CN107881149A - Application of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted - Google Patents
Application of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted Download PDFInfo
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Abstract
The invention discloses a kind of application of DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted, wherein, the tetrahedral four single stranded sequences such as SEQ ID NO of DNA:Shown in 14.The inventive method can be effectively promoted propagation and the differentiation of NSC.
Description
Technical field
The invention belongs to cell proliferation and differentiation technical field, and in particular to a kind of DNA tetrahedrons are promoting NSC
Application during Proliferation, Differentiation.
Background technology
Mouse neural stem cells (NE-4C), the cell line derive from ATCC cell banks, are a kind of preferably in vitro study god
One of model through system.NE-4C cell lines can maintain the characteristics of NSC under certain circumstances, but specific
After induction processing, the neuron or Deiter's cells of maturation can be divided into.At present, in the research on NSC
In, Main way is research medicine or material to nerve stem cell proliferation and the influence of differentiation, such as prostaglandin nerve
Influence in stem cells hyperplasia and atomization；And cell therapy of the NSC in some maincenter retrogression pathological changes is made
With, such as therapeutic action of the NSC in Alzheimer's disease animal model.
DNA tetrahedrons nano material (TDNs) is a kind of new DNA nano materials, is had at present in biomedical sector
Quite varied research and huge potential use prospect.Found in current research, used in Proliferation, Differentiation research process
Medicine or material may be certain to cell toxic action, i.e., with biocompatibility, biological safety, biodegradable
The features such as property is poor.TDNs be by four ss DNA it is single-stranded be self-assembly of under given conditions there is three-dimensional structure
DNA nano materials, the base sequence of four ss DNA (S1, S2, S3, S4) are to have followed strictly " base pair complementarity principle " to enter
And it is accurate, dexterously design.TDNs simple synthetic methods, yield are higher, to specific or Non-specific nuclease
The more common linear DNA of tolerance is good, and has good biocompatibility, biological safety and biodegradability.This
Outside, though the research on TDNs is more, the research that it influences on cell items physiological activity is seldom, especially promotes cell to increase
The research for growing atomization is less.
The content of the invention
For above-mentioned deficiency of the prior art, the present invention provides a kind of DNA tetrahedrons and is promoting neural NSC
Application during Proliferation, Differentiation, the DNA tetrahedrons base related to Notch signal paths by influenceing Wnt/ β-catenin
The expression change of cause and albumen, respectively reaches the purpose for promoting differentiation and proliferation of neural stem cells.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
Application of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted, D wherein, tetrahedral four lists of DNA
Chain-ordering such as SEQ ID NO:Shown in 1-4.
Further, it is by activating Wnt/ β-catenin signal paths that DNA tetrahedrons, which promote the propagation of NSC,
Play a role；DNA tetrahedrons promote the differentiation of NSC to be played a role by suppressing Notch signal paths.
Further, the process of nerve stem cell proliferation is promoted to include promoting β-catenin, Lef-1 and Cyclin-D eggs
White expression.
Further, the process of nerve stem cell proliferation is promoted to include promoting β-catenin, Lef-1 and Cyclin-D bases
The expression of cause.
Further, neural stem cell differentiating process is promoted to include reducing the table of Notch-1, Hes-1 and Hes-5 albumen
Reach.
Further, neural stem cell differentiating process is promoted to include promoting the expression of β-III-Tubulin albumen.
Further, neural stem cell differentiating process is promoted to include suppressing Notch-1, Hes-1 and Hes-5 gene respectively
Expression, and promote β-III-Tubulin genes expression.
Further, concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 50~500nM.
Further, concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 100~300nM.
Further, concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 250nM.
Beneficial effects of the present invention are：
1st, therefore, can be effectively because TDNs has good biocompatibility, biological safety and biodegradability
Solve the problems, such as that the biological property of conventional medicament or material is poor.DNA tetrahedrons (TDNs) nano material is in NSC
Promotion is played in propagation and atomization, while it is slower to solve nerve stem cell proliferation to a certain extent, differentiation and maturation speed
The problems such as slower, it is that NSC Experiment on therapy establishes certain Research foundation inside follow-up.
2nd, DNA tetrahedrons (TDNs) respectively facilitate its propagation by regulating and controlling Wnt/ β-catenin and Notch signal paths
And atomization, i.e., by promoting β-catenin, Lef-1 and Cyclin-D genes, albumen expression, it can reach and promote mouse
The purpose of nerve stem cell proliferation；By reducing the expression of Notch-1, Hes-1 and Hes-5 albumen, and promote β-III-
The expression of Tubulin genes, albumen, it can reach the purpose for promoting mouse neural stem cells differentiation.
Brief description of the drawings
Fig. 1 is four single-stranded synthesis TDNs schematic diagram.
Fig. 2 is TDNs polyacrylamide gel electrophoresis result schematic diagrams.
Fig. 3 is the schematic diagram of transmission electron microscope qualification result.
Fig. 4 is that the result identified using immunofluorescence technique the undifferentiated state of mouse neural stem cells is illustrated
Figure.
Fig. 5 is that mouse neural stem cells are illustrated for the result that TDNS intakes are detected using fluorescent tracer technique
Figure.
Fig. 6 is testing result schematic diagram of the TDNs concentration to mouse neural stem cells proliferative effect.
Fig. 7 is to be shown using the Flow cytometry mouse neural stem cells result that the cell cycle changes under TDNs effects
It is intended to.
Fig. 8 (a), (b) (c) are β-catenin, Lef-1 and Cyclin-D in mouse neural stem cells under TDNs is acted on
The detects schematic diagram of gene, expressing quantity.
Fig. 9 (a), (b) (c) are to survey β-III-Tubulin genes, albumen table in mouse neural stem cells under TDNs effects
Up to the detects schematic diagram of amount.
Figure 10,11 are the result schematic diagram that β-III-Tubulin expressing quantities are detected using immunofluorescence technique.
Figure 12 (a), (b), (c) be under TDNs effects Notch-1, Hes-1, Hes-5 gene in mouse neural stem cells,
The detects schematic diagram of expressing quantity.
Embodiment
The embodiment of the present invention is described below, in order to which those skilled in the art understand this hair
It is bright, it should be apparent that the invention is not restricted to the scope of embodiment, for those skilled in the art,
As long as various change in the spirit and scope of the present invention that appended claim limits and determines, these changes are aobvious and easy
See, all are using the innovation and creation of present inventive concept in the row of protection.
The synthesis and identification of embodiment 1DNA tetrahedrons (TDNs)
1st, TDNs synthesis
4 ss DNA single-stranded (S1, S2, S3, S4) of isoconcentration are added to the TM buffer (10mM containing 100 μ l
Tris-HCl, 50mM MgCl2, pH 8.0) 200 μ l EP pipes in, by reaction solution be heated to 95 DEG C maintenance 10min, then soon
Speed cools to 4 DEG C, and keeps 20min, obtains TDNs, and building-up process is as shown in Figure 1；Wherein, particular sequence single-stranded four DNA
It is as follows：
S1：
5’-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’
(SEQ ID NO:1)；
S2：
5’-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3’
(SEQ ID NO:2)；
S3：
5’-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3’
(SEQ ID NO:3)；
S4：
5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3’
(SEQ ID NO:4)。
2nd, TDNs identification
(1) polyacrylamide gel electrophoresis is identified
Polyacrylamide is prepared using 40% acrylamide, 10 × TAE, 10%APS solution, distilled water and TEMED
Gel；
Then the TDNs for taking 1 μ 6 × loading of L buffer and 5 μ L to be prepared is well mixed, respectively by itself and
In electrophoresis tank corresponding to marker additions, ice bath, constant pressure 100V, electrophoresis 60min；Then concentration ratio is used as 1:50 GelRed
And distilled water, under the conditions of lucifuge, shaking table handles 15~25min, then exposes, then is detected, and its result is shown in Fig. 2.
As shown in Fig. 2 TDNs four single-stranded S1, S2, S3 and S4 sizes are left in 60bp, 50bp, 50bp and 50bp respectively
The right side, TDNs size is about in 210bp or so.
(2) transmission electron microscope is identified
TDNs is identified using transmission electron microscope, its result is shown in Fig. 3；As shown in figure 3, TDNs (triangle mark) shape
Shape is in subtriangular shape under transmission electron microscope, and particle size is about in 10~15nM or so.Circle mark for polymer.
Embodiment 2 is using identification of the immunofluorescence technique to mouse neural stem cells
Mouse neural stem cells are identified using immunofluorescence technique, once comprised the following steps：
Cell suspending liquid is inoculated in the burnt capsule of copolymerization, is positioned in incubator and cultivates 24 hours.It is DMEM+ to suck component
The dual anti-culture medium of 10% serum+1%, PBS are washed 3 times, every time 5 minutes；
After 4% paraformaldehyde fixes 25 minutes, paraformaldehyde is sucked, PBS is washed 3 times, every time 5 minutes；
0.5%Triton-100 handles 20-25 minutes, sucks Triton-100, PBS is washed 3 times, every time 5 minutes；
Sheep blood serum is handled 1 hour, sucks sheep blood serum, PBS is washed 3 times, every time 5 minutes；
Primary antibody (anti-nestin antibody) processing, 4 DEG C, overnight.Second day, 37 DEG C of rewarmings 0.5 hour reclaimed primary antibody, and PBS is washed
3 times, every time 5 minutes.Two process resistant of fluorescence are carried, lucifuge, 37 DEG C, 1 hour, suck secondary antibody, PBS is washed 3 times, every time 5 minutes；
Phalloidine processing, lucifuge, 10-30 minutes, sucks phalloidine, PBS is washed 3 times, every time 5 minutes；
DAPI processing, lucifuge, 10 minutes, DAPI is sucked, PBS is washed 3 times, every time 5 minutes.10% glycerine approved sample, lucifuge, 4
DEG C preserve.Upper machine testing.
Testing result is shown in Fig. 4, as shown in figure 4, mouse neural stem cells show nestin antibody positives, represents that cell is still located
In undifferentiated state, tested available for follow-up Proliferation, Differentiation is carried out.
Intake of the embodiment 3 using fluorescent tracer technique detection mouse neural stem cells to TDNs
(1) the inoculating cell suspension (100 μ l/ holes) in burnt capsule is copolymerized, by culture plate in incubator preculture 24 hours
(37 DEG C, 5%CO2), then component is that the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% is dropped to by 10%
6%, cultivated in incubator 6 hours (37 DEG C, 5%CO2), then the serum-concentration in culture medium is dropped to 0 by 6%, in incubator
Culture 1 hour (37 DEG C, 5%CO2)。
(2) cell suspension of culture is divided into control group and experimental group, it is 250nM that concentration is added in control group, is used
The single-stranded S1 of DNA of Cy-5 modifications, it be 250nM that concentration is added in experimental group, using the TDNs of Cy-5 modifications, respectively at being trained in incubator
Foster 6 hours (37 DEG C, 5%CO2)。
(3) culture medium of experimental group and control group is sucked respectively, and PBS is washed 3 times, every time 5 minutes.Again with 4% paraformaldehyde
After fixing 25 minutes, paraformaldehyde is sucked, PBS washes 3 times, 5 minutes every time, and then phalloidine is handled, lucifuge, 10-30 minutes,
Phalloidine is sucked, PBS is washed 3 times, every time 5 minutes；DAPI processing is used again, lucifuge, 10 minutes, sucks DAPI, PBS is washed 3 times,
5 minutes every time；Finally with 10% glycerine approved sample, lucifuge, 4 DEG C of preservations.Upper machine testing, its testing result are shown in Fig. 5.
As shown in figure 5, single-stranded S1 absorbed by NSC it is less；NSC TDNs is absorbed it is more, and
TDNs into cell is largely gathered in the endochylema of cell, into the less of nucleus.
Embodiment 4TDNs promotes propagation and the detection of mouse neural stem cells
1st, breed
(1) inoculating cell suspension (the 100 μ l/ holes) in 96 orifice plates, preculture 24 hours (37 in incubator are placed in by culture plate
DEG C, 5%CO2), then component is dropped to 6% for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% 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%, trained in incubator
Foster 1 hour (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 μ l/ holes) is added into experimental group and control group respectively, 1~4h is then incubated in incubator
(37 DEG C, 5%CO2), then the absorbance per hole is detected at 450nm, its result is shown in Fig. 6.
As shown in fig. 6, when TDNs concentration is 62.5nM, 125nM, 250nM, compared with control group, mouse in experimental group
The breeding of NSC, all receives TDNs facilitation to a certain extent, and 250nM is optium concentration, table
Bright TDNs has the function that to promote mouse neural stem cells propagation.
2nd, bred using Flow cytometry cell
(1) the inoculating cell suspension in 25ml blake bottles, blake bottle is placed in incubator preculture 24 hours (37 DEG C, 5%
CO2), then component is dropped to 6% for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% 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%, 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) digested respectively using 0.25% trypsase and collect cellular control unit and experimental group cell, be placed in 15ml centrifugations
In pipe (2000rpm, 5 minutes), supernatant is abandoned, PBS washings, centrifugation (2000rpm, 5 minutes), add the μ l of ice ethanol 500 and fix
Cell, 4 DEG C overnight, adds within second day PBS centrifugations, abandons supernatant, adds PBS washings, centrifugation, abandons supernatant, then add 100 μ l
RNase, 37 DEG C of water-baths, 30 minutes, add 400 μ l PI dyeing and mix, 4 DEG C of lucifuges, 30 minutes.Cell is transferred to streaming pipe
In, upper machine testing, and data analysis is carried out, its result is shown in Fig. 7.
As shown in fig. 7, compared with control group, the cell number in the S phases (DNA synthesizes the phase) in experimental group substantially increases,
Illustrate that TDNs changes the cell cycle of NSC, have the function that to promote its propagation.
Embodiment 5TDNs promotes the mechanism of mouse neural stem cells propagation
1st, the mechanism of mouse neural stem cells propagation is promoted using immunoblotting detection TDNs
(1) inoculating cell suspension (the 100 μ l/ holes) in 6 orifice plates, preculture 24 hours (37 in incubator are placed in by culture plate
DEG C, 5%CO2), then component is dropped to 6% for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% 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%, trained in incubator
Foster 1 hour (37 DEG C, 5%CO2)。
(2) component is used to be cultivated for culture medium dual anti-DMEM+1% cell suspension obtained by step (1), by cell
Suspension is divided into control group and experimental group, and changes culture medium simultaneously daily；And it is 250nM's that concentration is added in experimental group
TDNs, the PBS of equivalent is added in control group, then respectively after 24 hours of incubation, carried respectively using holoprotein extracts kit
Take control group and experiment histone.
(3) SDS-PAGE electrophoresis is carried out respectively to control group obtained by step (2) and experiment histone, its detailed process is such as
Under：Encapsulating → loading → electrophoresis → transferring film → confining liquid shakes 4 DEG C of 1 hour → primary antibody of closing and stays overnight → recovery primary antibody, and TBST is washed
Wash 3 times, each 5-10 minutes → secondary antibody, 1 hour → abandon secondary antibody, TBST was washed 3 times, and each 5-10 minutes → exposure, detection is simultaneously
Data processing is carried out, its result is shown in Fig. 8 (a) and Fig. 8 (b).
It is related to nerve cord stem cells hyperplasia process in experimental group compared with control group as shown in Fig. 8 (a) and Fig. 8 (b)
Wnt/ β-catenin signal paths on three albumen, be β-catenin, Lef-1 and Cyclin-D respectively, three kinds of albumen
And expression quantity β-catenin, Lef-1 and the Cyclin-D of corresponding control gene increased, and further illustrate that TDNs promotes
The propagation of NSC.
2nd, the mechanism of mouse neural stem cells propagation is promoted using quantitative fluorescent PCR (Q-PCR) detection TDNs
(1) inoculating cell suspension (the 100 μ l/ holes) in 6 orifice plates, preculture 24 hours (37 in incubator are placed in by culture plate
DEG C, 5%CO2), then component is dropped to 6% for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% 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%, trained in incubator
Foster 1 hour (37 DEG C, 5%CO2)。
(2) suspension cell is divided into control group and experimental group, and the TDNs that concentration is 250nM is added in experimental group,
In control group add equivalent PBS, then cultivated in incubator 24 hours (37 DEG C, 5%CO2), then extracted and tried using gene
Agent box, control group and experimental group gene are extracted respectively, then passes through high-purity total serum IgE rapid extraction kit and reverse transcription reagents again
Box obtains stable cDNA.
(3)Q-PCR：Per hole add 20 μ l reaction system (2 μ l cDNA, 10 μ l SYBR, 0.8 μ l primers Fs orward,
0.8 μ l primers Reserve, 6.4 μ l ddH2O), upper machine testing, and data processing is carried out, its result is shown in Fig. 8 (c).
As shown in Fig. 8 (c), compared with control group, the Wnt/ β related to nerve stem cell proliferation process in experimental group-
Three albumen on catenin signal paths, are β-catenin, Lef-1 and Cyclin-D respectively, and three hatching egg bletillas are accordingly controlled
Gene β-catenin processed, Lef-1 and Cyclin-D expression quantity increased, and further illustrate that TDNs promotes nerve cord
The propagation of cell.
Embodiment 6TDNs promotes mouse neural stem cells differentiation and its detection of differentiation mechanism
1st, the phenomenon and its mechanism of mouse neural stem cells differentiation are promoted using immunoblotting detection TDNs
(1) inoculating cell suspension (the 100 μ l/ holes) in 6 orifice plates, preculture 24 hours (37 in incubator are placed in by culture plate
DEG C, 5%CO2), then component is dropped to 6% for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% 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%, trained in incubator
Foster 1 hour (37 DEG C, 5%CO2)。
(2) component is used to enter for the dual anti-+ 1%B27 of DMEM/F-12+1% culture medium to cell suspension obtained by step (1)
Row culture, and cell suspension is divided into control group and experimental group, and change culture medium simultaneously daily；And added in experimental group dense
The TDNs for 250nM is spent, the PBS of equivalent is added in control group, then uses holoprotein after culture 1 day, 3 days and 7 days respectively
Extracts kit extracts control group and experiment histone respectively.
(3) SDS-PAGE electrophoresis is carried out respectively to control group obtained by step (2) and experiment histone, its detailed process is such as
Under：Encapsulating → loading → electrophoresis → transferring film → confining liquid shakes 4 DEG C of 1 hour → primary antibody of closing and stays overnight → recovery primary antibody, and TBST is washed
Wash 3 times, each 5-10 minutes → secondary antibody, 1 hour → abandon secondary antibody, TBST was washed 3 times, and each 5-10 minutes → exposure, detection is simultaneously
Data processing is carried out, its result is shown in Fig. 9 (a), Fig. 9 (b), Figure 12 (a) and Figure 12 (b).
Break up as shown in Fig. 9 (a), Fig. 9 (b), Figure 12 (a) and Figure 12 (b), in experimental group correlation destination protein (β-
III-Tubulin expression quantity) is above control group, and breaks up related Notch signal paths compared with control group, in experimental group
On three albumen, be respectively Notch-1, Hes-1, Hes-5 expression quantity reduce, show that TDNs can promote mouse Nerve to do
The differentiation and maturation of cell.
2nd, the phenomenon and mechanism of mouse neural stem cells differentiation are promoted using quantitative fluorescent PCR (Q-PCR) detection TDNs
(1) (1) inoculating cell suspension (the 100 μ l/ holes) in 6 orifice plates, preculture 24 hours in incubator are placed in by culture plate
(37 DEG C, 5%CO2), then component is dropped to for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% by 10%
6%, cultivated in incubator 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, in incubator
It is middle culture 1 hour (37 DEG C, 5%CO2)。
(2) component is used to enter for the dual anti-+ 1%B27 of DMEM/F-12+1% culture medium to cell suspension obtained by step (1)
Row culture, and cell suspension is divided into control group and experimental group, and change culture medium simultaneously daily；And added in experimental group dense
The TDNs for 250nM is spent, the PBS of equivalent is added in control group, is then carried respectively after culture 1 day, 3 days and 7 days using gene
Kit is taken, control group and experimental group gene is extracted respectively, then passes through high-purity total serum IgE rapid extraction kit and reverse transcription again
Kit obtains stable cDNA.
(3)Q-PCR：Per hole add 20 μ l reaction system (2 μ l cDNA, 10 μ l SYBR, 0.8 μ l primers Fs orward,
0.8 μ l primers Reserve, 6.4 μ l ddH2O), upper machine testing, and data processing is carried out, its result is shown in Fig. 9 (c) and Figure 12
(c)。
As shown in Fig. 9 (c) and Figure 12 (c), the target gene (β-III- of correlation are broken up compared with control group, in experimental group
Tubulin expression quantity) is higher, and three eggs broken up compared with control group, in experimental group on related Notch signal paths
In vain, it is respectively Notch-1, Hes-1, Hes-5, the expression quantity of gene Notch-1, Hes-1, Hes-5 corresponding to it reduce, table
Bright TDNs can promote the differentiation and maturation of mouse neural stem cells.
3rd, immunofluorescence technique
(1) the inoculating cell suspension (100 μ l/ holes) in burnt capsule is copolymerized, it is small to be placed in preculture 24 in incubator by culture plate
When (37 DEG C, 5%CO2), then component is dropped to for the serum-concentration in the dual anti-culture medium of DMEM+10% serum+1% by 10%
6%, cultivated in incubator 6 hours (37 DEG C, 5%CO2), the serum-concentration in culture medium is then dropped to 0 by 6%, in incubator
It is middle culture 1 hour (37 DEG C, 5%CO2)。
(2) component is used to enter for the dual anti-+ 1%B27 of DMEM/F-12+1% culture medium to cell suspension obtained by step (1)
Row culture, cell suspension is divided into control group and experimental group, and changes culture medium simultaneously daily；And concentration is added in experimental group
For 250nM TDNs, the PBS of equivalent is added in control group, is cultivated 1 day, 7 days.
(3) culture medium of experimental group and control group is sucked respectively, and PBS is washed 3 times, every time 5 minutes.Again with 4% paraformaldehyde
After fixing 25 minutes, paraformaldehyde is sucked, PBS is washed 3 times, 5 minutes every time, 0.5%Triton-100 processing 20-25 minutes, inhaled
Triton-100, PBS is gone to wash 3 times, every time 5 minutes.Sheep blood serum is handled 1 hour, sucks sheep blood serum, PBS is washed 3 times, every time 5 points
Clock.The processing of primary antibody β-III-Tubulin antibody, 4 DEG C, overnight.Second day, 37 DEG C of rewarmings 0.5 hour reclaimed primary antibody, and PBS washes 3
It is secondary, 5 minutes every time.Two process resistant of fluorescence are carried, lucifuge, 37 DEG C, 1 hour, suck secondary antibody, PBS is washed 3 times, every time 5 minutes.
DAPI processing, lucifuge, 10 minutes, DAPI is sucked, PBS is washed 3 times, every time 5 minutes.10% glycerine approved sample, lucifuge, 4 DEG C of preservations.On
Machine testing.Its result is shown in Figure 10 and Figure 11.
As shown in Figure 10 and Figure 11, behind 1 day and 7 days, compared with control group, the fluorescence intensity (β-III- in experimental group
Tubulin it is) higher, and the form of cell is preferable.
Sequence table
<110>Sichuan University
<120>Application of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60
gaa 63
<210> 2
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
<210> 3
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60
tcc 63
<210> 4
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (10)
- Application of the 1.DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted, wherein, tetrahedral four of DNA is single-stranded Sequence such as SEQ ID NO:Shown in 1-4.
- 2. application of the DNA tetrahedrons according to claim 1 during differentiation and proliferation of neural stem cells is promoted, its feature It is, the DNA tetrahedrons, which promote the propagation of NSC, to be played a role by activating Wnt/ β-catenin signal paths 's；The DNA tetrahedrons promote the differentiation of NSC to be played a role by suppressing Notch signal paths.
- 3. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, promotes the process of nerve stem cell proliferation to include promoting β-catenin, Lef-1 and Cyclin-D albumen expression.
- 4. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, promotes the process of nerve stem cell proliferation to include promoting β-catenin, Lef-1 and Cyclin-D genes expression.
- 5. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, promotes neural stem cell differentiating process to include reducing the expression of Notch-1, Hes-1 and Hes-5 albumen.
- 6. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, promotes neural stem cell differentiating process to include promoting the expression of β-III-Tubulin albumen.
- 7. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, promotes neural stem cell differentiating process to include the expression for suppressing Notch-1, Hes-1 and Hes-5 gene respectively, and Promote the expression of β-III-Tubulin genes.
- 8. application of the DNA tetrahedrons according to claim 2 during differentiation and proliferation of neural stem cells is promoted, its feature It is, concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 50~500nM.
- 9. application of the DNA tetrahedrons according to claim 8 during differentiation and proliferation of neural stem cells is promoted, its feature It is, concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 100~300nM.
- 10. application of the DNA tetrahedrons according to claim 8 during differentiation and proliferation of neural stem cells is promoted, it is special Sign is that concentration of the DNA tetrahedrons during differentiation and proliferation of neural stem cells is promoted is 250nM.
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| PCT/CN2018/094561 WO2019100726A1 (en) | 2017-11-22 | 2018-07-04 | Application of d dna tetrahedron in promoting proliferation and differentiation of neural stem cells |
| CN201811396540.0A CN109806275B (en) | 2017-11-22 | 2018-11-21 | Application of DNA tetrahedron in preparation of nerve repair promoting medicine |
| PCT/CN2018/116847 WO2019101116A1 (en) | 2017-11-22 | 2018-11-22 | Uses of dna tetrahedron for preparing drug for promoting neural repair |
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| CN109646450A (en) * | 2019-01-31 | 2019-04-19 | 四川大学 | Purposes of the DNA tetrahedron in preparation treatment corneal injury drug |
| WO2019101116A1 (en) * | 2017-11-22 | 2019-05-31 | 四川大学 | Uses of dna tetrahedron for preparing drug for promoting neural repair |
| WO2019100726A1 (en) * | 2017-11-22 | 2019-05-31 | 四川大学 | Application of d dna tetrahedron in promoting proliferation and differentiation of neural stem cells |
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| WO2019100726A1 (en) * | 2017-11-22 | 2019-05-31 | 四川大学 | Application of d dna tetrahedron in promoting proliferation and differentiation of neural stem cells |
| CN109646450A (en) * | 2019-01-31 | 2019-04-19 | 四川大学 | Purposes of the DNA tetrahedron in preparation treatment corneal injury drug |
| CN111991412A (en) * | 2020-09-30 | 2020-11-27 | 四川大学 | Medicine for treating Parkinson's disease |
| CN111991412B (en) * | 2020-09-30 | 2021-11-05 | 四川大学 | Medicine for treating Parkinson's disease |
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