WO2019101115A1 - Dna四面体在制备治疗阿尔兹海默症药物中的用途 - Google Patents

Dna四面体在制备治疗阿尔兹海默症药物中的用途 Download PDF

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WO2019101115A1
WO2019101115A1 PCT/CN2018/116846 CN2018116846W WO2019101115A1 WO 2019101115 A1 WO2019101115 A1 WO 2019101115A1 CN 2018116846 W CN2018116846 W CN 2018116846W WO 2019101115 A1 WO2019101115 A1 WO 2019101115A1
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drug
dna
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tdns
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林云锋
邵晓茹
蔡潇潇
马文娟
谢雪萍
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四川大学
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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
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links

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  • the invention relates to the field of nerve repair, and in particular to the use of DNA tetrahedron in the preparation of a medicament for treating Alzheimer's disease.
  • AD Alzheimer's disease
  • apoptotic neuronal death may be an important component of AD
  • neuronal apoptosis has been discovered in early AD
  • a large number of apoptotic neurons in the cerebral cortex and hippocampus have also been shown in autopsy of AD patients. .
  • a ⁇ deposition in the brain is the central link of AD pathological changes, can trigger a series of pathological processes, which further promote A ⁇ deposition, thereby forming a cascade amplification reaction, ultimately producing cytotoxicity, including toxicity of mitochondria Pathway apoptosis.
  • Bax protein In the apoptosis of mitochondrial pathway, Bax protein is one of the major apoptotic genes, which can enhance the permeability of mitochondria, release cytochrome C from mitochondria, mediate the formation of apoptotic complex, and activate Caspase 3/6/7. It exerts hydrolysis and cleaves more than 100 substrates such as ⁇ -tubulin, Actin, PARPA, and Lamin in cells, eventually leading to apoptosis. Bcl-2 can form a dimer with Bax protein, reduce mitochondrial permeability and inhibit apoptosis.
  • TDNs DNA tetrahedrons
  • TDNs are DNA nanomaterials with a three-dimensional structure formed by four DNA single strands through strict base complementary pairing principles.
  • TDNs have good biocompatibility, biosafety and biodegradability and are simple in preparation, high in yield and good in nuclease resistance.
  • the present invention provides the use of a DNA tetrahedron for the preparation of a medicament for treating Alzheimer's disease.
  • the DNA tetrahedron is assembled into a tetrahedral structure by four single-stranded DNAs by base complementary pairing; each of the tetrahedrons is a double-stranded DNA structure.
  • the DNA tetrahedron is prepared by heating an aqueous solution containing the four single-stranded DNAs at an equal concentration, so that the hydrogen bonds between the bases are completely disconnected, maintaining for 5 to 20 minutes, and then rapidly cooling to 0 to 10 ° C, keeping at least 15 min; the aqueous solution further contained 10 mM Tris-HCl, 50 mM MgCl 2 ; the pH of the aqueous solution before heating was 8.
  • the heating maintenance time is 10 min
  • the specific temperature of the cooling is 4 ° C
  • the holding time after the cooling is 20 min.
  • sequences of the four single-stranded DNAs are shown in SEQ ID NOS. 1-4, respectively.
  • the drug is a drug that promotes proliferation of nerve cells.
  • the drug is a drug which reduces damage of nerve cells by ⁇ -amyloid.
  • the medicament inhibits neuronal apoptosis.
  • the drug is a drug that reduces the expression of Bax, Caspase 3 and/or cytochrome C protein.
  • the drug is a drug that increases expression of a Bcl-2 protein.
  • the present invention also provides a medicament for treating Alzheimer's disease, which is prepared by using the aforementioned DNA tetrahedron as an active substance together with a pharmaceutically acceptable adjuvant or auxiliary component.
  • DNA tetrahedrons protect PC12 cells from their high concentrations of exogenous beta amyloid in the environment.
  • DNA tetrahedra can also promote the proliferation of PC12 cells.
  • DNA tetrahedron can inhibit apoptosis by decreasing the expression of apoptosis-positive proteins Bax, Caspase 3, cytochrome C, and increasing the expression of the negative correlation protein Bcl-2.
  • TDNs in the brain of rats with Alzheimer's disease can significantly reduce the protein level of Bax and activated Caspase3 to near normal values, and can significantly increase the protein level of Bcl-2.
  • DNA tetrahedrons can also significantly reduce the number of apoptosis in the brain of rats with Alzheimer's disease.
  • the invention also proves that the DNA tetrahedron can improve the learning and memory ability of rats through the positioning navigation experiment and the space exploration experiment.
  • DNA tetrahedron is completely free of toxicity to cells because its metabolic end products are free nucleotides, which is safer than existing Alzheimer's treatment drugs.
  • DNA tetrahedron can be used as a medicament for the preparation of a treatment for Alzheimer's disease.
  • FIG. 1 is a schematic representation of four single-stranded synthetic TDNs.
  • Figure 2 is a graph showing the results of 8% non-denaturing polyacrylamide gel electrophoresis (PAGE).
  • Figure 3 is a schematic diagram showing the results of transmission electron microscopy.
  • Figure 4 is a schematic diagram showing the results of atomic force microscopy.
  • Fig. 5 is a graph showing the results of examining the effect of the concentration of A ⁇ 25-35 on the activity of PC12 cells.
  • Figure 6 is a graph showing the results of detection of PC12 cell activity under TDNs and/or A ⁇ treatment.
  • Figure 7 is a graph showing the results of detection of apoptosis of PC12 cells treated with TDNs and/or A?.
  • Figure 8 is a graph showing the results of detection of apoptosis of PC12 cells treated with another TDNs and/or A ⁇ .
  • Figure 9 is a graph showing the results of cell cycle detection of PC12 cells treated with TDNs and/or A ⁇ by flow cytometry.
  • Figure 10 is a graph showing the results of detection of apoptosis of PC12 cells treated with TDNs and/or A ⁇ by flow cytometry.
  • Figure 11 is a graph showing the results of detection of the expression of apoptosis-positive proteins Bax, Caspase 3, cytochrome C, and apoptosis-related protein Bcl-2 in PC12 cells treated with TDNs and/or A ⁇ .
  • Figure 12 is a graph showing the results of detection of apoptosis-positive genes Bax, Caspase 3, and apoptosis-related gene Bcl-2 in PC12 cells treated with TDNs and/or A ⁇ .
  • Figure 13 is a graph showing the results of detection of the expression of apoptosis-related protein Bcl-2 in PC12 cells treated with TDNs and/or A ⁇ .
  • Figure 14 shows the escape latency results for each group of rat positioning cruise experiments: *p ⁇ 0.05, **p ⁇ 0.01 indicates significant difference compared with the DNA group; #p ⁇ 0.05, ##p ⁇ 0.01 indicates that the model group The ratio was significantly different; &p ⁇ 0.05, &&p ⁇ 0.01 indicates a significant difference from the first day.
  • Figure 15 is a trajectory diagram of the second quadrant into the water on the 5th day of the cruise test in rats.
  • Figure 16 is a graph showing the percentage of total time in the target quadrant of each group of rat space exploration experiments.
  • Figure 17 shows the results of the number of crossing platforms in each group of rat space exploration experiments.
  • Figure 18 shows the latency results of the first time the rats in the space exploration experiment group found the platform.
  • Figure 19 is a trajectory diagram of the rat space exploration experiment.
  • Figure 20 shows the protein expression levels of active Caspase 3, Bax and Bcl-2 in hippocampus of each group: Control is a blank control group; sham is a sham operation group; AD+PBS is an Alzheimer's disease model (AD)+ The saline group; the AD+DNA group was the AD+DNA material group; *p ⁇ 0.05, **p ⁇ 0.01 indicates significant difference compared with the control group; ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01 means and false There was a significant difference between the surgical groups; &p ⁇ 0.05, &&p ⁇ 0.01 indicates a significant difference compared to the AD model + PBS group.
  • AD Alzheimer's disease model
  • Figure 21 is a statistical diagram showing the expression level of caspase-3 in hippocampus, and the group name and statistically significant difference are the same as Fig. 20.
  • Figure 22 is a statistical diagram showing the expression level of Bcl-2 in hippocampus tissue, and the group name and statistically significant difference are the same as Fig. 20.
  • Figure 23 is a statistical diagram showing the expression level of Bax in hippocampus tissue, and the group name and statistically significant difference are the same as Fig. 20.
  • Fig. 24 is a fluorescence diagram showing the apoptosis of hippocampus in each group by TUNEL staining, and the group name is the same as Fig. 20.
  • Figure 25 is a statistical diagram showing the positive cells of apoptosis in hippocampus of each group by Tunel staining.
  • the group name is the same as Fig. 20.
  • 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 was prepared by using 4.2 mL of distilled water, 1.2 mL of 40% acrylamide, 0.6 mL of 10 ⁇ TAE, 60 ⁇ L of 10% Aps and 6 ⁇ L of TEMED;
  • the results are shown in Fig. 2.
  • the red circle is labeled as the synthesized tetrahedron, and the band of about 500 bp is the polymer formed during the synthesis of the tetrahedron, indicating that the DNA tetrahedron is successfully synthesized.
  • 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.
  • Atomic force microscopy was used to detect TDNs.
  • the TDNs have a particle size of about 10 to 15 nM, which is consistent with the results of transmission electron microscopy, which further indicates that the DNA tetrahedron is successfully synthesized.
  • the cultured cell suspension was divided into control group and experimental group.
  • the experimental group was added with different concentrations of A ⁇ 25-35 .
  • the control group was added with the same amount of serum, and then cultured in the incubator for 24 hours (37 ° C, 5% CO). 2 ), after the end of the culture, add 10 ⁇ L of CCK-8 solution to each well, and avoid the production of air bubbles, then culture in the incubator for 1 to 4 hours (37 ° C, 5% CO 2 ) after the end of the incubation, at 450 nm
  • the absorbance of each well and data processing was performed by the incubator for 24 hours (37 ° C, 5% CO).
  • the concentrations of A ⁇ 25-35 were 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M and 100 ⁇ M, respectively, and when the concentration of A ⁇ 25-35 was 25 ⁇ M, the cell activity was less than 70%.
  • the cultured cell suspension was divided into blank control group, TDNs group, AD cell model group and TDNs pretreated AD cell model group; among them, blank control group did not add A ⁇ 25-35 ; TDNs pretreated AD cells
  • the model group was pretreated with 250 nM TDNs for 6 h, then A ⁇ 25-35 was added at a concentration of 25 ⁇ M; the AD cell model group was pretreated with serum-free medium for 6 h, then A ⁇ 25-35 at a concentration of 25 ⁇ M was added; Groups, TDNs group, AD cell model group and TDNs pretreated AD cell model group were placed in the incubator for 24h (37 ° C, 5% CO 2 ).
  • TDNs+A ⁇ 25-35 group pretreated with TDNs showed higher cell activity than A ⁇ 25-35 group, indicating that TDNs can enhance Alzheimer's model cells.
  • the PC12 cell suspension was inoculated into a confocal dish, and grouped according to the step (2) in Experimental Example 2, and correspondingly treated, and then the component was DMEM + 10% serum + 1% double antibody.
  • the medium was washed three times with PBS for 15 minutes each time;
  • Sheep serum was incubated for 1 hour at room temperature, washed with PBS three times each time;
  • the tetrahedral group had better spreading than the blank control group.
  • the cells in the AD cell model group showed obvious apoptosis, the number of cells decreased, the nucleus appeared to be lysed, and pyknosis.
  • the number of cells in the TDNs pretreated AD cell model group was reduced, the cell spreading was worse than that in the blank control group, but the nucleus was intact, and no obvious nuclear pyknosis occurred.
  • the apoptosis phenomenon was significantly lower than that of the AD cell model group.
  • step (2) grouped according to step (2) in Experimental Example 2, and correspondingly treated, then induced apoptosis, and a negative control group was established, and the cells were collected;
  • the red fluorescent cells were collected by centrifugation with 0.25% trypsin in a 15 mL centrifuge tube (2000 rpm, 5 minutes), the supernatant was discarded, washed with PBS, and centrifuged (2000 rpm, 5 minutes). 500 ul of fixed cells were added to ice ethanol and allowed to stand overnight at 4 °C. The next day, PBS was added for centrifugation, the supernatant was discarded, washed with PBS, centrifuged, and the supernatant was discarded. Add 100 ⁇ L of RNase in a 37 ° C water bath for 30 minutes. Add 400 ⁇ L of PI stain and mix well, avoiding light at 4 ° C for 30 minutes. The cells were transferred to a flow tube, tested on the machine, and analyzed using WinMDI 2.9 and WinCycle 32 software.
  • the number of cells in the S phase (DNA synthesis phase) in the TDNs group increased significantly, indicating that TDNs changed the cell cycle of PC12 and promoted its value-added.
  • the PC12 cells pretreated with TDNs had no obvious apoptotic peaks compared with the AD cell model group, and the number of cells in the S phase (DNA synthesis phase) increased significantly, indicating that TDNs changed the cell cycle of PC12 cells.
  • step (2) grouping according to step (2) in Experimental Example 2, and performing corresponding processing;
  • the cells were separately digested and collected in a 15 mL centrifuge tube (300 g, 5 minutes), the supernatant was discarded, washed with PBS, and centrifuged (300 g, 5 minutes).
  • the cells were suspended in 400 ⁇ L of Annexin V binding solution at a concentration of approximately 1 x 106 cells/mL.
  • 5 ⁇ L of Annexin V-FITC staining solution was added to the cell suspension, and gently mixed at 4 ° C for 15 minutes in the dark.
  • Add 5 ⁇ L of PI staining solution gently mix and incubate at 4 ° C for 5 minutes in the dark. Transfer the cells to a flow tube, perform on-machine detection, and analyze the data.
  • the proportion of apoptosis in the TDNs group was reduced compared to the blank control group. This indicates that TDNs have a certain inhibitory effect on PC12 cell apoptosis.
  • the T12s pretreated PC12 cells showed a significant decrease in the apoptotic ratio, indicating that TDNs had a significant inhibitory effect on A ⁇ -induced apoptosis.
  • TDNs can inhibit the apoptosis of PC12 cells, especially to prevent apoptosis caused by A ⁇ .
  • Bax protein In the apoptosis of mitochondrial pathway, Bax protein is one of the major apoptotic genes, which can enhance the permeability of mitochondria, release cytochrome C from mitochondria, mediate the formation of apoptotic complex, and activate Caspase 3/6/7. It exerts hydrolysis and leads to apoptosis.
  • Caspase 3 plays a role in apoptosis of the mitochondrial pathway and also plays a role in apoptosis of other pathways (death receptor ligand pathway and endoplasmic reticulum pathway).
  • step (2) grouping according to step (2) in Experimental Example 2, and extracting proteins in the blank control group, the TDNs group, the AD cell model group, and the TDNs pretreated AD cell model group, respectively, using the whole protein extraction kit;
  • TDNs treated PC12 cells expressed more Bcl-2 protein and fewer Caspase-3 proteins than the blank group.
  • TDNs pretreatment significantly increased the expression of cytochrome C, Bax and Caspase-3 proteins.
  • TDNs can enhance the anti-apoptotic ability of PC12 cells and can prevent apoptosis induced by A ⁇ .
  • step (2) grouping according to step (2) in Experimental Example 2, and performing corresponding processing;
  • TDNs-treated PC12 cells significantly increased the expression of Caspase-3 and Bax genes, and significantly decreased the expression of Bcl-2 gene.
  • TDNs pretreatment significantly increased the expression of Bax and Caspase-3 genes, and significantly increased the expression of Bcl-2 gene, which was consistent with protein expression.
  • TDNs can enhance the anti-apoptotic ability of PC12 cells and can prevent apoptosis induced by A ⁇ .
  • step (2) grouping according to step (2) in Experimental Example 2, and performing corresponding processing;
  • step (3) sucking the medium of step (2), washing 3 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. Fluorescence confocal microscopy.
  • the expression of the apoptotic negative correlation protein Bcl-2 was increased in the TDNs group compared with the blank control group, thus further indicating that TDNs can enhance the anti-apoptotic ability of PC12 cells.
  • the TDNs pretreated AD cell model group increased the expression of the apoptotic negative correlation protein Bcl-2, which further indicated that TDNs had a significant inhibitory effect on A ⁇ -induced apoptosis.
  • TDNs can prevent apoptosis of PC12 cells by reducing the expression of cytochrome C, Bax and Caspase-3 proteins and increasing the expression of Bcl-2 protein, especially to prevent apoptosis induced by A ⁇ .
  • TDNs can neutralize the damage of A ⁇ on nerve cells, maintain the activity of nerve cells, promote the proliferation of nerve cells, and prevent the apoptosis of nerve cells.
  • the following animal experiments were used to further verify that TDNs have an activity for treating Alzheimer's disease and can be used in the preparation of related drugs.
  • AD Alzheimer's disease model
  • SD model establishment method SD rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium 40 ⁇ g/g, anesthetized and then fixed on the brain stereotaxic instrument. According to the stereotactic map of the rat brain, the previous sputum was the starting point, the puncture point was 3.5 mm after the anterior iliac crest, and the right side of the midline was 2 mm, and then the dental drill was drilled to open the skull. The microneedle was used to vertically insert the needle 3 mm from the brain surface.
  • the bilateral hippocampal CA1 area was slowly and uniformly injected with 10 ⁇ g (1 ⁇ L) of A ⁇ 1-40, and the needle was left for 5min. The wound was sutured after the needle was withdrawn.
  • the rats were randomly divided into two groups. One group started the tail vein injection and injected normal saline. The rats were administered 100 ⁇ l/time/d for 21 days. Another group was injected with DNA material in the tail vein, and each rat was administered with 100 ⁇ l/time/d for 21 days. In the sham operation group, the brain was stereotactically injected with the same amount of normal saline, and the wound was sutured after the needle was withdrawn. The rest of the treatment was the same. The blank group does not do any processing.
  • the Morris water maze test was performed 21 days after administration. The test was divided into a positioning navigation experiment and a space exploration experiment.
  • Figures 14 and 15 are experimental results and trajectories of five consecutive days of directional navigation experiments. From the results, it was shown that during the directional navigation experiment, compared with the first day experiment, the escape latency of each group of rats was reduced, among which The difference in the results of the 5th day between the normal group, the sham operation group (Sharm) and the TDNs material injection group (DNA) was extremely significant (P ⁇ 0.01). From the second day of directional navigation, compared with the blank group, the escape latency of the model group rats was significantly increased (P ⁇ 0.05), and the escape latency of the DNA material injection group was significantly lower (P ⁇ 0.05).
  • Figure 16-19 shows the results of the space exploration experiment and the trajectory on the 6th day.
  • the results in Figure 16 show that compared with the model group, the blank group, the sham operation group, and the DNA material injection group have increased the time spent in the target quadrant, but The difference was not statistically significant (P>0.05).
  • Figure 17 shows that compared with the model group, the number of crossings in the blank group, the sham operation group, and the DNA material injection group increased, but the difference was not statistically significant (P>0.05).
  • the results in Figure 18 show that compared with the model group, the latency of the first search for the platform was reduced in the blank group, the sham operation group, and the DNA material injection group, but the difference was not statistically significant (P>0.05).
  • the rats in each group were removed from the whole brain, the cerebellum was peeled off on ice, the remaining part was cut to the left and right, half was placed in paraformaldehyde for pathological examination, and the other half was taken for PCR and hippocampus. Protein detection.
  • 0.5 mg/mL standard bovine serum albumin was added to a standard well of a 96-well plate at 0 ⁇ L, 1 ⁇ L, 2 ⁇ L, 4 ⁇ L, 8 ⁇ L, 12 ⁇ L, 16 ⁇ L, 20 ⁇ L, and supplemented with a standard dilution to 20 ⁇ L.
  • the sample was diluted to a certain concentration with a standard dilution and 20 ⁇ L was added to a 96-well plate.
  • BCA working solution Prepares an appropriate amount of BCA working solution according to 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), mix well, add 200 ⁇ L of BCA working solution to each well, and let stand at 37 ° C for 30 min.
  • the absorbance value at 562 nm was measured, and the protein concentration of the sample was calculated from the standard curve.
  • Gluing Clean the glass plate, fix it on the bracket, pour 10% separation glue prepared according to the above table between the glass plates, immediately close the surface with deionized water and polymerize at room temperature. When a clear interface occurs between the water seal layer and the separation layer, it indicates that the polymerization of the separation gel has been completed. Discard the ionized water, blot it with filter paper, pour 4% of the concentrated gel, insert the comb, and after the gel is solidified, carefully remove the comb and set aside.
  • Sample preparation before loading Add 5 ⁇ loading buffer, mix well, boil in boiling water for 5 min, and cool rapidly in ice bath.
  • the loading amount is 30 ⁇ g per lane.
  • Electrophoresis Add electrophoresis buffer into the electrophoresis tank, turn on the power, and electrophoresis at 80V to bromophenol blue to finish the concentrated layer. The time is about 30min. The electrophoresis voltage of the separation gel is 120V. When the bromophenol blue migrates to the lower edge of the separation gel, the power is turned off and the electrophoresis is stopped.
  • the PVDF membrane was placed in a dish, and a blocking solution containing 5% skim milk powder was added, and the shaker was shaken for 1.5 to 2 hours;
  • the membrane was washed with TBST for 10 min ⁇ 3 times;
  • the membrane was placed in a plate containing primary antibody dilution Bcl-2 (diluted 1:1000), active Caspase 3 (diluted 1:2000), Bax (diluted 1:1080) and GAPDH (diluted 1:3000). Incubate overnight at 4 ° C shaker shake;
  • the secondary antibody After the end of the secondary antibody reaction, the secondary antibody is recovered.
  • the membrane was then washed with TBST for 5 to 10 min x 3 times.
  • Figure 20 shows the protein expression levels of active Caspase 3, Bcl-2, and Bax in hippocampus of each group of rats.
  • the results showed that there was no difference in the expression levels of active Caspase 3, Bcl-2 and Bax between control group and Sham group; the expression levels of active Caspase 3 and Bax in AD+PBS group were significantly higher than that in control group and Sham group, Bcl- The expression level of 2 was significantly lower than that of control group and Sham group; the expression level of active Caspase 3 in AD+DNA material group was no different from that in control group and Sham group, but it was significantly lower than AD+PBS group; AD+DNA material group Bax The expression level was not different from that of the control group and the Sham group, but it was significantly lower than that of the AD+PBS group.
  • the expression level of Bcl-2 in the AD+DNA material group was not different between the control group and the Sham group, but it was significantly higher than that of the AD+PBS group.
  • the primer sequences used in RT-PCR are as follows:
  • Primer sequence (5'-3') Caspase-3-F 5 CGGACCTGTGGACCTGAAAA
  • Caspase-3-R 6 TAACCGGGTGCGGTAGAGTA
  • Bcl-2-F 7 CTGGTGGACAACATCGCTCT
  • Bcl-2-R 8 GCATGCTGGGGCCATATAGT
  • Bax-F 9 CACTAAAGTGCCCGAGCTGA
  • GAPDH-F 11 TTCCTACCCCCAATGTATCCG GAPDH-R 12 CATGAGGTCCACCACCCTGTT
  • Dissolution curve 75 ° C ⁇ 95 ° C, 1 ° C per 20 s temperature
  • CT target gene, sample to be tested
  • CT internal standard gene, sample to be tested
  • Anti-quenching seal tablets and DAPI 1:500 diluted mounts stored in a -20 ° C refrigerator, and photographed by fluorescence microscopy or laser confocal microscopy.
  • the apoptotic rate was calculated by collecting and analyzing the relevant parts of the sample through a microscope.
  • Figure 24 shows the apoptosis of hippocampus cells in each group by TUNEL staining.
  • Figure 25 shows the statistical analysis of Tunel positive cells in hippocampus cells of each group by TUNEL staining.
  • Con group was blank control group; Con group was blank control group; sham group was sham operation group; AD group was Alzheimer's disease model (AD) + saline group; AD+DNA group was AD+DNA material group.
  • This experimental example demonstrates that TDNs can inhibit the apoptosis of rat hippocampus cells in the AD model.
  • Experimental Examples 1 to 8 demonstrate that DNA tetrahedral materials have significant activity for preventing and treating Alzheimer's disease, and DNA tetrahedrons can be used for the preparation of a medicament for the prevention and treatment of Alzheimer's disease.

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Abstract

DNA四面体在促神经修复药物制备中的用途，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，所述DNA四面体能够保护β类淀粉样蛋白环境下的神经细胞，抑制其凋亡。

Description

DNA四面体在制备治疗阿尔兹海默症药物中的用途 技术领域
本发明涉及神经修复领域，具体涉及DNA四面体在制备治疗阿尔兹海默症药物中的用途。
背景技术
阿尔兹海默病(AD)是常见的神经退行性疾病之一，具有年龄相关性，其发病特点为在疾病初期主要表现为轻微的认知功能障碍，而后出现神经元的损伤，最后演变为痴呆。有研究指出，凋亡性神经元死亡可能是AD的重要组成部分，在AD早期就已经发现了神经元凋亡，而在AD患者的尸检中也显示大脑皮层和海马中有大量凋亡神经元。
阿尔兹海默病的发病机制目前尚不明确，影响较广的是β类淀粉样蛋白(Aβ)级联假说。该假说认为Aβ在脑内沉积是AD病理改变的中心环节，可引发一系列病理过程，这些病理过程又进一步促进Aβ沉积，从而形成级联式放大反应，最终产生细胞毒性，其毒性包括引发线粒体途径的凋亡。
线粒体途径的凋亡中，Bax蛋白是主要的凋亡基因之一，它可以增强线粒体的通透性，使线粒体释放细胞色素C，介导凋亡复合体形成，进而激活Caspase 3/6/7发挥水解作用，切割细胞中如α-tubulin、Actin、PARPA、Lamin等超过100种的底物，最终导致细胞凋亡。而Bcl-2能与Bax蛋白形成二聚体，降低线粒体通透性，抑制凋亡。
目前有研究表明，一些治疗阿尔兹海默症的药物可能导致细胞毒性。
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具有广泛的应用前景。TDNs是由四条DNA单链通过严格碱基互补配对原则形成的具有三维结构的DNA纳米材料。TDNs具有良好的生物相容性、生物安全性和生物可降解性且制备方法简单，产率较高，具有较好核酸酶耐受性。关于TDNs的研究虽多，但它对细胞各项生理活动影响的研究却很少，尤其是它治疗阿尔兹海默症的研究更是没有。
发明内容
为了解决上述问题，本发提供了DNA四面体在制备药物中的用途，所述药物是治疗阿尔兹海默症的药物。
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。
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。
进一步地，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。
进一步地，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细胞增殖的药物。
前述的用途中，所述药物是降低β类淀粉样蛋白对神经细胞的损伤的药物。
前述的用途中，所述药物抑制神经细胞凋亡的药物。
进一步地，所述药物是降低Bax、Caspase 3和/或细胞色素C蛋白表达的药物。
进一步地，所述药物是升高Bcl-2蛋白表达的药物。
本发明还提供了一种治疗阿尔兹海默症的药物，它是使用前述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：
DNA四面体可以使保护PC12细胞，维持其高浓度外源β类淀粉样蛋白环境中的活性。
DNA四面体还可以促进PC12细胞的增殖。
DNA四面体可以通过降低凋亡正相关蛋白Bax、Caspase 3、细胞色素C的表达，以及升高负相关蛋白Bcl-2的表达，来抑制细胞凋亡。
动物模型实验也证实了患有阿尔兹海默症的大鼠大脑中，TDNs可以显著降低Bax和激活的Caspase3的蛋白水平到接近正常值，同时能显著提高Bcl-2的蛋白水平。
DNA四面体同样能显著减少患有阿尔兹海默症的大鼠大脑中细胞凋亡的数量。
本发明的还通过定位航行实验和空间探索实验证明了DNA四面体能改善大鼠的学习记忆能力。
DNA四面体因为其代谢终产物为游离核苷酸，对细胞完全没有毒性，相比现有阿尔兹海默症治疗药物具有很好的安全性。
上述有益效果进一步表明，DNA四面体可以用作制备治疗阿尔兹海默症的药物。
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8％非变性聚丙烯酰胺凝胶电泳(PAGE)鉴定结果图。
图3为透射电镜鉴定结果示意图。
图4为原子力显微镜鉴定结果示意图。
图5为Aβ 25-35的浓度对PC12细胞活性影响的检测结果示意图。
图6为TDNs和/或Aβ处理下的PC12细胞活性的检测结果图。
图7为TDNs和/或Aβ处理下的PC12细胞凋亡的检测结果图。
图8为另一个TDNs和/或Aβ处理下的PC12细胞凋亡的检测结果图。
图9为采用流式细胞术检测TDNs和/或Aβ处理下的PC12细胞的细胞周期检测结果图。
图10为采用流式细胞术检测TDNs和/或Aβ处理下的PC12细胞凋亡的检测结果图。
图11为TDNs和/或Aβ处理下的PC12细胞中凋亡正相关蛋白Bax、Caspase 3、细胞色素C，以及凋亡负相关蛋白Bcl-2表达量的检测结果图。
图12为TDNs和/或Aβ处理下的PC12细胞中凋亡正相关基因Bax、Caspase 3，以及凋亡负相关基因Bcl-2表达量的检测结果图。
图13为为TDNs和/或Aβ处理下的PC12细胞中凋亡负相关蛋白Bcl-2表达量的检测结果图。
图14为大鼠定位巡航实验的各组逃避潜伏期结果：*p<0.05,**p<0.01表示和DNA组相比具有显著差异；#p<0.05,##p<0.01表示和模型组相比具有显著差异；&p<0.05,&&p<0.01表示和第一天比具有显著差异。
图15为大鼠第5天定位巡航实验的第二象限入水的轨迹图
图16为大鼠空间探索实验各组在靶象限的时间占总时间的百分比结果
图17为大鼠空间探索实验各组穿越平台数结果
图18为大鼠空间探索实验各组第一次寻找到平台的潜伏时间结果
图19为大鼠空间探索实验轨迹图
图20为各组大鼠海马组织中active Caspase 3、Bax、Bcl-2的蛋白表达水平：Control为空白对照组；sham为假手术组；AD+PBS为阿尔茨海默病模型(AD)+生理盐水组；AD+DNA组为缺AD+DNA材料组；*p<0.05,**p<0.01表示与对照组相比具有显著性差异；^ p<0.05,^^p<0.01表示与假手术组相比具有显著差异；&p<0.05,&&p<0.01表示与AD模型+PBS组相比具有显著性差异。
图21为海马组织中caspase-3的表达水平统计图，组别名称和统计学显著差异同图20。
图22为海马组织中Bcl-2的表达水平统计图，组别名称和统计学显著差异同图20。
图23为海马组织中Bax的表达水平统计图，组别名称和统计学显著差异同图20。
图24为TUNEL染色检测各组海马组织凋亡的荧光图，组别名称同图20。
图25为Tunel染色检测各组海马组织凋亡的阳性细胞统计图，组别名称同图20。
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
Figure PCTCN2018116846-appb-000001
2.鉴定
2.1凝胶电泳
采用4.2mL蒸馏水、1.2mL 40％丙烯酰胺、0.6mL 10×TAE、60μL 10％Aps以及6μL TEMED制备得到聚丙烯酰胺凝胶；
然后取1μL 6×loading buffer和5μL制备得到的TDNs混合均匀，分别将其和marker加入对应的电泳槽中，冰浴、恒压100V，电泳60min；然后采用浓度比为1:50的GelRed和蒸馏水，在避光条件下，摇床处理15～25min，然后曝光，再进行检测，其结果见图2。
结果如图2所示，红色圆圈标注为所合成的四面体，500bp左右的条带为合成四面体过程中形成的多聚物，表明成功合成了DNA四面体。
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原子力显微镜
使用原子力显微镜检测TDNs。
如图4所示，TDNs粒径在10～15nM左右，与透射电镜结果吻合，进一步表明成功合成了DNA四面体。
为了证明TDNs确实有助于阿尔兹海默症的治疗，以下将用实验例的方式进一步说明。
实验例1 Aβ处理PC12细胞可以模拟阿尔兹海默症
1.方法
(1)在96孔板中接种PC12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，实验组加入不同浓度的Aβ 25-35，对照组加入等量的血清，然后在孵箱中培养24h(37℃，5％CO 2)，培养结束后，向每孔中加入10μL的CCK-8溶液，并避免生产气泡，然后在孵箱中培养1～4h(37℃，5％CO 2)培育结束后，在450nm处检测每孔的吸光度，并进行数据处理。
2.结果
如图5所示，Aβ 25-35的浓度分别为6.25μM、12.5μM、25μM，50μM和100μM，当Aβ 25-35的浓度为25μM时，细胞活性低于70％。
结合Aβ积累是导致阿尔兹海默症的主要原因这一主流理论，本实验表明外源的Aβ 25-35处理PC12细胞可以模拟阿尔兹海默症的发生。
实验例2 TDNs减少Aβ对PC12细胞的活性损伤
1.方法
(1)在96孔板中接种PC12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空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组；其中，空白对照组不加Aβ 25-35；TDNs预处理的AD细胞模型组先用250nM的TDNs预处理6h，然后加入浓度为25μM的Aβ 25-35；AD细胞模型组用无血清培养基预处理6h，然后再加入浓度为25μM的Aβ 25-35；然后空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组均置于孵箱中培养24h(37℃，5％CO 2)。
(3)在450nm处检测每孔的吸光度，并进行数据处理。
2.结果
如图6所示，加入四面体后，细胞有明显增殖，经TDNs预处理的TDNs+Aβ 25-35组，细胞活性较Aβ 25-35组有提高，表明TDNs可提高阿尔兹海默模型细胞的细胞活性。
实验例3 TDNs预防Aβ导致的细胞凋亡
1、采用免疫荧光技术检测TDNs对细胞凋亡的影响
1.1方法
(1)将PC12细胞悬浮液接种于共聚焦小皿中，并按实验例2中步骤(2)进行分组，并进行相应处理，然后吸去组分为DMEM+10％血清+1％双抗的培养基，PBS反复洗三遍，每遍15分钟；
(2)使用4％多聚甲醛在4℃固定15分钟，而后空气中放置5分钟，PBS洗三遍，每遍15分钟；
(3)0.5％tritonX-100试剂37℃打孔15分钟，PBS洗三遍，每遍15分钟；
(4)羊血清室温孵育1小时，PBS洗，每次三遍；
(5)孵育所要观察的一抗(抗nestin抗体)，4℃过夜；
(6)37℃复温30分钟，PBS洗三遍，每次15分钟；
(7)37℃孵育二抗1小时，PBS洗三遍，每次15分钟；
(8)细胞核染料DAPI处理10分钟，再用PBS清洗；
(9)用试剂鬼笔环肽进行细胞骨架染色10分钟，PBS洗；
(10)镜检，共聚焦显微镜观察细胞免疫荧光染色的情况。
1.2结果
如图7和图8所示，四面体组较空白对照组，细胞有更好的铺展。AD细胞模型组细胞出现明显凋亡，细胞数目减少，细胞核出现裂解，固缩。TDNs预处理的AD细胞模型组虽然细胞数目有所减少，细胞铺展较空白对照组差，但细胞核完整，未出现明显核固缩的现象，凋亡现象较AD细胞模型组有了大幅的降低。
2、采用流式细胞术检测细胞周期
2.1方法
(1)在25mL培养瓶中接种PC12细胞悬液，将培养瓶置于孵箱中预培养24小时(37℃，5％CO2)，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％CO2)，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％CO2)；
(2)按实验例2中步骤(2)进行分组，并进行相应处理，然后诱导细胞凋亡，同时设立阴性对照组，并收集细胞；
(3)用PBS洗涤细胞一次，收集并调整细胞浓度为1×106/mL；然后将制备的单细胞悬液用体积分数为70％乙醇固定，4℃保存，染色前用PBS洗去固定液；
(4)向步骤(3)所得细胞悬液中加入RNase A，37℃，水浴30min，再加入PI染色混匀，于4℃下避光30min，然后上机检测，并记录激发波长为488nm处的红色荧光；
(5)用0.25％胰蛋白酶消化收集红色荧光处细胞于15mL离心管中(2000rpm、5分钟)，弃上清，PBS洗涤，离心(2000rpm、5分钟)。加入冰乙醇500ul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。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，使用WinMDI 2.9和WinCycle 32软件进行数据分析。
2.2结果
如图9所示，与空白对照组相比，TDNs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PC12的细胞周期，促进其增值。TDNs预处理的PC12细胞较AD细胞模型组，无明显凋亡峰，且处于S期(DNA合成期)的细胞数目明显增加，说明TDNs改变了PC12细胞的细胞周期。
3、采用流式细胞术检测TDNs对阿尔兹海默细胞凋亡的抑制作用
3.1方法
(1)在25mL培养瓶中接种PC12细胞悬液，将培养瓶置于孵箱中预培养24小时(37℃，5％CO2)，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％CO2)，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％CO2)；
(2)按实验例2中步骤(2)进行分组，并进行相应处理；
(3)使用不含EDTA的胰蛋白酶，分别消化收集细胞于15mL离心管中(300g、5分钟)，弃上清，PBS洗涤，离心(300g、5分钟)。用400μL Annexin V结合液悬浮细胞，浓度大约为1×106cells/mL。在细胞悬液中加入5μL Annexin V-FITC染色液，轻轻混匀于4℃避光条件下孵育15分钟。加入5μL PI染色液，轻轻混匀于4℃避光条件下孵育5分钟。将细胞转移至流式管中，上机检测，数据分析。
3.2结果
如图10所示，空白对照组相比，TDNs组中凋亡比例有所降低。说明TDNs对于PC12细胞凋亡具有一定抑制作用。TDNs预处理的PC12细胞较AD细胞模型组，凋亡比例明显降低，说明TDNs对于Aβ导致的细胞凋亡现象具有明显的抑制作用。
综上，TDNs能抑制PC12细胞的凋亡，尤其能预防Aβ导致的细胞凋亡。
实验例4 TDNs抑制细胞凋亡的机制
线粒体途径的凋亡中，Bax蛋白是主要的凋亡基因之一，它可以增强线粒体的通透性， 使线粒体释放细胞色素C，介导凋亡复合体形成，进而激活Caspase 3/6/7发挥水解作用，进而导致凋亡。其中Caspase 3除了在线粒体途径的凋亡中发挥作用，在其他途径(死亡受体配体途径和内质网途径)的凋亡中也发挥作用。
本实验例通过检测Bax、Caspase 3、细胞色素C和Bcl-2的表达，来探究TDNs对阿尔兹海默细胞模型的凋亡抑制作用。
1、采用蛋白质印迹法检测Bax、Caspase 3、细胞色素C和Bcl-2的蛋白水平
1.1方法
(1)在6孔板中接种PC12细胞悬液(100μL/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO2)，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％CO2)，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％CO2)；
(2)按实验例2中步骤(2)进行分组处理，并使用全蛋白提取试剂盒，分别提取空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组中的蛋白；
(3)然后再分别进行SDS-PAGE电泳，其具体过程为：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，1小时→弃二抗，TBST洗涤3次，每次5-10分钟→曝光，检测并进行数据处理。
1.2结果
如图11所示，相比空白组，TDNs处理的PC12细胞表达了更多的Bcl-2蛋白，以及更少的Caspase-3蛋白。而Aβ处理的PC12细胞组别中，TDNs预处理会显著升高细胞色素C、Bax和Caspase-3蛋白的表达。
表明TDNs能增强PC12细胞抗凋亡的能力，并确实能预防Aβ导致的细胞凋亡。
2、采用荧光定量PCR(Q-PCR)检测Bax、Caspase 3、细胞色素C和Bcl-2的转录水平
2.1方法
(1)在6孔板中接种PC12细胞悬液(100μL/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO2)，再将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％CO2)，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％CO2)；
(2)按实验例2中步骤(2)进行分组，并进行相应处理；
(3)Q-PCR检测：每孔加入20μl的反应体系(2μl cDNA、10μl SYBR、0.8μl引物Forward、0.8μl引物Reserve、6.4μl ddH2O)，上机检测，并进行数据处理。
2.2结果
如图12所示，相比空白对照组，TDNs处理的PC12细胞显著提高Caspase-3、Bax基因的表达，显著降低了Bcl-2基因的表达。而Aβ处理的PC12细胞组别中，TDNs预处理会显著升高Bax和Caspase-3基因的表达，非显著地提高Bcl-2基因的表达，与蛋白表达结果基本一致。
表明TDNs能增强PC12细胞抗凋亡的能力，并确实能预防Aβ导致的细胞凋亡。
3、采用免疫荧光技术检测Bcl-2
3.1方法
(1)在共聚焦小皿中接种PC12细胞悬液(100μl/孔)，将培养板在孵箱中预培养24小时(37℃，5％CO2)，然后将组分为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％CO2)，再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％CO2)；
(2)按实验例2中步骤(2)进行分组，并进行相应处理；
(3)然后吸去步骤(2)所述培养基，PBS洗3次，每次5分钟；
(4)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；
(5)0.5％Triton-100处理20-25分钟，吸去Triton-100，PBS洗3次，每次5分钟；
(6)羊血清处理1小时，吸去羊血清，PBS洗3次，每次5分钟；
(7)一抗Bcl-2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；
(8)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。荧光共聚焦显微镜检测。
3.2结果
如图13所示，与空白对照组相比，TDNs组中凋亡负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs能增强PC12细胞抗凋亡的能力。TDNs预处理的AD细胞模型组较AD细胞模型组，凋亡负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs对于Aβ导致的细胞凋亡具有明显的抑制作用。
综上，TDNs可以通过降低细胞色素C、Bax和Caspase-3蛋白的表达，以及提高Bcl-2蛋白的表达，来预防PC12细胞的凋亡，特别是预防Aβ导致的细胞凋亡。
以上各实验例证明，TDNs可以中和Aβ对神经细胞的伤害，维持神经细胞的活性，促进神经细胞的增殖，预防神经细胞的凋亡。以下使用动物实验进一步验证TDNs具有治疗阿尔兹海默症的活性，可应用在相关药物制备中。
实验例5 大鼠行为学检测
1.方法
1.1实验分组(每组6只)
A：空白对照组
B：假手术组
C：阿尔茨海默病模型(AD)+生理盐水组
D：AD+TDNs材料组
AD模型建立方法SD大鼠均采用1％戊巴比妥钠40μg/g腹腔内注射麻醉，麻醉后继而固定于脑立体定位仪上。按照大鼠脑立体定位图谱，以前囟为零起点，前囟后3.5mm处为穿刺点，中线右侧旁开2mm，而后牙科钻钻开颅骨，采用微量注射器自脑表面垂直进针3mm，即：(AP＝-3.5mm,ML＝2.0mm,DV＝3.0mm)，双侧海马CA1区缓慢匀速注射Aβ1-40各10μg(1μL)，留针5min，退针后缝合伤口。
AD模型造模成功后3d，将大鼠随机分为两组，一组开始尾静脉注射，注射生理盐水，大鼠每只给药100μl/次/d，连续给药21d。另一组尾静脉注射DNA材料，大鼠每只给药100μl/次/d，连续给药21d。假手术组脑立体定位注射等量的生理盐水，退针后缝合伤口，其余处理相同。而空白组不进行任何处理。
1.2行为学检测
所有组别，于给药后21d，进行Morris水迷宫试验，试验分为定位航行实验和空间探索实验。
定位航行实验：大鼠连续接受5天的训练，每天4次，每次时间间隔30min，记录下大鼠从4个入水点和入水并找到平台所需要的时间，即逃避潜伏期。4次潜伏期的平均成绩作为当日的最终结果进入到最后统计。
空间探索实验：实验的第6天，撤去平台，从距离平台的最远端入水后，将大鼠放入水中，记录下30s内大鼠的游泳轨迹，并观察分析大鼠在目标象限的停留时间，它的穿越平台的次数以及它第一次寻找到平台的潜伏时间。
1.3统计学处理
采用SPSS 16.0统计软件进行数据分析，所有数据以均值±标准差
Figure PCTCN2018116846-appb-000002
表示，所有组间比较采用采用多变量方差分析方法(MANOVA)，同一组不同时间点采用单因素方差分析(One-way ANOVA)中的LSD法(最小显著性法)，p<0.05为有显著性差异的统计学 意义，p<0.01为有极显著性差异的统计学意义。
2.结果
图14和15是连续进行5天定向航行实验的实验结果和轨迹图，从结果中显示定向航行实验期间，与第1天实验相比，各组大鼠的逃避潜伏期都有所降低，其中空白组(Normal)、假手术组(Sharm)以及TDNs材料注射组(DNA)的第5天实验结果差异具有极显著性(P<0.01)。从定向航行第2天开始,与空白组相比，模型组(Model)大鼠的逃避潜伏期显著升高(P<0.05)，DNA材料注射组大鼠的逃避潜伏期显著降低(P<0.05)。之后这种趋势更加显著,定向航行第5天，与模型组相比，空白组、假手术组以及DNA材料注射组大鼠的逃避潜伏期显著降低(P<0.05)，其中DNA材料注射组差异最显著(P<0.01)。
图16-19是第6天进行空间探索实验结果和轨迹图，图16结果显示，与模型组相比，空白组、假手术组以及DNA材料注射组在目标象限的所停留的时间增加，但差异不具有统计学意义(P>0.05)。图17结果显示，与模型组相比，空白组、假手术组以及DNA材料注射组在穿越平台次数增加，但差异不具有统计学意义(P>0.05)。图18结果显示，与模型组相比，空白组、假手术组以及DNA材料注射组的第一次寻找到平台的潜伏期减少，但差异不具有统计学意义(P>0.05)。
以上结果提示，DNA材料注射后能改善大鼠学习记忆能力。
行为学结束后，将各组大鼠摘取全脑，冰上剥去小脑，将剩余部分左右对切，一半放入多聚甲醛固定，用于病理学检测，另一半取海马用于pcr和蛋白检测。
实验例6 实验动物蛋白检测
1.方法
1.1蛋白样品制备
根据组织量加入200μL蛋白裂解液，重悬细胞后用移液器将裂解液移至1.5mL离心管中，4℃裂解30min，期间摇晃数次。
4℃，12000rpm离心10min，将上清转移到1.5mL离心管中并于-80℃保存。
1.2 BCA法测定蛋白浓度
将0.5mg/mL标准牛血清蛋白按0μL、1μL、2μL、4μL、8μL、12μL、16μL、20μL加到96孔板的标准品孔中，加标准品稀释液补足到20μL。
样品用标准品稀释液稀释一定浓度后加20μL到96孔板中。
按50体积BCA试剂A加1体积BCA试剂B(50：1)配制适量BCA工作液，充分混匀，各孔加入200μL BCA工作液，37℃放置30min。
测定562nm处的吸光度值，根据标准曲线计算样品的蛋白浓度。
1.3 SDS-PAGE电泳和转膜
制胶：清洗干净玻璃板，固定在支架上，在玻璃板之间灌入按上表配制的10％的分离胶，立即用去离子水封闭表面，于室温下聚合。当水封层与分离胶层之间出现明显界面时，表示分离胶的聚合已完成。弃去离子水，用滤纸吸干，再灌入4％的浓缩胶，插入梳子，待胶凝固后，小心拔去梳子，备用。
上样前样品的处理：加入5×上样缓冲液混匀，沸水中煮沸5min，迅速冰浴中冷却，上样量为每泳道30μg。
电泳：在电泳槽内加入电泳缓冲液，接通电源，80V恒压电泳至溴酚蓝跑完浓缩胶层，时间大约为30min。分离胶电泳电压为120V，当溴酚蓝迁移至分离胶下缘时，关掉电源，停止电泳。
预先配置1L转移缓冲液并冷却至4℃。先将PVDF膜在甲醇中浸泡约30s，再在转移缓冲液浸泡10min，后与滤纸、凝胶一起放入电转移缓冲液中。按夹心法依次将海绵、滤纸、凝胶、PVDF膜、滤纸、海绵按顺序放好，小心去除所有气泡，PVDF膜位于阳极侧，凝胶位于阴极侧，插入电泳槽中，倒入转膜缓冲液，200mA恒流转移，根据所转蛋白分 子量，约1min/kd。
1.4封闭及抗原抗体反应
封闭：将PVDF膜放入平皿中，加入含5％脱脂奶粉的封闭液，摇床振荡1.5～2h；
封闭结束后，用TBST洗膜10min×3次；
将膜放入含一抗稀释液Bcl-2(稀释比例1：1000)，active Caspase 3(稀释比例1：2000),Bax(稀释比例1:1080)和GAPDH(稀释比例1：3000)的平皿中，4℃摇床振荡孵育过夜；
第二天取出，室温振荡30min，吸弃一抗，TBST洗10min×3次；
用TBST稀释二抗(稀释比例1：5000)，室温摇床振荡反应1～2h；
二抗反应结束后，回收二抗。然后用TBST洗膜5～10min×3次。
1.5显色曝光
将ECL试剂中增强液与稳定的过氧化物酶溶液按1：1比例混匀，滴加工作液于PVDF膜上，反应数分钟待荧光带明显后，用滤纸吸去多余的底物液，覆上保鲜膜，X光胶片压片后依次放入显影液显影、定影液定影，冲洗胶片。
1.6统计学处理
采用SPSS 16.0统计软件进行数据分析，所有数据以均值±标准差
Figure PCTCN2018116846-appb-000003
表示，不同组间两两比较采用单因素方差分析(One-way ANOVA)中的LSD法(最小显著性法)，p<0.05为差异有统计学意义。
2.结果
图20为各组大鼠海马组织中active Caspase 3、Bcl-2、Bax的蛋白表达水平。结果显示，control组和Sham组的active Caspase 3、Bcl-2、Bax的蛋白表达水平无差异；AD+PBS组的active Caspase 3和Bax的表达水平极显著高于control组和Sham组，Bcl-2的表达水平显著低于control组和Sham组；AD+DNA材料组的active Caspase 3的表达水平与control组和Sham组无差异，但是极显著低于AD+PBS组；AD+DNA材料组Bax的表达水平与control组和Sham组无差异，但极显著低于AD+PBS组；AD+DNA材料组Bcl-2的表达水平control组和Sham组无差异，但是极显著高于AD+PBS组。
以上结果表明：空白对照组和假手术组凋亡水平没有差异；而AD模型组凋亡水平显著升高，DNA材料治疗后，能够有效地抑制凋亡水平。
实验例7 荧光定量PCR检测
1.方法
1.1引物
RT-PCR所用引物序列如下表：
引物名称 SEQ ID NO. 引物序列(5'-3')
Caspase-3-F 5 CGGACCTGTGGACCTGAAAA
Caspase-3-R 6 TAACCGGGTGCGGTAGAGTA
Bcl-2-F 7 CTGGTGGACAACATCGCTCT
Bcl-2-R 8 GCATGCTGGGGCCATATAGT
Bax-F 9 CACTAAAGTGCCCGAGCTGA
Bax-R 10 CTTCCAGATGGTGAGCGAGG
GAPDH-F 11 TTCCTACCCCCAATGTATCCG
GAPDH-R 12 CATGAGGTCCACCACCCTGTT
1.2实时荧光定量PCR检测
取0.2mL PCR管，配制如下反应体系
Figure PCTCN2018116846-appb-000004
Figure PCTCN2018116846-appb-000005
PCR扩增
预变性       95℃，10min
循环(40次)   95℃，15s→60℃，60s
溶解曲线     75℃→95℃，每20s升温1℃
1.3数据处理
数据以2-△△Ct进行处理：
A＝CT(目的基因，待测样本)-CT(内标基因，待测样本)
B＝CT(目的基因，对照样本)-CT(内标基因，对照样本)
K＝A-B
表达倍数＝2-K
采用SPSS 16.0统计软件进行数据分析，所有数据以均值±标准差
Figure PCTCN2018116846-appb-000006
表示，不同组间两两比较采用单因素方差分析(One-way ANOVA)中的LSD法(最小显著性法)，仅两组间比较采用t检验，p<0.05为有显著性差异的统计学意义，p<0.01为有极显著性差异的统计学意义。
2.结果
如图21-23所示，control组和sham组之间的caspase-3、Bcl-2、Bax表达水平无显著性差异(P>0.05)；AD+PBS组的caspase-3、Bax表达水平较control组和sham组均极显著性升高(P<0.01)，Bcl-2极显著性降低(P<0.01)；AD+DNA组的caspase-3、Bax表达水平较control组和sham组极显著性升高(P<0.01)，而Bcl-2极显著性降低(P<0.01)。与AD+PBS组相比，AD+DNA组的caspase-3、Bax表达水平极显著性降低(P<0.01)，Bcl-2表达水平极显著性升高(P<0.01)。
以上结果表明，空白对照组和假手术组凋亡水平没有差异；而AD模型凋亡水平显著性升高，DNA材料治疗后，能够有效地抑制凋亡水平。
实验例8 Tunel荧光染色实验
1.方法
1)冰冻切片组织用4％多聚甲醛固定组织20分钟
2)PBS清洗30min，置于透性处理液中冰上孵育2min。
3)制备TUNEL反应混合液。
4)加50μL TUNEL反应混合液于标本上，加盖玻片或封口膜在暗湿盒中反应37℃反应1h；PBS洗3min×3次。
5)防淬灭封片剂与DAPI 1:500稀释封片，-20℃冰箱保存，荧光显微镜或激光共聚焦显微镜拍片。
6)图像采集。通过显微镜采集分析样本相关部位，计算凋亡率。
2.结果
图24为TUNEL染色检测各组大鼠海马组织细胞凋亡情况。图25为TUNEL染色检测各组大鼠海马组织细胞Tunel阳性细胞统计情况。Con组为空白对照组；Con组为空白对照组；sham组为假手术组；AD组为阿尔茨海默病模型(AD)+生理盐水组；AD+DNA组为AD+DNA材料组。
从图24和25可以看出，与Con组相比，Sham组海马组织凋亡水平无显著性统计学差异；AD组、AD+DNA组大鼠海马组织凋亡水平明显升高，其中AD组组织细胞凋亡水平高于AD+DNA组组织细胞凋亡水平。
本实验例表明TDNs材料可以抑制AD模型中大鼠海马组织细胞凋亡的发生。
实验例1～8证明了DNA四面体材料具有显著的预防和治疗阿尔兹海默症的活性，DNA四面体可以用于制备预防和治疗阿尔兹海默症药物。

Claims (11)

  1. DNA四面体在制备药物中的用途，其特征在于，所述药物是治疗阿尔兹海默症的药物。
  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细胞增殖的药物。
  7. 如权利要求1所述的用途，其特征在于，所述药物是降低β类淀粉样蛋白对神经细胞的损伤的药物。
  8. 如权利要求1所述的用途，其特征在于，所述药物抑制神经细胞凋亡的药物。
  9. 如权利要求7或8所述的用途，其特征在于，所述药物是降低Bax、Caspase 3和/或细胞色素C蛋白表达的药物。
  10. 如权利要求6或7所述的用途，其特征在于，所述药物是升高Bcl-2蛋白表达的药物。
  11. 一种治疗阿尔兹海默症的药物，其特征在于，它是使用权利要求1～10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。
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CN110292644A (zh) * 2019-07-23 2019-10-01 四川大学 一种防治心肌缺血再灌注损伤或治疗心脏缺血性疾病的药物
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CN112156104B (zh) * 2020-11-10 2021-12-31 四川大学 一种治疗抑郁症的药物
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