CN109806275B

CN109806275B - Application of DNA tetrahedron in preparation of nerve repair promoting medicine

Info

Publication number: CN109806275B
Application number: CN201811396540.0A
Authority: CN
Inventors: 林云锋; 马文娟; 蔡潇潇; 邵晓茹; 谢雪萍
Original assignee: Chengdu Tengda Tree Nano Biotechnology Co ltd
Current assignee: Chengdu jingrunze Gene Technology Co.,Ltd.
Priority date: 2017-11-22
Filing date: 2018-11-21
Publication date: 2021-04-23
Anticipated expiration: 2038-11-21
Also published as: CN109806275A; WO2019101116A1

Abstract

The invention provides an application of a DNA tetrahedron in preparation of a nerve repair promoting drug, wherein the DNA tetrahedron is assembled into a tetrahedron structure by four single-stranded DNAs in a base complementary pairing mode; each edge of the tetrahedron is a double-stranded DNA structure. The DNA tetrahedron can obviously promote the proliferation, differentiation and migration of mouse neural stem cells, and has good biocompatibility and bioavailability.

Description

Application of DNA tetrahedron in preparation of nerve repair promoting medicine

Technical Field

The invention relates to the field of nerve repair, in particular to application of a DNA tetrahedron in preparation of a nerve repair promoting medicine.

Background

Currently, neurological diseases, such as neurodegenerative diseases, nerve injuries, etc., are a medical problem. Because of the difficulty in self-repair and renewal of nerve cells, Neural Stem Cell (NSCs) therapy is receiving increasing attention. Although there are technical challenges to the clinical application of neural stem cell therapy, it is currently most critical that the implanted cells themselves be unable to proliferate, differentiate and migrate.

Researchers have found that some drug molecules (e.g., prostaglandin E2, tenuigenin, etc.) can enhance the proliferation and differentiation ability of NSCs, but their biocompatibility and availability are not high, and thus, they have limitations in the treatment of neurological diseases.

DNA Tetrahedrons (TDNs) are a novel DNA nano material, and have very extensive research and huge potential application prospects in the field of biomedicine at present. The TDNs are DNA nano-materials with three-dimensional structures formed by self-assembling four single-stranded DNA single-strands under specific conditions, and the base sequences of the four single-stranded DNA strictly follow the base complementary pairing principle, so that the TDNs are accurately and skillfully designed. The TDNs have the advantages of simple synthesis method, high yield, better tolerance to specific or non-specific nuclease than common linear DNA, and good biocompatibility, biosafety and biodegradability.

TDNs have appeared in some studies in the role of drug carriers at present, but the application of TDNs in nerve repair is still to be developed.

Disclosure of Invention

The invention aims to provide a novel medicine capable of promoting nerve repair.

In order to solve this technical problem, the present invention provides the use of a DNA tetrahedron for the preparation of a medicament for promoting nerve repair.

In the application, the DNA tetrahedron is assembled by four single-stranded DNAs in a base complementary pairing mode to form a tetrahedron structure; each edge of the tetrahedron is a double-stranded DNA structure.

In the above application, the DNA tetrahedron is prepared by: heating the aqueous solution containing the four single-stranded DNAs with equal concentrations to completely break hydrogen bonds between bases, maintaining for 5-20 min, then rapidly cooling to 0-10 ℃, and keeping for at least 15 min; the aqueous solution also contains 10mM Tris-HCl and 50mM MgCl 2; the pH of the aqueous solution before heating was 8.

In the application, the maintaining time after heating is 10min, the specific temperature for cooling is 4 ℃, and the maintaining time after cooling is 20 min.

In the application, the sequences of the four single-stranded DNAs are respectively shown in SEQ ID NO. 1-4.

In the aforementioned use, the drug is a drug that promotes proliferation, differentiation and/or migration of neural stem cells.

Further, the drug is a drug that activates the Wnt/β -Catenin pathway.

Further, the drug is a drug that inhibits the Notch signaling pathway.

Further, the drug is a drug that activates the RHOA/ROCK2 signaling pathway.

The invention also provides a medicament for promoting nerve repair, which is characterized by being prepared by using the DNA tetrahedron of claims 1-9 as an active substance and adding pharmaceutically acceptable auxiliary materials or auxiliary components.

The invention has the following beneficial effects:

the TDNs can be taken by mouse neural stem cells in a large amount, and the taking rate of the TDNs is greatly higher than that of single-stranded DNA.

The TDNs can obviously increase the proliferation, differentiation and migration of mouse neural stem cells and have good nerve repair promoting capacity.

The TDNs are composed of nucleic acids, so the metabolism of the TDNs does not generate toxicity to cells, and the TDNs have good biocompatibility.

The TDNs can be prepared into the medicine for promoting nerve repair.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention. The present invention is described in further detail with reference to the following embodiments, but the present invention is not limited thereto, and various other modifications, substitutions and alterations can be made without departing from the basic technical idea of the present invention based on the above-mentioned contents of the present invention and common technical knowledge and conventional means in the art.

Drawings

FIG. 1 is a schematic diagram of four single-stranded synthetic TDNs.

FIG. 2 is a diagram showing the result of TDNs polyacrylamide gel electrophoresis.

FIG. 3 is a schematic diagram of the transmission electron microscope identification results.

FIG. 4 is a graph showing a distribution of single-stranded DNA particles, and b is a distribution of TDNs particles.

FIG. 5 is a diagram illustrating the results of the identification of the undifferentiated state of the mouse neural stem cells by immunofluorescence technique.

FIG. 6 is a graph showing the results of flow cytometry treatment of TDNs uptake by mouse neural stem cells.

FIG. 7 is a fluorescent tracing diagram of mouse neural stem cells.

FIG. 8 is a diagram showing the test results of the effect of TDNs concentration on mouse neural stem cell proliferation.

FIG. 9 is a diagram showing the results of detecting cell cycle changes of mouse neural stem cells under the action of TDNs by flow cytometry.

FIG. 10a is a blot of β -catenin, Lef-1 and Cyclin-D in mouse neural stem cells under the action of TDNs.

FIG. 10b is a bar graph showing the relative intensity of the beta-catenin, Lef-1 and Cyclin-D western blot signals in mouse neural stem cells under the action of TDNs.

FIG. 10c is a bar graph of relative gene expression levels of beta-catenin, Lef-1 and Cyclin-D in mouse neural stem cells under the action of TDNs.

FIG. 11a is a Western blot of β -III-Tubulin in mouse neural stem cells under the action of TDNs.

FIG. 11b is a bar graph showing the relative intensity of the beta-III-Tubulin Western blot signal in mouse neural stem cells under the action of TDNs.

FIG. 11c is a histogram of the relative expression level of β -III-Tubulin gene in mouse neural stem cells under the effect of TDNs.

FIG. 12a is a blot of proteins associated with Notch signaling pathway in mouse neural stem cells under the action of TDNs.

FIG. 12b is a bar graph showing the relative intensity of the Notch signaling pathway-associated Western blot signals in mouse neural stem cells under the action of TDNs.

FIG. 12c is a graph showing the relative expression level of Notch signaling pathway-related genes in mouse neural stem cells under the effect of TDNs.

FIG. 13 is an immunofluorescence map of β -III-Tubulin protein 1 day after TDNs treatment.

FIG. 14 is a photograph of immunofluorescence of β -III-Tubulin protein 7 days after TDNs treatment.

FIG. 15 is a graph showing the results of a mouse neural stem cell scratch test.

FIG. 16 is a diagram showing the results of a Transwell experiment on mouse neural stem cells.

FIG. 17a is an electrophoresis chart of the RhoA amplified band in mouse neural stem cells under the action of TDNs.

FIG. 17b is a graph showing the change of RhoA gene expression fold in mouse neural stem cells under the action of TDNs.

FIG. 17c is a graph showing the relative quantification of RhoA gene expression in mouse neural stem cells under the action of TDNs.

FIG. 17d is a photograph of the imprinting of RhoA protein in mouse neural stem cells with TDNs.

FIG. 17e is a bar graph showing the relative intensity of the RhoA western blot signal in mouse neural stem cells under the action of TDNs.

FIG. 17f is an immunofluorescence mapping of RhoA protein in mouse neural stem cells with TDNs.

FIG. 17g is a graph showing the immunofluorescence signal intensity of RhoA protein in mouse neural stem cells under the action of TDNs.

FIG. 18a is an electrophoresis diagram of an amplification band of Rock2 in mouse neural stem cells under the action of TDNs.

FIG. 18b is a graph showing the change of the expression fold of Rock2 gene in mouse neural stem cells under the action of TDNs.

FIG. 18c is a graph showing the relative quantification of Rock2 gene expression in mouse neural stem cells under the action of TDNs.

FIG. 18d is a photograph of the imprinting of Rock2 protein in mouse neural stem cells by TDNs.

FIG. 18e is a bar graph showing the relative intensity of the Rock2 Western blot signal in mouse neural stem cells under the action of TDNs.

FIG. 18f is an immunofluorescence chart of Rock2 protein in mouse neural stem cells under the action of TDNs.

FIG. 18g is a graph showing the immunofluorescence signal intensity of Rock2 protein in mouse neural stem cells under the action of TDNs.

FIG. 19a is an electrophoresis diagram of vinculin amplification bands in mouse neural stem cells under the action of TDNs.

FIG. 19b is a graph showing the change of the fold of vinculin gene expression in mouse neural stem cells under the action of TDNs.

FIG. 19c is a graph showing the relative quantification of vinculin gene expression in mouse neural stem cells under the action of TDNs.

FIG. 19d is a photograph of vinculin protein imprinting in mouse neural stem cells under the effect of TDNs.

FIG. 19e is a bar graph showing the relative intensity of vinculin western blot signals in mouse neural stem cells under the action of TDNs.

FIG. 19f is an immunofluorescence chart of vinculin protein in mouse neural stem cells under the action of TDNs.

FIG. 19g is a graph showing the immunofluorescence signal intensity of vinculin protein in mouse neural stem cells under the action of TDNs.

Detailed Description

EXAMPLES Synthesis of TDNs

1. Synthesis of

Four DNA single strands (S1, S2, S3, S4) were dissolved at the same final concentration in a TM buffer solution containing Tris-HCl and MgCl2 at concentrations of 10mM and 50mM, respectively, and the pH of the solution was adjusted to 8.0. And then, placing the mixture into a PCR instrument after vortex, uniform mixing and centrifugation, rapidly increasing the temperature to 95 ℃, stabilizing for 10-15 min, and then cooling to 4 ℃, and stabilizing for 20 min. I.e. a tetrahedral structure as shown in figure 1 is synthesized.

The sequences of the DNA single strands are shown in Table 1.

TABLE 1 sequence of single strands of TDNs

2. Identification

2.1 gel electrophoresis

a. Preparing polyacrylamide gel: mixing 40 wt% acrylamide solution, 10 XTAE, 10 wt% ammonium sulfate solution, distilled water and tetramethyl ethylene diamine according to the volume ratio of 1-2: 1 to prepare polyacrylamide gel.

b. Sample adding and electrophoresis: 1ul of 6 × loading buffer was mixed with 5ul of sample and marker, and then added to the corresponding electrophoresis tank. Electrophoresis was carried out for 1 hour under an ice bath at a constant pressure of 100V.

c. GelRed staining and exposure: and placing the polyacrylamide gel into a mixed liquid obtained by mixing GelRed and distilled water according to the ratio of 1: 50, keeping out of the sun, and shaking for 15-25 minutes. And (6) exposing.

As a result: as shown in FIG. 2, lanes 1-8 are Marker, 1-S1, 2-S2, 3-S3, 4-S4, 5-Sl + S2, 6-S1+ S2+ S3, 7-S1+ S2+ S3+ S4(7-TDNs), respectively. As can be seen from the figure, the sizes of the ss DNA single strands S1, S2, S3 and S4 are about 60bp, 50bp and 50bp respectively, and the sizes of the TDNs prepared by the invention are about 210 bp.

2.2 Transmission Electron microscopy

And (3) taking a proper amount of sample liquid on a metal sheet, irradiating for 5-10 minutes under infrared to dry the sample liquid, and detecting on a machine.

As a result: as shown in FIG. 3, the TDNs are in an approximately triangular shape under a transmission electron microscope, and the particle size is within a range of 10-15 nM. Circles are labeled polymers.

2.3 dynamic light Scattering

And (3) taking a proper amount of ssDNA and TDNs solution, and placing the ssDNA and TDNs solution in a dynamic light scattering detector for detection.

As a result: as shown in FIG. 4-a, the ssDNA particle size is about 48.255 nm. As shown in FIG. 4-b, the particle size of TDNs is about 16.801 nm.

To demonstrate that TDNs do contribute to neural repair, the following examples are further described, in which the mouse neural stem cells are NE-4C cells.

Experimental example 1 identification of neural Stem cells

The method adopts an immunofluorescence technique to identify the mouse neural stem cells, and comprises the following steps:

A. the cell suspension was inoculated into a confocal dish and placed in an incubator for 24 hours. Absorbing a culture medium with the components of DMEM + 10% serum + 1% double antibody, washing with PBS for 3 times, and washing for 5 minutes each time;

B. after fixing with 4% paraformaldehyde for 25 minutes, removing paraformaldehyde by suction, washing with PBS for 3 times, 5 minutes each time;

C. treating with 0.5% Triton-100 for 20-25 min, removing Triton-100, washing with PBS for 3 times (5 min each);

D. treating sheep serum for 1 hr, sucking out sheep serum, washing with PBS for 3 times, each for 5 min;

E. primary anti (anti-nestin antibody) treatment, 4 ℃, overnight. The next day, rewarming at 37 ℃ for 0.5 hour, recovering the primary antibody, washing 3 times with PBS, 5 minutes each time. Treating the secondary antibody carrying fluorescence at 37 ℃ for 1 hour in a dark place, absorbing the secondary antibody, and washing with PBS for 3 times for 5 minutes each time;

E. treating phalloidin in dark for 10-30 min, removing phalloidin, washing with PBS for 3 times (5 min each);

F. DAPI treatment, protected from light for 10min, blotted off DAPI, washed 3 times with PBS for 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.

The detection result is shown in fig. 5, and the mouse neural stem cells show nestin antibody positive, which indicates that the cells are still in an undifferentiated state, and can be used for carrying out subsequent experiments.

The "cells" referred to in the following experimental examples were all the mouse neural stem cells in the same undifferentiated state as in experimental example 1.

Experimental example 2 neural Stem cell uptake assay

Can be taken up by cells in large quantities, which is the prerequisite for the therapeutic action of most drugs. This experimental example examined the uptake ability of neural stem cells for TDNs.

(1) Flow cytometry

a. Inoculating cell suspension into a 6-well plate, and pre-culturing in an incubator for 24 hours (37 ℃, 5% (v/v) CO 2); then the serum concentration in the culture medium is reduced from 10% to 6%, and the culture is continued in an incubator for 6 hours (37 ℃, 5% (v/v) CO 2); the serum concentration in the medium was then reduced from 6% to 0, and the incubation was continued for 1 hour (37 ℃ C., 5% (v/v) CO 2).

b. The negative control group was not treated, the positive control group was added with Cy 5-modified single-stranded DNA S1 at a concentration of 250nM, and the experimental group was added with Cy 5-modified TDNs at a concentration of 250nM, and incubated in an incubator for 12 hours (37 ℃, 5% (v/v) CO 2).

c. The cells were collected, centrifuged at 1000r/min for 5min, resuspended in PBS, repeated three times, and tested on the machine.

As a result: as shown in FIGS. 6-a and 6-b, uptake of TNDs by neural stem cells was significantly greater than that of ssDNA. (please explain FIG. 6-a)

The above experiments demonstrate that TNDs can be well taken up by mouse neural stem cells.

(2) Fluorescent tracing technique

a. Inoculating mouse neural stem cell suspension into a confocal dish, and pre-culturing for 24 hours (37 ℃, 5% (v/v) CO2) in an incubator; then the serum concentration in the culture medium is reduced from 10% to 6%, and the culture is continued in an incubator for 6 hours (37 ℃, 5% (v/v) CO 2); the serum concentration in the medium was then reduced from 6% to 0, and the incubation was continued for 1 hour (37 ℃ C., 5% (v/v) CO 2).

b. The control group was added with Cy 5-modified S1 at a concentration of 250nM, and the experimental group was added with Cy 5-modified TDNs at a concentration of 250nM and incubated in an incubator for 12 hours (37 ℃, 5% (v/v) CO 2).

c. The medium was aspirated off, washed three times with PBS, 5 minutes each; fixing with 4 wt% paraformaldehyde for 25 min, removing paraformaldehyde by suction, and washing with PBS for 5min three times; then processing with phalloidin, keeping out of the sun for 10-30 minutes, sucking off the phalloidin, and washing with PBS for three times, 5 minutes each time; then treating with DAPI, keeping out of the sun for 10 minutes, removing DAPI by suction, and washing with PBS for three times, 5 minutes each time; sealing with 10 wt% glycerol, protecting from light, storing at 4 deg.C, and detecting on machine.

As a result: as in fig. 7-a and 7-b, ssDNA was less taken up by neural stem cells; neural stem cells take up more TDNs, and TDNs entering cells accumulate mostly in the cytoplasm of cells and enter the nucleus less.

Indicating that the TDNs can be well taken up by the neural stem cells.

Experimental example 3 proliferation and detection of mouse neural stem cells promoted by TDNs

Proliferation of CCK-8 assay

(1) The cell suspension (100. mu.l/well) was inoculated into a 96-well plate, and the plate was pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium consisting of DMEM + 10% serum + 1% double antibody was reduced from 10% to 6%, and the medium was incubated in an incubator for 6 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium was reduced from 6% to 0, and the medium was incubated 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 PBS was added in an equal amount to the control group, followed by culturing in an incubator for 24 hours (37 ℃, 5% CO)₂)。

(3) Adding CCK-8 solution (10 mu l/well) into the experimental group and the control group respectively, and then incubating in an incubator for 1-4 h (37 ℃, 5% CO)₂) Then, the absorbance at 450nm was measured in each well, and the results are shown in FIG. 8.

As shown in FIG. 8, when the concentrations of TDNs are 62.5nM, 125nM and 250nM, the proliferation process of the mouse neural stem cells in the experimental group is promoted by TDNs to a certain extent compared with the control group, and 250nM is the optimal concentration, which indicates that TDNs have the effect of promoting the proliferation of the mouse neural stem cells.

2. Detection of cell proliferation by flow cytometry

(1) The cell suspension was inoculated into a 25ml flask, and the flask was pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium consisting of DMEM + 10% serum + 1% double antibody was reduced from 10% to 6%, and the medium was incubated in an incubator for 6 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium was reduced from 6% to 0, and the medium was incubated in an incubator for 1 hour (37 ℃ C., 5% CO)₂)。

(3) The cells of the control group and the cells of the experimental group were digested and collected with 0.25% trypsin, and then placed in a 15ml centrifuge tube (2000rpm, 5 minutes), the supernatant was discarded, washed with PBS, centrifuged (2000rpm, 5 minutes), 500. mu.l of iced ethanol was added, 4 ℃ overnight, PBS was added the next day, centrifuged, the supernatant was discarded, PBS was added, washed, centrifuged, the supernatant was discarded, 100. mu.l of RNase was added, 37 ℃ water bath was used, 30 minutes was added, 400. mu.l of PI was added for staining and mixing, and the mixture was kept at 4 ℃ in the dark for 30 minutes. The cells were transferred to a flow tube, tested on a machine, and subjected to data analysis, the results of which are shown in FIG. 9.

As shown in fig. 9, the number of cells in S phase (DNA synthesis phase) was significantly increased in the experimental group compared to the control group, indicating that TDNs changed the cell cycle of neural stem cells and had the effect of promoting their proliferation.

In conclusion, TDNs can promote the proliferation of neural stem cells.

Experimental example 4 Wnt/beta-catenin signal pathway-related gene expression detection

The Wnt/beta-catenin signal pathway is an important pathway for cell cycle regulation, and the activation of the Wnt/beta-catenin signal pathway can obviously promote the proliferation of stem cells. The experimental example detects the expression condition of Wnt/beta-catenin signal channel related genes of NSC under TDNs treatment.

1. Sample processing

(1) The 6-well plate was inoculated with a cell suspension (100. mu.l/well), and the plate was pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium consisting of DMEM + 10% serum + 1% double antibody was reduced from 10% to 6%, and the medium was incubated in an incubator for 6 hours (37 ℃ C., 5% CO)₂) Then, the serum concentration in the medium was reduced from 6% to 0, and the medium was incubated in an incubator for 1 hour (37 ℃ C., 5% CO)₂)。

(2) Culturing the cell suspension obtained in the step (1) by adopting a culture medium with the components of DMEM + 1% double antibody, dividing the cell suspension into a control group and an experimental group, and simultaneously replacing the culture medium every day; TDNs were added to the experimental group at a concentration of 250nM, while an equal amount of PBS was added to the control group.

2. Protein detection

After the experimental group is treated for 24 hours, extracting the holoproteins of the experimental group and the control group, and detecting three proteins on a Wnt/beta-catenin signal path related to the proliferation process of the neural stem cells by Western blot: beta-catenin, Lef-1 and Cyclin-D.

The brief detection procedure is as follows: pouring the gel → loading → electrophoresis → transferring the membrane → sealing the solution by shaking for 1 hour → first antibody 4 ℃ overnight → recovering the first antibody → TBST washing for 3 times (5-10 minutes each time) → incubation for second antibody for 1 hour → discarding the second antibody, TBST washing for 3 times (5-10 minutes each time) → exposure.

As shown in FIG. 10a and FIG. 10b, after the mouse neural stem cells are treated by TDNs for 24 hours, the contents of beta-catenin, Lef-1 and Cyclin-D proteins are obviously higher than those of a control group.

3. Gene expression assay

After the experimental group is treated for 24 hours, extracting RNA of the experimental group and the control group, obtaining cDNA through a reverse transcription kit, and then carrying out fluorescent quantitative PCR detection on three genes on a Wnt/beta-catenin signal path related to the proliferation process of the neural stem cells by using a dye method: beta-catenin, Lef-1 and Cyclin-D.

As shown in FIG. 10c, after TDNs treatment for 24h, the expression levels of beta-catenin, Lef-1 and Cyclin-D genes are significantly higher than those of the control group.

The experimental example shows that the TDNs can promote the proliferation of the neural stem cells by up-regulating the expression of three genes on a Wnt/beta-catenin signal path.

Experimental example 5 detection of expression of beta-III-Tubulin and Notch Signal pathway-related Gene

beta-III-Tubulin protein is a neuron marker, and when neural stem cells are differentiated into neurons, the expression of beta-III-Tubulin is up-regulated. The Notch signaling pathway plays an important role in the development of the nervous system, which is manifested in an inhibitory state during differentiation of adult neural stem cells. The experimental example shows that TDNs have the effect of promoting differentiation on NSC by detecting the expression of beta-III-Tubulin and Notch signal pathway related genes.

1. Sample processing

2. Protein detection

After the experimental group is treated for 1 day, 3 days and 7 days respectively, extracting the whole proteins of the experimental group and the control group, and detecting the proteins related to the neural stem cell differentiation through Western blot: beta-III-Tubulin, Notch-1, Hes-1 and Hes-5.

As shown in FIGS. 11a, 11b, 12a and 12b, the expression levels of the marker protein (. beta. -III-Tubulin) for neural stem cell differentiation in the experimental group were all higher than those in the control group, and the expression levels of three proteins on the differentiation-related Notch signaling pathway, Notch-1, Hes-1 and Hes-5, in the experimental group were decreased compared with those in the control group, indicating that TDNs can promote the differentiation and maturation of mouse neural stem cells.

3. Gene expression assay

After the experimental group is treated for 1 day, 3 days and 7 days respectively, extracting RNA of the experimental group and the control group, obtaining cDNA through a reverse transcription kit, and then carrying out fluorescence quantitative PCR detection on the protein related to neural stem cell differentiation by using a dye method: beta-III-Tubulin, Notch-1, Hes-1 and Hes-5.

As shown in FIGS. 11c and 12c, the expression levels of the differentiation-related target gene (. beta. -III-Tubulin) were higher in the experimental group than in the control group, and the expression levels of the corresponding genes Notch-1, Nos-1 and Nos-5 were decreased in the experimental group than in the control group, indicating that TDNs can promote the differentiation and maturation of mouse neural stem cells, and that the differentiation thereof is related to the inhibition of the Notch pathway, wherein the three proteins on the differentiation-related Notch signaling pathway are Notch-1, Nos-1 and Nos-5.

4. Immunofluorescence technique

(1) Samples were processed according to the method of section 1 of this example, replacing only the 6-well plate with a confocal cuvette.

(2) After 1 and 7 days of TDNs treatment, the culture medium of the experimental and control groups was aspirated, washed 3 times with PBS, each for 5 minutes. After further fixation with 4% paraformaldehyde for 25 minutes, paraformaldehyde was aspirated and washed with PBS 3 times for 5 minutes each, 0.5% Triton-100 was treated for 20-25 minutes, Triton-100 was aspirated and washed with PBS 3 times for 5 minutes each. Sheep serum was treated for 1 hour, aspirated and washed 3 times with PBS for 5 minutes each. anti-beta-III-Tubulin antibody treatment was performed overnight at 4 ℃. The next day, rewarming at 37 ℃ for 0.5 hour, recovering the primary antibody, washing 3 times with PBS, 5 minutes each time. The secondary antibody carrying fluorescence was treated in the dark at 37 ℃ for 1 hour, the secondary antibody was aspirated and washed 3 times with PBS for 5 minutes each. DAPI treatment, protected from light for 10min, blotted off DAPI, washed 3 times with PBS for 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.

As a result: as shown in FIGS. 13 and 14, the fluorescence intensity (. beta. -III-Tubulin) was higher in the experimental group and the morphology of the cells was closer to neurons than in the control group after 1 day and 7 days.

In conclusion, TDNs promote differentiation of neural stem cells by inhibiting Notch signaling pathways.

Example 6 TDNs promote migration of mouse neural Stem cells

1. Scratch test

a. Inoculating mouse neural stem cell suspension into a 6-well plate, and pre-culturing the culture plate in an incubator for 24 hours (37 ℃, 5% (v/v) CO 2); then reducing the serum concentration in the culture medium from 10% to 6%, and continuing culturing in an incubator for 6 hours (37 ℃, 5% (v/v) CO 2); then the serum concentration in the culture medium is reduced from 6% to 0, and the culture is continued for 1 hour in an incubator (37 ℃, 5% (v/v) CO 2); the monolayer cells were then scored along a straight line in a culture plate using a sterile gun tip and washed three times with PBS.

b. No TDNs were added to the control group, corresponding concentrations of TDNs were added to the experimental group, and the resulting mixture was incubated in an incubator (37 ℃ C., 5% (v/v) CO 2). Pictures were taken under light glasses after 0, 12, 24 hours, respectively, and the scratch change was recorded.

c. As a result: as shown in fig. 15, TDNs significantly promoted the lateral migration of mouse neural stem cells compared to the control group.

Transwell experiment

a. Placing a Transwell (with the aperture of 0.8 μm) chamber in a 6-well plate, inoculating the mouse neural stem cell suspension in the chamber, and pre-culturing the culture plate in an incubator for 24 hours (37 ℃, 5% (v/v) CO 2); then the serum concentration in the culture medium is reduced from 10% to 6%, and the culture is continued in an incubator for 6 hours (37 ℃, 5% (v/v) CO 2); the serum concentration in the medium was then reduced from 6% to 0, and the incubation was continued for 1 hour (37 ℃ C., 5% (v/v) CO 2).

b. No TDNs were added to the control group, TDNs were added to the experimental group at a concentration of 250nM, and the resulting mixture was incubated in an incubator for 12 hours (37 ℃ C., 5% (v/v) CO 2).

c. The medium was aspirated off, washed three times with PBS, 5 minutes each; fixing with 4 wt% paraformaldehyde for 25 minutes, removing paraformaldehyde by suction, and washing with PBS for 5 minutes for three times; treating with DAPI, keeping out of the sun for 10min, removing DAPI by suction, and washing with PBS for 5min three times; storing in dark at 4 deg.C, and detecting on machine.

d. As a result: as shown in fig. 16, the number of cells passing through the Transwell chamber was significantly increased in the experimental group compared to the control group, i.e., TDNs promoted the vertical migration of neural stem cells.

Scratch experiments and Transwell experiments prove that the TDNs have good promotion effects on transverse and longitudinal migration of mouse neural stem cells.

Example 7 detection of RHOA/ROCK2 Signal pathway related proteins

1. Sample processing

(1) Inoculating mouse neural stem cell suspension into a 6-well plate, and pre-culturing the culture plate in an incubator for 24 hours (37 ℃, 5% (v/v) CO 2); then the serum concentration in the culture medium is reduced from 10% to 6%, and the culture is continued in an incubator for 6 hours (37 ℃, 5% (v/v) CO 2); the serum concentration in the medium was then reduced from 6% to 0, and the incubation was continued for 1 hour (37 ℃ C., 5% (v/v) CO 2).

(2) No TDNs were added to the control group, and TDNs at a concentration of 250nM were added to the experimental group and incubated in an incubator.

2. Fluorescent quantitative PCR detection

After TDNs are treated for 24 hours, total RNA of an experimental group and a control group is extracted, reverse transcription is carried out, then fluorescent quantitative PCR reaction is carried out, and expression of RhoA, Rock2 and Vincultin is detected.

As a result: as shown in fig. 17a to 17c, fig. 18a to 18c, and fig. 19a to 19c, in the semiquantitative PCR and the quantitative PCR, the expression level of the corresponding gene was increased by RHOA, ROCK2, and Vinculin, which are three proteins on the migration-related RHOA/ROCK2 signal pathway in the experimental group, compared to the control group. The TDNs are shown to promote the migration of neural stem cells by activating the RHOA/ROCK2 signal path.

3. Western blot detection

After TDNs are treated for 24 hours, total proteins of the experimental group and the control group are extracted, and the Western blot is used for detecting RhoA, Rock2 and Vincultin proteins.

As a result: as shown in fig. 17d to 17e, fig. 18d to 18e, and fig. 19d to 19e, the expression levels of three proteins, RHOA, ROCK2, and Vinculin, on the migration-related RHOA/ROCK2 signal pathway in the experimental group were increased as compared to the control group. The TDNs are shown to promote the migration of neural stem cells by activating the RHOA/ROCK2 signal path.

4. Immunofluorescence detection

The 6-well plate in section 1 of this example was changed to a confocal dish for sample treatment. After TDNs are treated for 24 hours, the culture medium is aspirated, and the culture medium is washed with PBS for three times, 5 minutes each time; fixing with 4 wt% paraformaldehyde for 25 minutes, removing paraformaldehyde by suction, and washing with PBS for 5 minutes three times; treating with 0.5% Triton-100 for 20-25 min, sucking out Triton-100, and washing with PBS for three times, 5min each time; the goat serum was then treated for 1 hour, aspirated and washed three times with PBS for 5 minutes each. anti-RhoA, Rock2, Vincultin antibody treatment, 4 degrees C, overnight. The next day, rewarming at 37 ℃ for 0.5 hour, recovering the primary antibody, washing 3 times with PBS, 5 minutes each time. The secondary antibody carrying fluorescence was treated in the dark at 37 ℃ for 1 hour, the secondary antibody was aspirated and washed 3 times with PBS for 5 minutes each. DAPI treatment, protected from light for 10min, blotted off DAPI, washed 3 times with PBS for 5min each. Sealing with 10% glycerol, protecting from light, storing at 4 deg.C, and detecting on machine.

As a result: as shown in fig. 17f to 17g, fig. 18f to 18g, and fig. 19f to 19g, the fluorescence intensity of the proteins (RhoA, Rock2, Vinculin) was higher in the experimental group than in the control group, and further, it was demonstrated that TDNs promote migration of neural stem cells by activating the RhoA/Rock2 signaling pathway.

The above experiments further demonstrated that TDNs can promote neural stem cell migration.

In conclusion, the DNA tetrahedron of the invention is easy to be absorbed by nerve cells, can obviously increase the proliferation, differentiation and migration of mouse nerve stem cells, has good nerve repair promotion capability and has good biocompatibility. The DNA tetrahedron can be used for preparing medicaments for promoting nerve repair.

SEQUENCE LISTING

<110> Sichuan university

Application of <120> DNA tetrahedron in preparation of nerve repair promoting medicine

<130> GY007-18P1697

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> S1

<400> 1

atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60

gaa 63

<210> 2

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> S2

<400> 2

acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60

tcg 63

<210> 3

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> S3

<400> 3

actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60

tcc 63

<210> 4

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> S4

<400> 4

acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60

ccg 63

Claims

Use of a DNA tetrahedron in the preparation of a medicament, wherein the medicament is a neurorestorative medicament;

the DNA tetrahedron is assembled into a tetrahedron structure by four single-stranded DNAs in a base complementary pairing mode; each edge of the tetrahedron is a double-stranded DNA structure;

the sequences of the four single-stranded DNAs are respectively shown in SEQ ID NO. 1-4.
2. The use according to claim 1, wherein the DNA tetrahedron is prepared by a method comprising: heating the aqueous solution containing the four single-stranded DNAs with equal concentrations to completely break hydrogen bonds between bases, maintaining for 5-20 min, then rapidly cooling to 0-10 ℃, and keeping for at least 15 min; the aqueous solution also contains 10mM Tris-HCl and 50mM MgCl 2; the pH of the aqueous solution before heating was 8.
3. Use according to claim 2, wherein the holding time after heating is 10min, the specific temperature for cooling is 4 ℃ and the holding time after cooling is 20 min.
4. The use according to claim 1, wherein the medicament is a medicament for promoting proliferation, differentiation and/or migration of neural stem cells.
5. The use according to claim 4, wherein the medicament is a medicament that activates the Wnt/β -Catenin pathway.
6. The use of claim 4, wherein the medicament is a medicament that inhibits the Notch signaling pathway.
7. The use of claim 4, wherein the medicament is a medicament which activates the RHOA/ROCK2 signalling pathway.