WO2019101116A1 - Dna四面体在促神经修复药物制备中的用途 - Google Patents

Dna四面体在促神经修复药物制备中的用途 Download PDF

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WO2019101116A1
WO2019101116A1 PCT/CN2018/116847 CN2018116847W WO2019101116A1 WO 2019101116 A1 WO2019101116 A1 WO 2019101116A1 CN 2018116847 W CN2018116847 W CN 2018116847W WO 2019101116 A1 WO2019101116 A1 WO 2019101116A1
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drug
tdns
neural stem
stem cells
minutes
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林云锋
马文娟
蔡潇潇
邵晓茹
谢雪萍
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Sichuan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the invention relates to the field of nerve repair, and in particular to the use of DNA tetrahedrons in the preparation of nerve prosthetic drugs.
  • neural stem cells NSCs
  • Some researchers have found that some drug molecules (such as prostaglandin E2, tenuigenin, etc.) can enhance the proliferation and differentiation of NSC, but its biocompatibility and utilization are not high, there are limitations in the treatment of nervous system diseases. .
  • TDNs DNA tetrahedrons
  • DNA tetrahedrons are a new type of DNA nanomaterials that currently have extensive research and potential applications in the biomedical field.
  • TDNs are DNA nanomaterials with three-dimensional structure formed by self-assembly of four single-stranded DNA single strands under specific conditions, and the base sequences of four single-stranded DNAs strictly follow the principle of "base complementary pairing", and thus accurate, Cleverly designed.
  • the synthesis method of TDNs is simple, the yield is high, and the tolerance to specific or non-specific nucleases is better than that of ordinary linear DNA, and it has good biocompatibility, biosafety and biodegradability.
  • TDNs have appeared in some research as a drug carrier, but its application in nerve repair has yet to be developed.
  • the present invention aims to provide a new drug that promotes nerve repair.
  • the present invention provides the use of a DNA tetrahedron for the preparation of a medicament, which is a drug for promoting nerve repair.
  • the DNA tetrahedron is assembled into a tetrahedral structure by base-pair complementary pairing of four single-stranded DNAs; each of the ribs of the tetrahedron is a double-stranded DNA structure.
  • the DNA tetrahedron is prepared by heating an aqueous solution containing the four single-stranded DNAs at equal concentrations, so that the hydrogen bonds between the bases are completely broken, maintaining for 5 to 20 minutes, and then rapidly cooling to 0 to 0. 10 ° C, maintained for at least 15 min; the aqueous solution also contained 10 mM Tris-HCl, 50 mM MgCl 2 ; the pH of the aqueous solution before heating was 8.
  • the maintenance time after heating is 10 minutes
  • the specific temperature of the temperature reduction is 4 ° C
  • the retention time after the temperature reduction is 20 minutes.
  • sequences of the four single-stranded DNAs are shown in SEQ ID NOS. 1 to 4, respectively.
  • the drug is a drug that promotes proliferation, differentiation and/or migration of neural stem cells.
  • the drug is a drug that activates the Wnt/ ⁇ -Catenin pathway.
  • the drug is a drug that inhibits the Notch signaling pathway.
  • the drug is a drug that activates the RHOA/ROCK2 signaling pathway.
  • the present invention also provides a drug for promoting nerve repair, which is prepared by using the DNA tetrahedron according to any one of claims 1 to 9 as an active material, together with a pharmaceutically acceptable adjuvant or auxiliary component.
  • the TDNs of the present invention can be ingested in large amounts by mouse neural stem cells, and their uptake rate is much higher than that of single-stranded DNA.
  • the TDNs of the invention can significantly increase the proliferation, differentiation and migration of mouse neural stem cells, and has a good nerve repairing ability.
  • TDNs of the present invention are composed of nucleic acids, their metabolism is not toxic to cells and has good biocompatibility.
  • the TDNs of the present invention can be prepared as a drug for promoting nerve repair.
  • FIG. 1 is a schematic representation of four single-stranded synthetic TDNs.
  • FIG. 2 is a schematic diagram showing the results of TDNs polyacrylamide gel electrophoresis.
  • Figure 3 is a schematic illustration of the results of transmission electron microscopy identification.
  • Figure 4 is a particle size distribution diagram, where a is the particle size distribution of single-stranded DNA and b is the particle size distribution of TDNs.
  • Fig. 5 is a graph showing the results of identifying the undifferentiated state of mouse neural stem cells by immunofluorescence technique.
  • Figure 6 is a graph showing the results of flow cytometry treatment of TDNs uptake by mouse neural stem cells.
  • Figure 7 is a fluorescent trace of mouse neural stem cells.
  • Figure 8 is a graphical representation of the results of the effect of TDNs concentration on the proliferation of mouse neural stem cells.
  • Figure 9 is a graph showing the results of cell cycle changes of mouse neural stem cells under the action of TDNs by flow cytometry.
  • Figure 10a is a blot of ⁇ -catenin, Lef-1 and Cyclin-D in mouse neural stem cells under the action of TDNs.
  • Figure 10b is a bar graph showing the relative intensities of ⁇ -catenin, Lef-1 and Cyclin-D protein blots in mouse neural stem cells under the action of TDNs.
  • Figure 10c is a bar graph showing the relative gene expression levels of ⁇ -catenin, Lef-1 and Cyclin-D in mouse neural stem cells under the action of TDNs.
  • Figure 11a is a Western blot of ⁇ -III-Tubulin in mouse neural stem cells under the action of TDNs.
  • Figure 11b is a bar graph showing the relative intensity of ⁇ -III-Tubulin western blot signal in mouse neural stem cells under the action of TDNs.
  • Figure 11c is a bar graph showing the relative expression levels of ⁇ -III-Tubulin gene in mouse neural stem cells under the action of TDNs.
  • Figure 12a is a blot of the Notch signaling pathway-associated protein in mouse neural stem cells under the action of TDNs.
  • Figure 12b is a bar graph showing the relative strength of the Notch signaling pathway-related Western blot signal in mouse neural stem cells under the action of TDNs.
  • Figure 12c is a graph showing the relative expression of the Notch signaling pathway-related genes in mouse neural stem cells under the action of TDNs.
  • Figure 13 is an immunofluorescence map of ⁇ -III-Tubulin protein 1 day after TDNs treatment.
  • Figure 14 is an immunofluorescence map of ⁇ -III-Tubulin protein after 7 days of TDNs treatment.
  • Figure 15 is a graph showing the results of a mouse neural stem cell scratch test.
  • Figure 16 is a graph showing the results of a mouse neural stem cell Transwell experiment.
  • Figure 17a is a diagram showing the RhoA amplified band electrophoresis of mouse neural stem cells under the action of TDNs.
  • Figure 17b is a graph showing the fold change of RhoA gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 17c is a relative quantitative diagram of RhoA gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 17d is a RhoA protein imprinting diagram of mouse neural stem cells under the action of TDNs.
  • Figure 17e is a bar graph showing the relative strength of RhoA Western blotting signals in mouse neural stem cells under the action of TDNs.
  • Figure 17f is an immunofluorescence map of RhoA protein in mouse neural stem cells under the action of TDNs.
  • Figure 17g is a graph showing the intensity of RhoA protein immunofluorescence signal in mouse neural stem cells under the action of TDNs.
  • Figure 18a is a magnetic resonance diagram of the Rock2 amplified band in mouse neural stem cells under the action of TDNs.
  • Figure 18b is a graph showing the fold change of Rock2 gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 18c is a relative quantitative diagram of Rock2 gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 18d is a map of Rock2 protein in mouse neural stem cells under the action of TDNs.
  • Figure 18e is a bar graph showing the relative strength of the Rock2 Western blot signal in mouse neural stem cells under the action of TDNs.
  • Figure 18f is a diagram showing the immunofluorescence of Rock2 protein in mouse neural stem cells under the action of TDNs.
  • Figure 18g is a graph showing the intensity of Rock2 protein immunofluorescence signal in mouse neural stem cells under the action of TDNs.
  • Figure 19a is a diagram showing the electrophoresis of vinculin amplification bands in mouse neural stem cells under the action of TDNs.
  • Figure 19b is a graph showing the fold change of vinculin gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 19c is a relative quantitative diagram of vinculin gene expression in mouse neural stem cells under the action of TDNs.
  • Figure 19d is a picogram of the vinculin protein in mouse neural stem cells under the action of TDNs.
  • Figure 19e is a bar graph showing the relative strength of the vinculin protein imprinting signal in mouse neural stem cells under the action of TDNs.
  • Figure 19f is an immunofluorescence map of vinculin protein in mouse neural stem cells under the action of TDNs.
  • Figure 19g is a graph showing the immunofluorescence signal intensity of vinculin protein in mouse neural stem cells under the action of TDNs.
  • the four DNA single strands (S1, S2, S3, S4) were dissolved in the TM buffer solution at the same final concentration, wherein the solute of the TM buffer solution was Tris-HCl and MgCl2, and their concentrations were 10 mM and 50 mM, respectively.
  • the pH of the solution was adjusted to 8.0.
  • the mixture was vortexed, mixed, centrifuged, and placed in a PCR machine. The temperature was rapidly raised to 95 ° C for 10 to 15 minutes, and then cooled to 4 ° C for 20 minutes. That is, a tetrahedral structure as shown in FIG. 1 is synthesized.
  • the sequence of the DNA single strand is shown in Table 1.
  • polyacrylamide gel 40 wt% acrylamide solution, 10 ⁇ TAE, 10 wt% ammonium sulfate solution, distilled water, tetramethylethylenediamine according to the volume of 1-2:1:1:1:1 The mixture is made into a polyacrylamide gel.
  • lanes 1-8 are Marker, 1-S1, 2-S2, 3-S3, 4-S4, 5-S1+S2, 6-S1+S2+S3, 7-S1+ S2+S3+S4 (7-TDNs).
  • the sizes of the ss DNA single strands S1, S2, S3, and S4 are about 60 bp, 50 bp, 50 bp, and 50 bp, respectively, and the size of the TDNs prepared by the present invention is about 210 bp.
  • results As shown in Fig. 3, the shape of TDNs was approximately triangular in shape under transmission electron microscopy, and the particle size ranged from 10 to 15 nM.
  • the circle is labeled as a polymer.
  • mice neural stem cells used in the experimental examples are all NE-4C cells.
  • the cell suspension was inoculated into a confocal dish and placed in an incubator for 24 hours.
  • the medium containing DMEM + 10% serum + 1% double antibody was aspirated, and washed three times with PBS for 5 minutes each time;
  • the test results are shown in Fig. 5.
  • the mouse neural stem cells showed positive for nestin antibody, indicating that the cells were still in an undifferentiated state and could be used for subsequent experiments.
  • mice neural stem cells of the same undifferentiated state in Experimental Example 1.
  • This experimental example measures the ability of neural stem cells to uptake TDNs.
  • the negative control group did not do any treatment.
  • the positive control group was supplemented with Cyn-modified single-stranded DNA S1 at a concentration of 250 nM.
  • the experimental group was supplemented with Cyn-modified TDNs at a concentration of 250 nM and cultured in an incubator for 12 hours (37 ° C, 5%). (v/v)CO2).
  • control group was added with Cy5 modified S1 at a concentration of 250 nM, and the experimental group was added with Cyn-modified TDNs at a concentration of 250 nM, and cultured in an incubator for 12 hours (37 ° C, 5% (v/v) CO 2 ).
  • RESULTS As shown in Figures 7-a and 7-b, ssDNA was less taken up by neural stem cells; neural stem cells took more uptake of TDNs, and most of the TDNs that entered the cells accumulated in the cytoplasm of the cells and entered the nucleus less. .
  • TDNs can be well taken up by neural stem cells.
  • the cultured cell suspension was divided into a control group and an experimental group, and TDNs were added to the experimental group, and an equal amount of PBS was added to the control group, followed by incubation in an incubator for 24 hours (37 ° C, 5% CO 2 ). ).
  • TDNs As shown in Fig. 8, when the concentration of TDNs was 62.5 nM, 125 nM, and 250 nM, the proliferation process of mouse neural stem cells in the experimental group was promoted to a certain extent by TDNs, and 250 nM was the most. Good concentration indicates that TDNs have the effect of promoting the proliferation of mouse neural stem cells.
  • the cultured cell suspension was divided into a control group and an experimental group, and TDNs were added to the experimental group, and an equal amount of PBS was added to the control group, followed by incubation in an incubator for 24 hours (37 ° C, 5% CO 2 ). ).
  • control cells and the experimental group cells were separately digested with 0.25% trypsin, placed in a 15 ml centrifuge tube (2000 rpm, 5 minutes), the supernatant was discarded, washed with PBS, centrifuged (2000 rpm, 5 minutes), and then added with ice. 500 ⁇ l of ethanol was fixed, and the cells were fixed at 4 ° C overnight. The next day, PBS was added to centrifuge, the supernatant was discarded, washed with PBS, centrifuged, and the supernatant was discarded. Then, 100 ⁇ l of RNase was added, and a 37 ° C water bath was added for 30 minutes. 400 ⁇ l of PI was added and mixed. Protected from light at 4 ° C for 30 minutes. The cells were transferred to a flow tube, detected by the machine, and analyzed by data. The results are shown in Fig. 9.
  • the number of cells in the S phase (DNA synthesis phase) in the experimental group was significantly increased, indicating that TDNs changed the cell cycle of the neural stem cells and promoted its proliferation.
  • TDNs can promote the proliferation of neural stem cells.
  • the Wnt/ ⁇ -catenin signaling pathway is an important pathway for cell cycle regulation, and its activation can significantly promote stem cell proliferation. This experiment was to detect the expression of Wnt/ ⁇ -catenin signaling pathway-related genes in NSCs treated with TDNs.
  • the brief detection procedure is as follows: filling ⁇ loading ⁇ electrophoresis ⁇ transfer membrane ⁇ blocking liquid shaking for 1 hour ⁇ primary antibody 4°C overnight ⁇ recovering primary antibody ⁇ TBST washing 3 times (5-10 minutes each time) ⁇ secondary antibody incubation 1 Hours ⁇ Discard secondary antibody, TBST wash 3 times (5-10 minutes each time) ⁇ Exposure.
  • RNA of the experimental group and the control group were extracted, and the cDNA was obtained by the reverse transcription kit, and then the fluorescent method was used to detect the Wnt/ ⁇ -catenin signaling pathway related to the neural stem cell proliferation process by fluorescent quantitative PCR.
  • TDNs promote the proliferation of neural stem cells by up-regulating the expression of three genes on the Wnt/ ⁇ -catenin signaling pathway.
  • the ⁇ -III-Tubulin protein is a neuronal marker, and when the neural stem cells differentiate into neurons, the expression of ⁇ -III-Tubulin is up-regulated.
  • the Notch signaling pathway plays an important role in the development of the nervous system, which manifests itself as an inhibitory state during adult neural stem cell differentiation.
  • the expression of ⁇ -III-Tubulin and Notch signaling pathway-related genes was examined to demonstrate the role of TDNs in promoting differentiation of NSCs.
  • the brief detection procedure is as follows: filling ⁇ loading ⁇ electrophoresis ⁇ transfer membrane ⁇ blocking liquid shaking for 1 hour ⁇ primary antibody 4°C overnight ⁇ recovering primary antibody ⁇ TBST washing 3 times (5-10 minutes each time) ⁇ secondary antibody incubation 1 Hours ⁇ Discard secondary antibody, TBST wash 3 times (5-10 minutes each time) ⁇ Exposure.
  • the expression levels of the marker protein ( ⁇ -III-Tubulin) of neural stem cell differentiation in the experimental group were higher than those in the control group, and compared with the control group, the differentiation in the experimental group was correlated.
  • the expression levels of Notch-1, Hes-1 and Hes-5 were decreased in the three proteins of Notch signaling pathway, indicating that TDNs can promote the differentiation and maturation of mouse neural stem cells.
  • RNA of the experimental group and the control group were extracted, the cDNA was obtained by the reverse transcription kit, and the protein related to neural stem cell differentiation was detected by fluorescent quantitative PCR using the dye method: ⁇ - III-Tubulin, Notch-1, Hes-1 and Hes-5.
  • the expression level of the differentiation-related gene ( ⁇ -III-Tubulin) in the experimental group was higher, and the differentiation-related Notch signal in the experimental group was compared with the control group.
  • the three proteins in the pathway are Notch-1, Hes-1, Hes-5, and the corresponding expressions of the genes Notch-1, Hes-1 and Hes-5 are decreased, indicating that TDNs can promote mouse neural stem cells.
  • the differentiation is mature, and its differentiation is related to the inhibition of the Notch pathway.
  • the temperature was rewarmed at 37 ° C for 0.5 hours, and the primary antibody was recovered and washed three times with PBS for 5 minutes each time.
  • Fluorescent secondary antibody treatment protected from light, 37 ° C, 1 hour, the secondary antibody was aspirated, washed three times with PBS for 5 minutes each time.
  • DAPI treatment protected from light, 10 minutes, aspirate DAPI, wash 3 times with PBS for 5 minutes each time.
  • 10% glycerol seal protected from light, stored at 4 ° C. Check on the machine.
  • TDNs promote the differentiation of neural stem cells by inhibiting the Notch signaling pathway.
  • the control group did not add TDNs.
  • the experimental group was added TDNs of the corresponding concentration and cultured in an incubator (37 ° C, 5% (v/v) CO 2 ). Photographs were taken under light microscope at 0, 12, and 24 hours, respectively, and scratch changes were recorded.
  • a. Transwell (0.8 ⁇ m pore size) chamber was placed in a 6-well plate, and the mouse neural stem cell suspension was inoculated in the chamber.
  • the culture plate was pre-incubated in the incubator for 24 hours (37 ° C, 5% (v/v). ) CO2); then reduce the serum concentration in the medium from 10% to 6%, continue to culture for 6 hours in the incubator (37 ° C, 5% (v / v) CO2); then the serum concentration in the medium 6% was reduced to 0 and incubation was continued for 1 hour in the incubator (37 ° C, 5% (v/v) CO 2 ).
  • the control group was not added with TDNs.
  • the experimental group was added with TDNs at a concentration of 250 nM and cultured in an incubator for 12 hours (37 ° C, 5% (v/v) CO 2 ).
  • the scratch test and the Transwell experiment demonstrated that the TDNs of the present invention do have a good promoting effect on the lateral and longitudinal migration of mouse neural stem cells.
  • TDNs were not added to the control group, and TDNs at a concentration of 250 nM were added to the experimental group and cultured in an incubator.
  • the 6-well plate in the first section of this experimental example was replaced with a confocal small dish for sample processing.
  • the medium was aspirated, washed three times with PBS for 5 minutes each time; after fixing with 4 wt% paraformaldehyde for 25 minutes, paraformaldehyde was aspirated, washed three times with PBS for 5 minutes each time; 0.5% again Triton-100 was treated for 20-25 minutes, and Triton-100 was aspirated, washed three times with PBS for 5 minutes each time; then treated with sheep serum for 1 hour, and the serum was removed by PBS and washed three times for 5 minutes each time.
  • the DNA tetrahedron of the present invention is easily taken up by nerve cells, can significantly increase the proliferation, differentiation and migration of mouse neural stem cells, has a good nerve-promoting ability, and has good biocompatibility.
  • the DNA tetrahedron of the present invention can be used for the preparation of a drug for promoting nerve repair.

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Abstract

提供了DNA四面体在促神经修复药物制备中的用途。该四面体由四条单链DNA通过碱基互补配对方式组装成四面体结构，其每条棱都是双链DNA结构。还提供了使用DNA四面体作为活性物质制备的促神经修复的药物。

Description

DNA四面体在促神经修复药物制备中的用途 技术领域
本发明涉及神经修复领域，具体涉及DNA四面体在促神经修复药物制备中的用途。
背景技术
目前，神经系统疾病，诸如神经退行性疾病、神经损伤等，是医学界难题。因为神经细胞的自我修复和更新很难，神经干细胞(neural stem cells,NSCs)治疗越来越备受关注。尽管神经干细胞治疗的临床应用还有一些技术难题需要攻克，目前最关键的还在于植入细胞自身能不能增殖、分化和迁移。
已经有研究者发现，一些药物分子(例如前列腺素E2、tenuigenin等)能够提升NSC的增殖和分化能力，然而其生物相容性和利用度都不高，在神经系统疾病的治疗上存在局限性。
DNA四面体(TDNs)是一种新型的DNA纳米材料，目前在生物医学领域有着十分广泛的研究和巨大的潜在运用前景。TDNs是由四条单链DNA单链在特定的条件下自组装形成的具有三维结构的DNA纳米材料，而四条单链DNA的碱基序列是严格遵循了“碱基互补配对原则”，进而精确、巧妙地设计出来的。TDNs合成方法简便、产率较高，对特异性或非特异性核酸酶的耐受性均较普通的线性DNA好，且具有良好的生物相容性、生物安全性和生物可降解性。
目前TDNs已经以药物载体的角色出现在一些研究中，但其在神经修复中的应用尚有待开发。
发明内容
本发明旨在提供一种新的能促进神经修复的药物。
为了解决这一技术问题，本发明提供了DNA四面体在制备药物中的用途，所述药物是促神经修复的药物。
前述的用途中，所述DNA四面体由四条单链DNA通过碱基互补配对方式组装成四面体结构；所述四面体的每条棱都是双链DNA结构。
前述的用途中，所述DNA四面体的制备方法是：将含有等浓度所述四条单链DNA的水溶液加热，使得碱基间氢键完全断开，维持5～20min，然后快速降温到0～10℃，保持至少15min；所述水溶液中还含有10mM Tris-HCl、50mM MgCl2；所述水溶液加热前的pH值为8。
前述的用途中，所述加热后维持时间为10min，所述降温的具体温度为4℃，所述降温后保持时间为20min。
前述的用途中，所述四条单链DNA的序列分别如SEQ ID NO.1～4所示。
前述的用途中，所述药物是促进神经干细胞的增殖、分化和/或迁移的药物。
进一步地，所述药物是激活Wnt/β-Catenin通路的药物。
进一步地，所述药物是抑制Notch信号通路的药物。
进一步地，所述药物是激活RHOA/ROCK2信号通路的药物。
本发明还提供了一种促神经修复的药物，其特征在于，它是使用权利要求1～9所述DNA四面体作为活性物质，加上药学上可接受的辅料或辅助性成分制备而成。
本发明具有以下有益效果：
本发明的TDNs可以被小鼠神经干细胞大量摄取，其摄取率大大高于单链DNA。
本发明的TDNs可以明显增加小鼠神经干细胞的增殖、分化和迁移，具有很好的促神经修复能力。
本发明的TDNs由于是核酸构成的，其代谢不会对细胞产生毒性，具有良好的生物相容性。
本发明的TDNs可以制备成为促神经修复的药物。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。下面通过具体实施方式对本发明做进一步详细说明，但是并不是对本发明的限制，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
附图说明
图1为四条单链合成TDNs的示意图。
图2为TDNs聚丙烯酰胺凝胶电泳结果示意图。
图3为透射电镜鉴定结果的示意图。
图4粒径分布图，其中a为单链DNA的粒径分布图，b为TDNs的粒径分布图。
图5为采用免疫荧光技术对小鼠神经干细胞的未分化状态进行鉴定的结 果示意图。
图6为小鼠神经干细胞对TDNs摄取情况流式细胞术处理后的结果图。
图7为小鼠神经干细胞荧光示踪图。
图8为TDNs浓度对小鼠神经干细胞增殖影响的检测结果示意图。
图9为采用流式细胞术检测小鼠神经干细胞在TDNs作用下细胞周期变化的结果示意图。
图10a为TDNs作用下小鼠神经干细胞中β-catenin、Lef-1和Cyclin-D印迹图。
图10b为TDNs作用下小鼠神经干细胞中β-catenin、Lef-1和Cyclin-D蛋白印迹信号相对强度柱形图。
图10c为TDNs作用下小鼠神经干细胞中β-catenin、Lef-1和Cyclin-D相对基因表达量柱形图。
图11a为TDNs作用下小鼠神经干细胞中β-III-Tubulin蛋白印迹图。
图11b为TDNs作用下小鼠神经干细胞中β-III-Tubulin蛋白印迹信号相对强度柱形图。
图11c为TDNs作用下小鼠神经干细胞中β-III-Tubulin基因的相对表达量柱形图。
图12a为TDNs作用下小鼠神经干细胞中Notch信号通路相关蛋白的印迹图。
图12b为TDNs作用下小鼠神经干细胞中Notch信号通路相关蛋白印迹信号相对强弱柱形图。
图12c为TDNs作用下小鼠神经干细胞中Notch信号通路相关基因的相对表达强弱图。
图13为TDNs处理1天后β-III-Tubulin蛋白免疫荧光图。
图14为TDNs处理7天后β-III-Tubulin蛋白免疫荧光图。
图15为小鼠神经干细胞划痕实验结果图。
图16为小鼠神经干细胞Transwell实验结果图。
图17a为TDNs作用下小鼠神经干细胞中RhoA扩增条带电泳图。
图17b为TDNs作用下小鼠神经干细胞中RhoA基因表达倍数变化图。
图17c为TDNs作用下小鼠神经干细胞中RhoA基因表达相对定量图。
图17d为TDNs作用下小鼠神经干细胞中RhoA蛋白印记图。
图17e为TDNs作用下小鼠神经干细胞中RhoA蛋白印迹信号相对强弱柱形图。
图17f为TDNs作用下小鼠神经干细胞中RhoA蛋白免疫荧光图。
图17g为TDNs作用下小鼠神经干细胞中RhoA蛋白免疫荧光信号强度 图。
图18a为TDNs作用下小鼠神经干细胞中Rock2扩增条带电泳图。
图18b为TDNs作用下小鼠神经干细胞中Rock2基因表达倍数变化图。
图18c为TDNs作用下小鼠神经干细胞中Rock2基因表达相对定量图。
图18d为TDNs作用下小鼠神经干细胞中Rock2蛋白印记图。
图18e为TDNs作用下小鼠神经干细胞中Rock2蛋白印迹信号相对强弱柱形图。
图18f为TDNs作用下小鼠神经干细胞中Rock2蛋白免疫荧光图。
图18g为TDNs作用下小鼠神经干细胞中Rock2蛋白免疫荧光信号强度图。
图19a为TDNs作用下小鼠神经干细胞中vinculin扩增条带电泳图。
图19b为TDNs作用下小鼠神经干细胞中vinculin基因表达倍数变化图。
图19c为TDNs作用下小鼠神经干细胞中vinculin基因表达相对定量图。
图19d为TDNs作用下小鼠神经干细胞中vinculin蛋白印记图。
图19e为TDNs作用下小鼠神经干细胞中vinculin蛋白印迹信号相对强弱柱形图。
图19f为TDNs作用下小鼠神经干细胞中vinculin蛋白免疫荧光图。
图19g为TDNs作用下小鼠神经干细胞中vinculin蛋白免疫荧光信号强度图。
具体实施方式
实施例TDNs的合成
1.合成
将四种DNA单链(S1、S2、S3、S4)以相同终浓度溶解到TM buffer溶液中，其中，TM buffer溶液的溶质为Tris-HCl和MgCl2，它们的浓度分别为10mM和50mM，并调节溶液的pH值为8.0。然后将混合物经过涡旋、混匀、离心后置于PCR仪内，将温度迅速升高到95℃稳定10～15min，再冷却至4℃稳定20min。即合成如图1所示的四面体结构。
所述DNA单链的序列如表1所示。
表1 TDNs单链的序列
名称 SEQ ID NO. 序列(5’→3’)
S1 1 atttatcacccgccatagtagacgtatcaccaggcagttgagacgaacattcctaagtctgaa
S2 2 acatgcgagggtccaataccgacgattacagcttgctacacgattcagacttaggaatgttcg
S3 3 actactatggcgggtgataaaacgtgtagcaagctgtaatcgacgggaagagcatgcccatcc
S4 4 acggtattggaccctcgcatgactcaactgcctggtgatacgaggatgggcatgctcttcccg
2.鉴定
2.1凝胶电泳
a、制备聚丙烯酰胺凝胶：将40wt％的丙烯酰胺溶液、10×TAE、10wt％的硫酸铵溶液、蒸馏水、四甲基乙二胺按照1～2:1:1:1:1的体积比混合，制成聚丙烯酰氨凝胶。
b、加样及电泳：将1ul 6×loading buffer分别与5ul的样品和marker混合均匀，然后分别加入对应的电泳槽中。在冰浴、恒压100V的条件下，电泳处理1小时。
c、GelRed染色及曝光：将聚丙烯酰氨凝胶放置于GelRed和蒸馏水按照1:50的比例混合的混合液液中，避光，摇床15～25分钟。曝光。
结果：如图2所示，泳道1～8分别为Marker，1-S1，2-S2，3-S3，4-S4，5-S1+S2，6-S1+S2+S3，7-S1+S2+S3+S4(7-TDNs)。从图中可以看出，ss DNA单链S1、S2、S3、S4的大小分别约为60bp、50bp、50bp、50bp，则本发明制备出的TDNs的大小约为210bp。
2.2透射电镜
取适量样本液于金属片上，红外下照射5～10分钟使其干燥，上机检测。
结果：如图3所示，TDNs的形状在透射电镜下呈近似三角形形状，粒径大小在10～15nM范围内。圆圈标注的为多聚物。
2.3动态光散射
取适量ssDNA和TDNs溶液，放置于动态光散射检测仪中，进行检测。
结果：如图4-a，ssDNA的粒径约为48.255nm。如图4-b，TDNs的粒径约为16.801nm。
为了证明TDNs确实有助于神经修复，以下将用实验例的方式进一步说明，实验例中用到的小鼠神经干细胞均为NE-4C细胞。
实验例1 神经干细胞的鉴定
采用免疫荧光技术对小鼠神经干细胞进行鉴定，一次包括以下步骤：
A、将细胞悬浮液接种于共聚焦小皿中，放置于孵箱中培养24小时。吸去组分为DMEM+10％血清+1％双抗的培养基，PBS洗3次，每次5分钟；
B、4％多聚甲醛固定25分钟后，吸去多聚甲醛，PBS洗3次，每次5分钟；
C、0.5％Triton-100处理20-25分钟，吸去Triton-100，PBS洗3次，每次5分钟；
D、羊血清处理1小时，吸去羊血清，PBS洗3次，每次5分钟；
E、一抗(抗nestin抗体)处理，4℃，过夜。第二天，37℃复温0.5小时，回收一抗，PBS洗3次，每次5分钟。携带荧光的二抗处理，避光，37℃，1小时，吸去二抗，PBS洗3次，每次5分钟；
E、鬼笔环肽处理，避光，10-30分钟，吸去鬼笔环肽，PBS洗3次，每次5分钟；
F、DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟。10％甘油封样，避光，4℃保存。上机检测。
检测结果如图5所示，小鼠神经干细胞显示nestin抗体阳性，表示细胞仍处于未分化的状态，可用于进行后续实验。
后面的实验例所涉及的“细胞”均为实验例1中同种未分化状态的小鼠神经干细胞。
实验例2 神经干细胞摄取实验
能被细胞大量摄取，是大多数药物起到治疗作用的前提。本实验例检测神经干细胞对TDNs的摄取能力。
(1)流式细胞术
a、在6孔板接种细胞悬液，先在孵箱预培养24小时(37℃，5％(v/v)CO2)；然后将培养基中的血清浓度由10％降到6％，于孵箱继续培养6小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由6％降到0，于孵箱继续培养1小时(37℃，5％(v/v)CO2)。
b、阴性对照组不做任何处理，阳性对照组加入浓度为250nM的Cy5修饰的单链DNA S1，实验组加入浓度为250nM的Cy5修饰的TDNs，于孵箱培养12小时(37℃，5％(v/v)CO2)。
c、将细胞收集起来，1000r/min下离心5min，PBS重悬，重复三次，上机检测。
结果：如图6-a和6-b，神经干细胞对TNDs的摄取明显多于对ssDNA的摄取。(请贵方解释图6-a)
上述实验证明，TNDs能够很好的被小鼠神经干细胞摄取。
(2)荧光示踪技术
a、在共聚焦小皿中接种小鼠神经干细胞悬液，先在孵箱预培养24小时(37℃，5％(v/v)CO2)；然后将培养基中的血清浓度由10％降到6％，于孵箱继续培养6小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由6％降到0，于孵箱继续培养1小时(37℃，5％(v/v)CO2)。
b、对照组加入浓度为250nM的Cy5修饰的S1，实验组加入浓度为250nM的Cy5修饰的TDNs，于孵箱培养12小时(37℃，5％(v/v)CO2)。
c、吸去培养基，用PBS清洗三次，每次5分钟；然后用4wt％的多聚甲醛固定25分钟，吸去多聚甲醛，用PBS清洗三次，每次5分钟；再用鬼笔环肽处理，避光10～30分钟，吸去鬼笔环肽，用PBS清洗三次，每次5分钟；然后用DAPI处理，避光10分钟，吸去DAPI，用PBS清洗三次，每次5分钟；再用10wt％的甘油封样，避光，4℃保存，并上机检测。
结果：如图7-a和7-b，ssDNA被神经干细胞摄取的较少；神经干细胞对TDNs摄取的较多，且进入细胞的TDNs大部分聚集在细胞的胞浆内，进入细胞核的较少。
表明TDNs可以很好地被神经干细胞所摄取。
实验例3 TDNs促进小鼠神经干细胞的增殖及检测
1.CCK-8检测增殖
(1)在96孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将培养的细胞悬液分为对照组和实验组，并在实验组中加入TDNs，对照组中加入等量的PBS，然后于孵箱中培养24小时(37℃，5％CO 2)。
(3)分别向实验组和对照组中加入CCK-8溶液(10μl/孔)，然后于孵箱中孵育1～4h(37℃，5％CO 2)，再在450nm处检测每孔的吸光度，其结果见图8。
如图8所示，当TDNs浓度为62.5nM、125nM、250nM时，与对照组相比，实验组中小鼠神经干细胞的增殖过程，在一定程度上都受到了TDNs的促进作用，且250nM是最佳浓度，表明TDNs具有促进小鼠神经干细胞增殖的作用。
2、采用流式细胞术检测细胞增殖
(1)在25ml培养瓶中接种细胞悬液，将培养瓶置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培 养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将培养的细胞悬液分为对照组和实验组，并在实验组中加入TDNs，对照组中加入等量的PBS，然后于孵箱中培养24小时(37℃，5％CO 2)。
(3)使用0.25％胰蛋白酶分别消化收集对照组细胞和实验组细胞，置于15ml离心管中(2000rpm、5分钟)，弃上清，PBS洗涤，离心(2000rpm、5分钟)，再加入冰乙醇500μl固定细胞，4℃过夜，第二天加入PBS离心，弃上清，再加入PBS洗涤，离心，弃上清，然后加入100μl RNase，37℃水浴，30分钟，加入400μl PI染色混匀，4℃避光，30分钟。将细胞转移至流式管中，上机检测，并进行数据分析，其结果见图9。
如图9所示，与对照组相比，实验组中处于S期(DNA合成期)的细胞数目明显增加，说明TDNs改变了神经干细胞的细胞周期，具有促进其增殖的作用。
综上，TDNs可以促进神经干细胞增殖。
实验例4 Wnt/β-catenin信号通路相关基因表达检测
Wnt/β-catenin信号通路是细胞周期调控的重要通路，它的激活能显著促进干细胞的增殖。本实验例检测TDNs处理下，NSC的Wnt/β-catenin信号通路相关基因的表达情况。
1.样品处理
(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM+1％双抗的培养基对步骤(1)所得细胞悬液进行培养，将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，同时，对照组中加入等量的PBS。
2.蛋白检测
在实验组处理24h后，提取实验组、对照组的全蛋白，再通过Western blot检测与神经干细胞增殖过程相关的Wnt/β-catenin信号通路上的三个蛋白：β-catenin、Lef-1和Cyclin-D。
简要检测流程如下：灌胶→上样→电泳→转膜→封闭液摇动封闭1小时→一抗4℃过夜→回收一抗→TBST洗涤3次(每次5-10分钟)→二抗孵育1小时→弃二抗、TBST洗涤3次(每次5-10分钟)→曝光。
如图10a和图10b所示，小鼠神经干细胞经TDNs处理24h后，β-catenin、Lef-1和Cyclin-D蛋白含量要显著高于对照组。
3.基因表达检测
在实验组处理24h后，提取实验组、对照组的RNA，通过逆转录试剂盒获得cDNA，再使用染料法进行荧光定量PCR检测与神经干细胞增殖过程相关的Wnt/β-catenin信号通路上的三个基因：β-catenin、Lef-1和Cyclin-D。
如图10c所示，小鼠神经干细胞TDNs处理24h后，β-catenin、Lef-1和Cyclin-D基因表达量要显著高于对照组。
本实验例表明，TDNs通过上调Wnt/β-catenin信号通路上的三个基因的表达来促进神经干细胞的增殖。
实验例5 β-III-Tubulin及Notch信号通路相关基因表达检测
β-III-Tubulin蛋白是一种神经元标记物，当神经干细胞分化成神经元时，β-III-Tubulin表达就会上调。Notch信号通路在神经系统发育过程扮演重要角色，其在成体神经干细胞分化过程中表现为抑制状态。本实验例通过检测β-III-Tubulin及Notch信号通路相关基因的表达来说明TDNs对NSC的促分化作用。
1.样品处理
(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM+1％双抗的培养基对步骤(1)所得细胞悬液进行培养，将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，同时，对照组中加入等量的PBS。
2.蛋白检测
在实验组分别处理1天、3天和7天后，提取实验组、对照组的全蛋白，再通过Western blot检测与神经干细胞分化相关的蛋白：β-III-Tubulin、Notch-1、Hes-1和Hes-5。
简要检测流程如下：灌胶→上样→电泳→转膜→封闭液摇动封闭1小时→一抗4℃过夜→回收一抗→TBST洗涤3次(每次5-10分钟)→二抗孵育1小时→弃二抗、TBST洗涤3次(每次5-10分钟)→曝光。
如图11a、11b、12a和12b所示，实验组中神经干细胞分化的标记物蛋白(β-III-Tubulin)的表达量均高于对照组，且与对照组相比，实验组中分化相关Notch信号通路上的三个蛋白，分别是Notch-1、Hes-1、Hes-5的表达量降低，表明TDNs能够促进小鼠神经干细胞的分化成熟。
3.基因表达检测
在实验组分别处理1天、3天和7天后，提取实验组、对照组的RNA，通 过逆转录试剂盒获得cDNA，再使用染料法进行荧光定量PCR检测与神经干细胞分化相关的蛋白：β-III-Tubulin、Notch-1、Hes-1和Hes-5。
如图11c和12c所示，与对照组相比，实验组中分化相关的目的基因(β-III-Tubulin)的表达量均较高，且与对照组相比，实验组中分化相关Notch信号通路上的三个蛋白，分别是Notch-1、Hes-1、Hes-5，其所对应的基因Notch-1、Hes-1、Hes-5的表达量降低，表明TDNs能够促进小鼠神经干细胞的分化成熟，其分化与Notch通路的抑制有关。
4.免疫荧光技术
(1)按照本实验例第1节的方法处理样品，仅将6孔板换成共聚焦小皿。
(2)TDNs处理1天和7天后，分别吸去实验组和对照组的培养基，PBS洗3次，每次5分钟。再用4％多聚甲醛固定25分钟后，吸去多聚甲醛，PBS洗3次，每次5分钟，0.5％Triton-100处理20-25分钟，吸去Triton-100，PBS洗3次，每次5分钟。羊血清处理1小时，吸去羊血清，PBS洗3次，每次5分钟。一抗β-III-Tubulin抗体处理，4℃，过夜。第二天，37℃复温0.5小时，回收一抗，PBS洗3次，每次5分钟。携带荧光的二抗处理，避光，37℃，1小时，吸去二抗，PBS洗3次，每次5分钟。DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟。10％甘油封样，避光，4℃保存。上机检测。
结果：如图13、14所示，于1天和7天后，与对照组相比，实验组中的荧光强度(β-III-Tubulin)较高，且细胞的形态更接近神经元。
综上，TDNs通过抑制Notch信号通路促进神经干细胞的分化。
实施例6 TDNs促进小鼠神经干细胞的迁移
1.划痕实验
a、在6孔板中接种小鼠神经干细胞悬液，先将培养板在孵箱预培养24小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由10％降到6％，于孵箱继续培养6小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由6％降到0，于孵箱继续培养1小时(37℃，5％(v/v)CO2)；然后使用无菌枪尖在培养板中，将单层细胞沿着直线做划痕，并用PBS洗三次。
b、对照组不加入TDNs，实验组加入对应浓度的TDNs，于孵箱培养(37℃，5％(v/v)CO2)。分别于0、12、24小时后于光镜下拍照，记录划痕变化。
c、结果：如图15，与对照组相比，TDNs明显促进小鼠神经干细胞的横线迁移。
2.Transwell实验
a、将Transwell(孔径为0.8μm)小室放置于6孔板中，在小室中接种小 鼠神经干细胞悬液，先将培养板在孵箱预培养24小时(37℃，5％(v/v)CO2)；然后将培养基中的血清浓度由10％降到6％，于孵箱继续培养6小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由6％降到0，于孵箱继续培养1小时(37℃，5％(v/v)CO2)。
b、对照组不加入TDNs，实验组加入浓度为250nM的TDNs，于孵箱培养12小时(37℃，5％(v/v)CO2)。
c、吸去培养基，用PBS清洗三次，每次5分钟；然后用4wt％的多聚甲醛固定25分钟后，吸去多聚甲醛，用PBS清洗三次，每次5分钟；再用DAPI处理，避光10分钟，吸去DAPI，用PBS清洗三次，每次5分钟；避光，4℃保存，并上机检测。
d、结果：如图16，与对照组相比，实验组中穿过Transwell小室的细胞明显增加，即TDNs促进了神经干细胞的垂直向迁移。
划痕实验和Transwell实验，证明了本发明的TDNs对小鼠神经干细胞的横向和纵向迁移确实具有良好的促进作用。
实施例7 RHOA/ROCK2信号通路相关蛋白的检测
1.样品处理
(1)在6孔板中接种小鼠神经干细胞悬液，先将培养板在孵箱预培养24小时(37℃，5％(v/v)CO2)；然后将培养基中的血清浓度由10％降到6％，于孵箱继续培养6小时(37℃，5％(v/v)CO2)；再将培养基中的血清浓度由6％降到0，于孵箱继续培养1小时(37℃，5％(v/v)CO2)。
(2)对照组不加入TDNs，实验组加入浓度为250nM的TDNs，于孵箱培养。
2.荧光定量PCR检测
TDNs处理24h后，提取实验组和对照组的总RNA，反转录后，进行荧光定量PCR反应，检测RhoA、Rock2、Vinculin的表达。
结果：如图9a～9c、图10a～10c、图11a～11c所示，在半定量PCR及定量PCR中，与对照组相比，实验组中迁移相关RHOA/ROCK2信号通路上的三个蛋白，分别是RhoA、Rock2、Vinculin，其对应的基因的表达量增加。说明TDNs促进神经干细胞的迁移，是通过激活RHOA/ROCK2信号通路来实现的。
3.蛋白印迹检测
TDNs处理24h后，提取实验组和对照组的总蛋白，使用Western blot检测RhoA、Rock2、Vinculin蛋白。
结果：如图17d～17e、图18d～18e、图19d～19e所示，与对照组相比，实验组中迁移相关RHOA/ROCK2信号通路上的三个蛋白，分别是RhoA、Rock2、 Vinculin，其对应的蛋白的表达量增加。说明TDNs促进神经干细胞的迁移，是通过激活RHOA/ROCK2信号通路来实现的。
4.免疫荧光检测
将本实验例第1节中的6孔板换成共聚焦小皿进行样品处理。TDNs处理24h后，吸去培养基，PBS清洗三次，每次5分钟；用4wt％的多聚甲醛固定25分钟后，吸去多聚甲醛，PBS清洗三次，每次5分钟；再用0.5％Triton-100处理20～25分钟，吸去Triton-100，PBS清洗三次，每次5分钟；然后用羊血清处理1小时，吸去羊血清，PBS清洗三次，每次5分钟。一抗RhoA、Rock2、Vinculin抗体处理，4℃，过夜。第二天，37℃复温0.5小时，回收一抗，PBS洗3次，每次5分钟。携带荧光的二抗处理，避光，37℃，1小时，吸去二抗，PBS洗3次，每次5分钟。DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟。10％甘油封样，避光，4℃保存，并上机检测。
结果：如图17d～17e、图18d～18e、图19d～19e所示，与对照组相比，实验组中蛋白的荧光的强度(RhoA、Rock2、Vinculin)较高，进一步说明了TDNs促进神经干细胞的迁移，是通过激活RHOA/ROCK2信号通路来实现的。
以上实验进一步地证明了TDNs可以促进神经干细胞迁移。
综上，本发明的DNA四面体容易被神经细胞摄取，可以明显增加小鼠神经干细胞的增殖、分化和迁移，具有很好的促神经修复能力，并且具有很好的生物相容性。本发明的DNA四面体可以用于制备促神经修复的药物。

Claims (10)

  1. DNA四面体在制备药物中的用途，其特征在于，所述药物是促神经修复的药物。
  2. 如权利要求1所述的用途，其特征在于，所述DNA四面体由四条单链DNA通过碱基互补配对方式组装成四面体结构；所述四面体的每条棱都是双链DNA结构。
  3. 如权利要求1所述的用途，其特征在于，所述DNA四面体的制备方法是：将含有等浓度所述四条单链DNA的水溶液加热，使得碱基间氢键完全断开，维持5～20min，然后快速降温到0～10℃，保持至少15min；所述水溶液中还含有10mM Tris-HCl、50mM MgCl 2；所述水溶液加热前的pH值为8。
  4. 如权利要求3所述的用途，其特征在于，所述加热后维持时间为10min，所述降温的具体温度为4℃，所述降温后保持时间为20min。
  5. 如权利要求1所述的用途，其特征在于，所述四条单链DNA的序列分别如SEQ ID NO.1～4所示。
  6. 如权利要求1所述的用途，其特征在于，所述药物是促进神经干细胞的增殖、分化和/或迁移的药物。
  7. 如权利要求6所述的用途，其特征在于，所述药物是激活Wnt/β-Catenin通路的药物。
  8. 如权利要求6所述的用途，其特征在于，所述药物是抑制Notch信号通路的药物。
  9. 如权利要求6所述的用途，其特征在于，所述药物是激活RHOA/ROCK2信号通路的药物。
  10. 一种促神经修复的药物，其特征在于，它是使用权利要求1～9所述DNA四面体作为活性物质，加上药学上可接受的辅料或辅助性成分制备而成。
PCT/CN2018/116847 2017-11-22 2018-11-22 Dna四面体在促神经修复药物制备中的用途 Ceased WO2019101116A1 (zh)

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