrahedron according to claim 1 for promoting proliferation and differentiation of neural stem cells, characterized in that said DNA tetrahedron promotes proliferation of neural stem cells by activating Wnt/β-catenin signaling pathway; The DNA tetrahedron promotes differentiation of neural stem cells by inhibiting the Notch signaling pathway.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the process of promoting proliferation of neural stem cells comprises promoting expression of β-catenin, Lef-1 and Cyclin-D proteins.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the process of promoting proliferation of neural stem cells comprises promoting expression of β-catenin, Lef-1 and Cyclin-D genes.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the process of promoting differentiation of neural stem cells comprises reducing the expression of Notch-1, Hes-1 and Hes-5 proteins.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the process of promoting differentiation of neural stem cells comprises promoting expression of β-III-Tubulin protein.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the process of promoting differentiation of neural stem cells comprises inhibiting expression of Notch-1, Hes-1 and Hes-5 genes, respectively. Promotes the expression of the β-III-Tubulin gene.
The use of the DNA tetrahedron according to claim 2 for promoting proliferation and differentiation of neural stem cells, characterized in that the concentration of the DNA tetrahedron in promoting proliferation and differentiation of neural stem cells is 50 to 500 nM.
The DNA tetrahedron according to claim 8, wherein the concentration of the DNA tetrahedron in promoting proliferation and differentiation of neural stem cells is 100 to 300 nM.
The use of the DNA tetrahedron according to claim 8 for promoting proliferation and differentiation of neural stem cells, characterized in that the concentration of the DNA tetrahedron in promoting proliferation and differentiation of neural stem cells is 250 nM.