CN109806275A - DNA tetrahedron is promoting the purposes in neural restoration medicine preparation - Google Patents
DNA tetrahedron is promoting the purposes in neural restoration medicine preparation Download PDFInfo
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Abstract
The present invention provides purposes of the DNA tetrahedron in rush neural restoration medicine preparation, the DNA tetrahedron is assembled into tetrahedral structure by base pair complementarity mode by four single stranded DNAs；Tetrahedral every rib is all double-stranded DNA structure.The four sides DNA physical efficiency in the present invention remarkably promotes proliferation, differentiation and the migration of mouse neural stem cells, and has good biocompatibility and bioavilability.
Description
Technical field
The present invention relates to neural restoration fields, and in particular to DNA tetrahedron is promoting the purposes in neural restoration medicine preparation.
Background technique
Currently, the nervous system disease, neurodegenerative disease, neurotrosis etc., are medical field problems.Because neural
The self-regeneration and update of cell are difficult, and neural stem cell (neural stem cells, NSCs) is treated increasingly by pass
Note.Although there are also some technical problems needs to capture for the clinical application of neural stem cell treatment, most critical also resides in plant at present
Entering cell itself can be proliferated, breaks up and migrate.
There is researcher's discovery, some drug molecules (such as prostaglandin E2, tenuigenin etc.) are able to ascend NSC
Proliferation and differentiation capability, however its biocompatibility and availability be not high, and there are offices in the treatment of the nervous system disease
It is sex-limited.
DNA tetrahedron (TDNs) is a kind of novel DNA nano material, is had at present in field of biomedicine very extensive
Research and it is huge it is potential use prospect.TDNs is self-assembly of under given conditions by four single stranded DNAs are single-stranded
DNA nano material with three-dimensional structure, and the base sequence of four single stranded DNAs is to have followed strictly " base pair complementarity original
Then ", and then accurately, dexterously it designs.TDNs simple synthetic method, yield are higher, to specificity or non-specific core
The more common linear DNA of the tolerance of sour enzyme is good, and has good biocompatibility, biological safety and biodegradable
Property.
TDNs is appeared in some researchs with the role of pharmaceutical carrier at present, but its application in neural restoration is still
Have to be developed.
Summary of the invention
The present invention is intended to provide a kind of new drug that can promote neural restoration.
In order to solve this technical problem, the present invention provides the purposes of DNA tetrahedron in medicine preparation, the drugs
It is the drug for promoting neural restoration.
Above-mentioned to use on the way, the DNA tetrahedron is assembled into four sides by base pair complementarity mode by four single stranded DNAs
Body structure；Tetrahedral every rib is all double-stranded DNA structure.
Above-mentioned to use on the way, the tetrahedral preparation method of DNA is: will be containing four single stranded DNAs described in isoconcentration
Aqueous solution heating maintains 5~20min so that hydrogen bond is fully disconnected between base, and then fast cooling is kept at least to 0~10 DEG C
15min；Also contain 10mM Tris-HCl, 50mM MgCl2 in the aqueous solution；PH value before the aqueous solution heating is 8.
It is above-mentioned on the way, held time after the heating as 10min, the actual temp of the cooling is 4 DEG C, the drop
The retention time is 20min after temperature.
Above-mentioned to use on the way, the sequence of four single stranded DNAs is respectively as shown in NO.1~4 SEQ ID.
Above-mentioned to use on the way, the drug is proliferation, differentiation and/or the drug of migration for promoting neural stem cell.
Further, the drug is the drug for activating Wnt/ β-Catenin access.
Further, the drug is the drug for inhibiting Notch signal path.
Further, the drug is the drug for activating RHOA/ROCK2 signal path.
The present invention also provides a kind of drugs for promoting neural restoration, which is characterized in that it is using claim 1~9 institute
DNA tetrahedron is stated as active material, in addition pharmaceutically acceptable auxiliary material or complementary ingredient are prepared.
The invention has the following advantages:
TDNs of the invention can be much higher than single stranded DNA by mouse neural stem cells huge uptake, uptake ratio.
TDNs of the invention can obviously increase proliferation, differentiation and the migration of mouse neural stem cells, have and promote well
Neural restoration ability.
For TDNs of the invention due to being that nucleic acid is constituted, metabolism will not generate toxicity to cell, have good biology
Compatibility.
TDNs of the invention can be prepared into the drug for promoting neural restoration.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.The present invention is described in further details below by specific embodiment, but is not to this
The limitation of invention, above content according to the present invention are not departing from this according to the ordinary technical knowledge and customary means of this field
Under the premise of inventing above-mentioned basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.
Detailed description of the invention
Fig. 1 is the schematic diagram of four single-stranded synthesis TDNs.
Fig. 2 is TDNs polyacrylamide gel electrophoresis result schematic diagram.
Fig. 3 is the schematic diagram of transmission electron microscope qualification result.
Fig. 4 grain size distribution, wherein a is the grain size distribution of single stranded DNA, and b is the grain size distribution of TDNs.
Fig. 5 is that the result identified using undifferentiated state of the immunofluorescence technique to mouse neural stem cells is illustrated
Figure.
Fig. 6 is that mouse neural stem cells absorb situation flow cytometry treated result figure to TDNs.
Fig. 7 is mouse neural stem cells fluorescent tracing figure.
Fig. 8 is testing result schematic diagram of the TDNs concentration to mouse neural stem cells proliferative effect.
Fig. 9 is to be shown using Flow cytometry mouse neural stem cells in the result that TDNs acts on the variation of lower cell cycle
It is intended to.
Figure 10 a is that TDNs acts on β-catenin, Lef-1 and Cyclin-D trace figure in lower mouse neural stem cells.
Figure 10 b is that TDNs acts on β-catenin, Lef-1 and Cyclin-D western blot letter in lower mouse neural stem cells
Number relative intensity column diagram.
Figure 10 c is that TDNs acts on β-catenin, Lef-1 and Cyclin-D Relative gene table in lower mouse neural stem cells
Up to amount column diagram.
Figure 11 a is that TDNs acts on β-III-Tubulin western blot figure in lower mouse neural stem cells.
Figure 11 b is that TDNs acts on β-III-Tubulin western blot signal relative intensity column in lower mouse neural stem cells
Shape figure.
Figure 11 c is the relative expression quantity column diagram that TDNs acts on β-III-Tubulin gene in lower mouse neural stem cells.
Figure 12 a is the trace figure that TDNs acts on Notch signal path GAP-associated protein GAP in lower mouse neural stem cells.
Figure 12 b is that Notch signal path GAP-associated protein GAP blot signals are relatively strong in mouse neural stem cells under TDNs is acted on
Weak column diagram.
Figure 12 c is relative expression's power that TDNs acts on Notch signal path related gene in lower mouse neural stem cells
Figure.
Figure 13 is β-III-Tubulin protein immunization fluorogram after TDNs is handled 1 day.
Figure 14 is β-III-Tubulin protein immunization fluorogram after TDNs is handled 7 days.
Figure 15 is mouse neural stem cells scratch experiment result figure.
Figure 16 is mouse neural stem cells Transwell experimental result picture.
Figure 17 a is that TDNs acts on RhoA amplified band electrophoretogram in lower mouse neural stem cells.
Figure 17 b is that TDNs acts on RhoA gene expression multiple variation diagram in lower mouse neural stem cells.
Figure 17 c is that TDNs acts on RhoA gene expression relative quantification figure in lower mouse neural stem cells.
Figure 17 d is that TDNs acts on RhoA protein blot figure in lower mouse neural stem cells.
Figure 17 e is that TDNs acts on the relatively strong and weak column diagram of RhoA western blot signal in lower mouse neural stem cells.
Figure 17 f is that TDNs acts on RhoA protein immunization fluorogram in lower mouse neural stem cells.
Figure 17 g is that TDNs acts on RhoA protein immunization fluorescence signal intensity figure in lower mouse neural stem cells.
Figure 18 a is that TDNs acts on Rock2 amplified band electrophoretogram in lower mouse neural stem cells.
Figure 18 b is that TDNs acts on Rock2 gene expression multiple variation diagram in lower mouse neural stem cells.
Figure 18 c is that TDNs acts on Rock2 gene expression relative quantification figure in lower mouse neural stem cells.
Figure 18 d is that TDNs acts on Rock2 protein blot figure in lower mouse neural stem cells.
Figure 18 e is that TDNs acts on the relatively strong and weak column diagram of Rock2 western blot signal in lower mouse neural stem cells.
Figure 18 f is that TDNs acts on Rock2 protein immunization fluorogram in lower mouse neural stem cells.
Figure 18 g is that TDNs acts on Rock2 protein immunization fluorescence signal intensity figure in lower mouse neural stem cells.
Figure 19 a is that TDNs acts on vinculin amplified band electrophoretogram in lower mouse neural stem cells.
Figure 19 b is that TDNs acts on vinculin gene expression multiple variation diagram in lower mouse neural stem cells.
Figure 19 c is that TDNs acts on vinculin gene expression relative quantification figure in lower mouse neural stem cells.
Figure 19 d is that TDNs acts on vinculin protein blot figure in lower mouse neural stem cells.
Figure 19 e is that TDNs acts on the relatively strong and weak column diagram of vinculin western blot signal in lower mouse neural stem cells.
Figure 19 f is that TDNs acts on vinculin protein immunization fluorogram in lower mouse neural stem cells.
Figure 19 g is that TDNs acts on vinculin protein immunization fluorescence signal intensity figure in lower mouse neural stem cells.
Specific embodiment
The synthesis of embodiment TDNs
1. synthesis
Four kinds of DNA single-stranded (S1, S2, S3, S4) are dissolved into TM buffer solution with identical final concentration, wherein TM
The solute of buffer solution is Tris-HCl and MgCl2, their concentration is respectively 10mM and 50mM, and adjusts the pH value of solution
It is 8.0.Then mixture is placed in PCR instrument by being vortexed, mixing, be centrifuged, temperature is quickly risen to 95 DEG C and stablizes 10
~15min is cooled to 4 DEG C of stable 20min.Synthesize tetrahedral structure as shown in Figure 1.
The DNA single-stranded sequence is as shown in table 1.
1 TDNs of table single-stranded sequence
2. identification
2.1 gel electrophoresis
A, polyacrylamide gel is prepared: the ammonium sulfate of the acrylamide solution of 40wt%, 10 × TAE, 10wt% is molten
Liquid, distilled water, tetramethylethylenediamine are mixed according to 1~2: 1: 1: 1: 1 volume ratio, and polyacrylamide gel is made.
B, sample-adding and electrophoresis: 6 × loading of 1ul buffer is uniformly mixed with the sample of 5ul and marker respectively,
It is then respectively adding in corresponding electrophoresis tank.Under conditions of ice bath, constant pressure 100V, electrophoretic process 1 hour.
C, polyacrylamide gel GelRed dyeing and exposure: is placed in GelRed and distilled water according to 1: 50 ratio
It in mixed mixed liquor liquid, is protected from light, shaking table 15~25 minutes.Exposure.
As a result: as shown in Fig. 2, swimming lane 1~8 is respectively Marker, 1-S1,2-S2,3-S3,4-S4,5-Sl+S2,6-S1+
S2+S3,7-S1+S2+S3+S4 (7-TDNs).It can be seen from the figure that the size of ss DNA single-stranded S1, S2, S3, S4 are respectively about
Size for 60bp, 50bp, 50bp, 50bp, the then TDNs that the present invention prepares is about 210bp.
2.2 transmission electron microscope
Take appropriate sample liquid on sheet metal, infrared lower irradiation makes it dry for 5~10 minutes, upper machine testing.
As a result: as shown in figure 3, the shape of TDNs is in subtriangular shape under transmission electron microscope, particle size 10~
Within the scope of 15nM.Circle mark is polymer.
2.3 dynamic light scattering
Appropriate ssDNA and TDNs solution is taken, is placed in dynamic light scattering detector, is detected.
As a result: such as Fig. 4-a, the partial size of ssDNA is about 48.255nm.Such as Fig. 4-b, the partial size of TDNs is about 16.801nm.
In order to prove that TDNs contributes positively to neural restoration, will be further illustrated below with the mode of experimental example, experimental example
In the mouse neural stem cells used be NE-4C cell.
The identification of 1 neural stem cell of experimental example
Mouse neural stem cells are identified using immunofluorescence technique, it is primary the following steps are included:
A, cell suspending liquid is inoculated in the burnt capsule of copolymerization, is placed in incubator and cultivates 24 hours.Group is sucked to be divided into
The dual anti-culture medium of DMEM+10% serum+1%, PBS are washed 3 times, every time 5 minutes；
B, after 4% paraformaldehyde fixes 25 minutes, paraformaldehyde is sucked, PBS is washed 3 times, every time 5 minutes；
C, 0.5%Triton-100 is handled 20-25 minutes, sucks Triton-100, PBS is washed 3 times, every time 5 minutes；
D, sheep blood serum is handled 1 hour, sucks sheep blood serum, PBS is washed 3 times, every time 5 minutes；
E, primary antibody (anti-nestin antibody) is handled, and 4 DEG C, overnight.Second day, 37 DEG C rewarming 0.5 hour, recycle primary antibody, PBS
It washes 3 times, every time 5 minutes.Two process resistant for carrying fluorescence, are protected from light, 37 DEG C, 1 hour, suck secondary antibody, PBS is washed 3 times, every time 5 points
Clock；
E, phalloidine is handled, and is protected from light, 10-30 minutes, is sucked phalloidine, PBS is washed 3 times, every time 5 minutes；
F, DAPI is handled, and is protected from light, 10 minutes, is sucked DAPI, PBS is washed 3 times, every time 5 minutes.10% glycerol approved sample, is protected from light,
4 DEG C of preservations.Upper machine testing.
Testing result indicates cell still in not dividing as shown in figure 5, mouse neural stem cells show nestin antibody positive
The state of change can be used for carrying out subsequent experimental.
" cell " involved in subsequent experimental example is that the mouse Nerve of undifferentiated state of the same race in experimental example 1 is dry thin
Born of the same parents.
The intake experiment of 2 neural stem cell of experimental example
It can be the premise that most drugs play therapeutic effect by cell huge uptake.It is thin that this experimental example detects nerve cord
Intake ability of the born of the same parents to TDNs.
(1) flow cytometry
A, in 6 orifice plates inoculating cell suspension, first at incubator preculture 24 hours (37 DEG C, 5% (v/v) CO2)；It then will training
The serum-concentration supported in base drops to 6% by 10%, continues to cultivate 6 hours (37 DEG C, 5% (v/v) CO2) in incubator；It again will culture
Serum-concentration in base drops to 0 by 6%, continues to cultivate 1 hour (37 DEG C, 5% (v/v) CO2) in incubator.
B, negative control group is without any processing, and the single stranded DNA that the Cy5 that concentration is 250nM is modified is added in positive controls
S1, the TDNs that the Cy5 that concentration is 250nM is modified is added in experimental group, in incubator culture 12 hours (37 DEG C, 5% (v/v) CO2).
C, cell being collected, 5min is centrifuged under 1000r/min, PBS is resuspended, in triplicate, upper machine testing.
As a result: such as Fig. 6-a and 6-b, neural stem cell is significantly more than the intake to ssDNA to the intake of TNDs.(ask you
Explanation figure 6-a)
It is above-mentioned it is demonstrated experimentally that TNDs can be good at being absorbed by mouse neural stem cells.
(2) fluorescent tracer technique
A, the Mice Inoculated neural stem cell suspension in being copolymerized burnt capsule, first incubator preculture 24 hours (37 DEG C, 5%
(v/v)CO2)；Then the serum-concentration in culture medium is dropped to 6% by 10%, in incubator continue culture 6 hours (37 DEG C, 5%
(v/v)CO2)；The serum-concentration in culture medium is dropped to 0 by 6% again, in incubator continue culture 1 hour (37 DEG C, 5% (v/v)
CO2)。
B, the S1 that the Cy5 that control group is added that concentration is 250nM is modified, experimental group are added what the Cy5 that concentration is 250nM was modified
TDNs, in incubator culture 12 hours (37 DEG C, 5% (v/v) CO2).
C, culture medium is sucked, is cleaned three times with PBS, 5 minutes every time；Then 25 minutes are fixed with the paraformaldehyde of 4wt%,
Paraformaldehyde is sucked, is cleaned three times with PBS, 5 minutes every time；It is handled again with phalloidine, is protected from light 10~30 minutes, sucks Phallus
Cyclic peptide is cleaned three times with PBS, and 5 minutes every time；Then it is handled with DAPI, is protected from light 10 minutes, sucks DAPI, clean three with PBS
It is secondary, 5 minutes every time；The glycerol approved sample for using 10wt% again, is protected from light, 4 DEG C of preservations, and upper machine testing.
As a result: such as Fig. 7-a and 7-b, ssDNA is absorbed less by neural stem cell；Neural stem cell absorbs TDNs
It is more, and the TDNs for entering cell is largely gathered in the endochylema of cell, into the less of nucleus.
Show that TDNs can be absorbed by neural stem cell well.
The proliferation and detection of 3 TDNs of experimental example promotion mouse neural stem cells
1.CCK-8 detection proliferation
(1) inoculating cell suspension (100 hole μ l/) in 96 orifice plates, is placed in preculture 24 hours (37 in incubator for culture plate
DEG C, 5%CO2), then group is divided into the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% and drops to 6% by 10%,
Cultivated in incubator 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, is trained in incubator
1 hour feeding (37 DEG C, 5%CO2)。
(2) cell suspension of culture is divided into control group and experimental group, and TDNs is added in experimental group, added in control group
Enter the PBS of equivalent, then cultivated in incubator 24 hours (37 DEG C, 5%CO2)。
(3) CCK-8 solution (10 hole μ l/) is added into experimental group and control group respectively, 1~4h is then incubated in incubator
(37 DEG C, 5%CO2), then the absorbance in every hole is detected at 450nm, the result is shown in Fig. 8.
As shown in figure 8, when TDNs concentration is 62.5nM, 125nM, 250nM, compared with the control group, mouse in experimental group
The breeding of neural stem cell all receives the facilitation of TDNs to a certain extent, and 250nM is optium concentration, table
Bright TDNs has the function of promoting mouse neural stem cells proliferation.
2, using Flow cytometry cell Proliferation
(1) the inoculating cell suspension in 25ml culture bottle, culture bottle is placed in incubator preculture 24 hours (37 DEG C, 5%
CO2), then group is divided into the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% and drops to 6% by 10%, in incubator
It is middle culture 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, is cultivated 1 hour in incubator
(37 DEG C, 5%CO2)。
(2) cell suspension of culture is divided into control group and experimental group, and TDNs is added in experimental group, added in control group
Enter the PBS of equivalent, then cultivated in incubator 24 hours (37 DEG C, 5%CO2)。
(3) it is digested respectively using 0.25% trypsase and collects cellular control unit and experimental group cell, be placed in 15ml centrifugation
In pipe (2000rpm, 5 minutes), supernatant is abandoned, PBS washing is centrifuged (2000rpm, 5 minutes), adds 500 μ l of ice ethyl alcohol and fix
Cell, 4 DEG C overnight, and addition PBS centrifugation in second day abandons supernatant, adds PBS washing, and centrifugation abandons supernatant, 100 μ l are then added
RNase, 37 DEG C of water-baths 30 minutes, are added 400 μ l PI dyeing and mix, and 4 DEG C are protected from light, and 30 minutes.Cell is transferred to streaming pipe
In, upper machine testing, and data analysis is carried out, the result is shown in Fig. 9.
As shown in figure 9, compared with the control group, the cell number in the S phase (DNA synthesizes the phase) in experimental group obviously increases,
Illustrate that TDNs changes the cell cycle of neural stem cell, has the function of promoting its proliferation.
To sum up, TDNs can promote nerve stem cell proliferation.
The detection of 4 Wnt/ β-catenin signal path related gene expression of experimental example
Wnt/ β-catenin signal path is the important access of cell cycle regulating, its activation energy remarkably promotes dry thin
The proliferation of born of the same parents.This experimental example detects under TDNs processing, the expression of the Wnt/ β-catenin signal path related gene of NSC.
1. sample treatment
(1) inoculating cell suspension (100 hole μ l/) in 6 orifice plates, is placed in preculture 24 hours (37 in incubator for culture plate
DEG C, 5%CO2), then group is divided into the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% and drops to 6% by 10%,
Cultivated in incubator 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, is trained in incubator
1 hour feeding (37 DEG C, 5%CO2)。
(2) it is divided into the dual anti-culture medium of DMEM+1% using group to cultivate cell suspension obtained by step (1), by cell
Suspension is divided into control group and experimental group, and replaces culture medium simultaneously daily；And it is 250nM's that concentration is added in experimental group
TDNs, meanwhile, the PBS of equivalent is added in control group.
2. Protein Detection
Experimental group processing for 24 hours after, extract experimental group, control group holoprotein, then by Western blot detection with
Three albumen on the relevant Wnt/ β-catenin signal path of nerve stem cell proliferation process: β-catenin, Lef-1 and
Cyclin-D。
Brief testing process is as follows: encapsulating → loading → electrophoresis → transferring film → confining liquid shakes 4 DEG C of 1 hour → primary antibody of closing
Overnight → recycling primary antibody → TBST washs 3 (5-10 minutes each) → secondary antibodies and is incubated for 1 hour → abandons secondary antibody, TBST is washed 3 times
(5-10 minutes each) → exposure.
As as-shown-in figures 10 a and 10b, mouse neural stem cells through TDNs processing for 24 hours after, β-catenin, Lef-1 and
Cyclin-D protein content will be significantly higher than control group.
3. gene expression detection
After experimental group processing for 24 hours, the RNA of experimental group, control group is extracted, cDNA is obtained by Reverse Transcriptase kit, then
Fluorescence quantitative PCR detection Wnt/ β-catenin signal path relevant to nerve stem cell proliferation process is carried out using dye method
On three genes: β-catenin, Lef-1 and Cyclin-D.
As shown in figure l0c, after mouse neural stem cells TDNs processing for 24 hours, β-catenin, Lef-1 and Cyclin-D gene
Expression quantity will be significantly higher than control group.
This experimental example shows TDNs by the expression of three genes on up-regulation Wnt/ β-catenin signal path to promote
Into the proliferation of neural stem cell.
The detection of experimental example 5 β-III-Tubulin and Notch signal path related gene expression
β-III-Tubulin albumen is a kind of neuron marker, when it is neural stem cell differentiating at neuron when, β-III-
Tubulin expression will raise.Notch signal path is played an important role in nervous system development process, in adult neural
Holddown is shown as in stem cell atomization.This experimental example passes through detection β-III-Tubulin and Notch signal path phase
The expression of correlation gene is to illustrate TDNs to the rush differentiation of NSC.
1. sample treatment
(1) inoculating cell suspension (100 hole μ l/) in 6 orifice plates, is placed in preculture 24 hours (37 in incubator for culture plate
DEG C, 5%CO2), then group is divided into the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% and drops to 6% by 10%,
Cultivated in incubator 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, is trained in incubator
1 hour feeding (37 DEG C, 5%CO2)。
(2) it is divided into the dual anti-culture medium of DMEM+1% using group to cultivate cell suspension obtained by step (1), by cell
Suspension is divided into control group and experimental group, and replaces culture medium simultaneously daily；And it is 250nM's that concentration is added in experimental group
TDNs, meanwhile, the PBS of equivalent is added in control group.
2. Protein Detection
After experimental group handles 1 day, 3 days and 7 days respectively, the holoprotein of experimental group, control group is extracted, then is passed through
Western blot is detected and neural stem cell differentiating relevant albumen: β-III-Tubulin, Notch-1, Hes-1 and Hes-
5。
Brief testing process is as follows: encapsulating → loading → electrophoresis → transferring film → confining liquid shakes 4 DEG C of 1 hour → primary antibody of closing
Overnight → recycling primary antibody → TBST washs 3 (5-10 minutes each) → secondary antibodies and is incubated for 1 hour → abandons secondary antibody, TBST is washed 3 times
(5-10 minutes each) → exposure.
As shown in Figure 11 a, 11b, 12a and 12b, neural stem cell differentiating marker albumen (β-III- in experimental group
Tubulin expression quantity) is above control group, and compared with the control group, breaks up on correlation Notch signal path in experimental group
Three albumen are that the expression quantity of Notch-1, Hes-1, Hes-5 reduce respectively, show that TDNs can promote mouse neural stem cells
Differentiation and maturation.
3. gene expression detection
After experimental group handles 1 day, 3 days and 7 days respectively, the RNA of experimental group, control group is extracted, reverse transcription reagents are passed through
Box obtains cDNA, reuses dye method and carries out fluorescence quantitative PCR detection and neural stem cell differentiating relevant albumen: β-III-
Tubulin, Notch-1, Hes-1 and Hes-5.
As shown in Figure 11 c and 12c, break up relevant target gene (β-III- compared with the control group, in experimental group
Tubulin expression quantity) is higher, and compared with the control group, and three eggs on correlation Notch signal path are broken up in experimental group
It is white, be Notch-1, Hes-1, Hes-5 respectively, corresponding to the expression quantity of gene Notch-1, Nes-1, Hes-5 reduce, table
Bright TDNs can promote the differentiation and maturation of mouse neural stem cells, break up related with the inhibition of Notch access.
4. immunofluorescence technique
(1) sample is handled according to the method for this experimental example Section 1, only changes 6 orifice plates into copolymerization burnt capsule.
(2) after TDNs is handled 1 day and 7 days, the culture medium of experimental group and control group is sucked respectively, PBS is washed 3 times, every time 5 points
Clock.After fixing 25 minutes with 4% paraformaldehyde again, paraformaldehyde is sucked, PBS washes 3 times, and 5 minutes every time, 0.5%Triton-
100 processing 20-25 minutes, suck Triton-100, PBS is washed 3 times, every time 5 minutes.Sheep blood serum is handled 1 hour, sucks sheep blood
Clearly, PBS is washed 3 times, every time 5 minutes.The processing of primary antibody β-III-Tubulin antibody, 4 DEG C, overnight.Second day, 37 DEG C of rewarmings 0.5 were small
When, primary antibody is recycled, PBS is washed 3 times, every time 5 minutes.Two process resistant for carrying fluorescence, are protected from light, 37 DEG C, 1 hour, suck secondary antibody,
PBS is washed 3 times, every time 5 minutes.DAPI processing, is protected from light, 10 minutes, sucks DAPI, PBS is washed 3 times, every time 5 minutes.10% glycerol
Approved sample is protected from light, 4 DEG C of preservations.Upper machine testing.
As a result: as shown in Figure 13,14, behind 1 day and 7 days, compared with the control group, the fluorescence intensity (β-in experimental group
III-Tubulin) higher, and the form of cell is closer to neuron.
To sum up, TDNs is by inhibiting Notch signal path to promote the differentiation of neural stem cell.
The migration of 6 TDNs of embodiment promotion mouse neural stem cells
1. scratch experiment
A, the Mice Inoculated neural stem cell suspension in 6 orifice plates, first by culture plate incubator preculture 24 hours (37 DEG C,
5% (v/v) CO2)；The serum-concentration in culture medium is dropped to 6% by 10% again, in incubator continue culture 6 hours (37 DEG C, 5%
(v/v)CO2)；The serum-concentration in culture medium is dropped to 0 by 6% again, in incubator continue culture 1 hour (37 DEG C, 5% (v/v)
CO2)；Then cell monolayer is done into scratch along straight line in culture plate using sterile rifle point, and is washed three times with PBS.
B, control group is added without TDNs, and the TDNs of corresponding concentration is added in experimental group, in incubator culture (37 DEG C, 5% (v/v)
CO2).It takes pictures under light microscopic after 0,12,24 hour, record scratch variation.
C, result: such as Figure 15, compared with the control group, TDNs is obviously promoted the horizontal line migration of mouse neural stem cells.
2.Transwell experiment
A, Transwell (aperture is 0.8 μm) cell is placed in 6 orifice plates, Mice Inoculated nerve cord is thin in cell
Born of the same parents' suspension, first by culture plate at incubator preculture 24 hours (37 DEG C, 5% (v/v) CO2)；Then the serum in culture medium is dense
Degree drops to 6% by 10%, continues to cultivate 6 hours (37 DEG C, 5% (v/v) CO2) in incubator；Again by the serum-concentration in culture medium
0 is dropped to by 6%, continues to cultivate 1 hour (37 DEG C, 5% (v/v) CO2) in incubator.
B, control group is added without TDNs, and the TDNs that concentration is 250nM is added in experimental group, in incubator culture 12 hours (37 DEG C,
5% (v/v) CO2).
C, culture medium is sucked, is cleaned three times with PBS, 5 minutes every time；Then 25 minutes are fixed with the paraformaldehyde of 4wt%
Afterwards, paraformaldehyde is sucked, is cleaned three times with PBS, 5 minutes every time；It is handled again with DAPI, is protected from light 10 minutes, sucks DAPI, used
PBS is cleaned three times, and 5 minutes every time；It is protected from light, 4 DEG C of preservations, and upper machine testing.
D, result: such as Figure 16, compared with the control group, the cell in experimental group across the cell Transwell is obviously increased, i.e.,
TDNs promotes the vertical to migration of neural stem cell.
Scratch experiment and Transwell experiment, it was demonstrated that TDNs of the invention is to the transverse direction of mouse neural stem cells and vertical
There is good facilitation really to migration.
The detection of 7 RHOA/ROCK2 signal path GAP-associated protein GAP of embodiment
1. sample treatment
(1) the Mice Inoculated neural stem cell suspension in 6 orifice plates, first by culture plate incubator preculture 24 hours (37 DEG C,
5% (v/v) CO2)；Then the serum-concentration in culture medium is dropped to 6% by 10%, in incubator continue culture 6 hours (37 DEG C,
5% (v/v) CO2)；The serum-concentration in culture medium is dropped to 0 by 6% again, in incubator continue culture 1 hour (37 DEG C, 5% (v/
v)CO2)。
(2) control group is added without TDNs, and the TDNs that concentration is 250nM is added in experimental group, in incubator culture.
2. fluorescence quantitative PCR detection
After TDNs processing for 24 hours, the total serum IgE of experimental group and control group is extracted, after reverse transcription, carries out quantitative fluorescent PCR reaction,
Detect the expression of RhoA, Rock2, Vinculin.
As a result: and right in semiquantitive PCR and quantitative PCR as shown in Fig. 9 a~9c, Figure 10 a~10c, Figure 11 a~11c
Compared according to group, in experimental group migrate correlation RHOA/ROCK2 signal path on three albumen, be respectively RhoA, Rock2,
The expression quantity of Vinculin, corresponding gene increase.Illustrate that TDNs promotes the migration of neural stem cell, is to pass through activation
RHOA/ROCK2 signal path is realized.
3. western blot detects
TDNs processing for 24 hours after, extract experimental group and control group total protein, using Western blot detection RhoA,
Rock2, Vinculin albumen.
As a result: as shown in Figure 17 d~17e, Figure 18 d~18e, Figure 19 d~19e, compared with the control group, being migrated in experimental group
Three albumen on related RHOA/ROCK2 signal path are RhoA, Rock2, Vinculin, the table of corresponding albumen respectively
Increase up to amount.It illustrates that TDNs promotes the migration of neural stem cell, is realized by activation RHOA/ROCK2 signal path.
4. Immunofluorescence test
It changes the 6 orifice plates in this experimental example Section 1 into copolymerization burnt capsule and carries out sample treatment.After TDNs processing for 24 hours, suck
Culture medium, PBS are cleaned three times, and 5 minutes every time；After fixing 25 minutes with the paraformaldehyde of 4wt%, paraformaldehyde is sucked, PBS is clear
It washes three times, 5 minutes every time；It is handled 20~25 minutes with 0.5%Triton-100 again, sucks Triton-100, PBS cleaning three
It is secondary, 5 minutes every time；Then it being handled 1 hour with sheep blood serum, sucks sheep blood serum, PBS is cleaned three times, and 5 minutes every time.Primary antibody RhoA,
The processing of Rock2, Vinculin antibody, 4 DEG C, overnight.Second day, 37 DEG C rewarming 0.5 hour, recycle primary antibody, PBS washes 3 times, every time
5 minutes.Two process resistant for carrying fluorescence, are protected from light, 37 DEG C, 1 hour, suck secondary antibody, PBS is washed 3 times, every time 5 minutes.At DAPI
Reason, is protected from light, 10 minutes, sucks DAPI, PBS is washed 3 times, every time 5 minutes.10% glycerol approved sample, is protected from light, 4 DEG C of preservations, and upper machine examination
It surveys.
As a result: as shown in Figure 17 d~17e, Figure 18 d~18e, Figure 19 d~19e, compared with the control group, albumen in experimental group
Fluorescence intensity (RhoA, Rock2, Vinculin) it is higher, further illustrate TDNs promote neural stem cell migration, be
It is realized by activation RHOA/ROCK2 signal path.
The above experiment, which further demonstrates TDNs, to promote neural stem cell to migrate.
To sum up, DNA tetrahedron of the invention is easy to be absorbed by nerve cell, can obviously increase mouse neural stem cells
Proliferation, differentiation and migration have and promote neural restoration ability well, and have good biocompatibility.DNA of the invention
Tetrahedron can be used for preparing the drug for promoting neural restoration.
SEQUENCE LISTING
<110>Sichuan University
<120>DNA tetrahedron is promoting the purposes in neural restoration medicine preparation
<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
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<212> DNA
<213> artificial sequence
<220>
<223> S4
<400> 4
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (10)
- The purposes of 1.DNA tetrahedron in medicine preparation, which is characterized in that the drug is the drug for promoting neural restoration.
- 2. purposes as described in claim 1, which is characterized in that the DNA tetrahedron passes through base complementrity by four single stranded DNAs Matching method is assembled into tetrahedral structure；Tetrahedral every rib is all double-stranded DNA structure.
- 3. purposes as described in claim 1, which is characterized in that the tetrahedral preparation method of DNA is: will contain isoconcentration The aqueous solution of four single stranded DNAs heats, so that hydrogen bond is fully disconnected between base, maintains 5~20min, then fast cooling To 0~10 DEG C, at least 15min is kept；Also contain 10mM Tris-HCl, 50mM MgCl in the aqueous solution2；The aqueous solution PH value before heating is 8.
- 4. purposes as claimed in claim 3, which is characterized in that held time after the heating as 10min, the tool of the cooling Temperature is 4 DEG C, and the retention time is 20min after the cooling.
- 5. purposes as described in claim 1, which is characterized in that the sequence of four single stranded DNAs is respectively such as SEQ ID NO.1 Shown in~4.
- 6. purposes as described in claim 1, which is characterized in that the drug be promote neural stem cell proliferation, differentiation and/ Or the drug of migration.
- 7. purposes as claimed in claim 6, which is characterized in that the drug is the drug for activating Wnt/ β-Catenin access.
- 8. purposes as claimed in claim 6, which is characterized in that the drug is the drug for inhibiting Notch signal path.
- 9. purposes as claimed in claim 6, which is characterized in that the drug is the medicine for activating RHOA/ROCK2 signal path Object.
- 10. it is a kind of promote neural restoration drug, which is characterized in that it be use the claim 1~9 DNA tetrahedron as Active material, in addition pharmaceutically acceptable auxiliary material or complementary ingredient are prepared.
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| CN201711173678.XA CN107881149A (en) | 2017-11-22 | 2017-11-22 | Application of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted |
| CN201711173678X | 2017-11-22 | ||
| CN201810351727.2A CN108546730A (en) | 2018-04-19 | 2018-04-19 | Application of the DNA tetrahedrons in promoting mouse neural stem cells migration |
| CN2018103517272 | 2018-04-19 |
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Cited By (10)
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| CN110404081A (en) * | 2019-08-26 | 2019-11-05 | 四川大学 | A kind of nano-complex of DNA tetrahedron and microRNA |
| CN111467364A (en) * | 2020-05-11 | 2020-07-31 | 四川大学 | Medicine for preventing drug-induced osteonecrosis of jaw |
| CN111494401A (en) * | 2019-08-22 | 2020-08-07 | 四川大学 | Use of DNA tetrahedron in preparation of medicine for promoting myoblast proliferation |
| CN111991412A (en) * | 2020-09-30 | 2020-11-27 | 四川大学 | Medicine for treating Parkinson's disease |
| CN112156104A (en) * | 2020-11-10 | 2021-01-01 | 四川大学 | Medicine for treating depression |
| CN112641796A (en) * | 2020-12-28 | 2021-04-13 | 四川大学 | Nerve protection medicine for cerebral arterial thrombosis |
| CN113509482A (en) * | 2021-04-30 | 2021-10-19 | 四川大学 | Application of DNA tetrahedron in preparation of medicine for preventing and treating Sjogren syndrome |
| CN114748604A (en) * | 2022-05-10 | 2022-07-15 | 四川大学 | Compound for bone marrow damage and/or inhibition |
| CN114767830A (en) * | 2022-04-28 | 2022-07-22 | 四川大学 | Compound for preventing osteonecrosis and/or promoting angiogenesis and pharmaceutical application thereof |
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| CN111494401A (en) * | 2019-08-22 | 2020-08-07 | 四川大学 | Use of DNA tetrahedron in preparation of medicine for promoting myoblast proliferation |
| CN110404081A (en) * | 2019-08-26 | 2019-11-05 | 四川大学 | A kind of nano-complex of DNA tetrahedron and microRNA |
| CN111467364A (en) * | 2020-05-11 | 2020-07-31 | 四川大学 | Medicine for preventing drug-induced osteonecrosis of jaw |
| CN111991412A (en) * | 2020-09-30 | 2020-11-27 | 四川大学 | Medicine for treating Parkinson's disease |
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| CN112156104B (en) * | 2020-11-10 | 2021-12-31 | 四川大学 | Medicine for treating depression |
| CN112641796A (en) * | 2020-12-28 | 2021-04-13 | 四川大学 | Nerve protection medicine for cerebral arterial thrombosis |
| WO2022194109A1 (en) | 2021-03-18 | 2022-09-22 | 成都景润泽基因科技有限公司 | Complex for treating optic nerve disease, and preparation method therefor and use thereof |
| CN113509482A (en) * | 2021-04-30 | 2021-10-19 | 四川大学 | Application of DNA tetrahedron in preparation of medicine for preventing and treating Sjogren syndrome |
| CN113509482B (en) * | 2021-04-30 | 2022-09-02 | 四川大学 | Application of DNA tetrahedron in preparation of medicine for preventing and treating Sjogren syndrome |
| CN114767830A (en) * | 2022-04-28 | 2022-07-22 | 四川大学 | Compound for preventing osteonecrosis and/or promoting angiogenesis and pharmaceutical application thereof |
| CN114748604A (en) * | 2022-05-10 | 2022-07-15 | 四川大学 | Compound for bone marrow damage and/or inhibition |
| CN114748604B (en) * | 2022-05-10 | 2023-04-07 | 四川大学 | Compound for bone marrow damage and/or inhibition |
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| WO2019101116A1 (en) | 2019-05-31 |
| CN109806275B (en) | 2021-04-23 |
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