Application of circCdyl in treatment of related neurodegenerative diseases

Abstract

The invention discloses an application of circCdl in preparation of a drug for treating related neurodegenerative diseases, the drug over-expresses circCdl and promotes proliferation and differentiation of hippocampus NSCs, and the application comprises the following steps: S1, constructing a circCdl over-expressed lentiviral vector; s2, detecting the influence of circCdyl on the proliferation of the hippocampus NSCs through an in-vitro experiment; s3, detecting the influence of circCdyl on the differentiation of the hippocampus NSCs through an in-vitro experiment; and S4, detecting the improvement condition of the learning and memory functions of the hippocampal hippocampus injury model of the cut rat after overexpression of circCdyl through an in-vivo experiment. The invention mainly aims to discover a new function of circCdyl in proliferation and differentiation of hippocampal NSCs and provide a corresponding recombinant vector so as to solve the problem of hippocampal nerve regeneration in neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of neurobiology, and specifically relates to the application of circCdyl in treating related neurodegenerative diseases. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with an insidious onset and progressive development. Clinical features include memory loss, confusion, speech dysfunction, loss of life skills, cognitive dysfunction, execution disorder, and personality and behavioral changes. Neural stem cells (NSCs) are a type of adult stem cells in the classification of stem cells. These cells not only have the ability to continuously self-renew, but can also differentiate into various types of cells that make up the tissue. NSCs can proliferate through unequal division to provide sufficient cell populations for terminal cells in the nervous system tissue. In addition, NSCs also have the ability to continuously renew themselves to eliminate aging or damaged NSCs, thereby maintaining the relative homeostasis of the NSC system. These NSCs found in the brain of mature mammals are mainly present in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Further studies on the involvement of NSCs in neural regeneration have found that transplanting NSCs or neural progenitor cells into the damaged nervous system can cause them to differentiate into new neurons. Therefore, inducing neurogenesis to compensate for neuronal loss and reshape degenerated neuronal networks is expected to become a new direction for AD treatment.

[0003] Circular RNA (circRNA) is a special type of non-coding RNA molecule with reverse splicing sites and a circular structure. It is more stable than linear RNA and is not easily degraded by enzymes. Studies have found that circRNA is involved in the development of diseases, especially tumor-related diseases, and can significantly inhibit tumor growth, but there are few reports on its mechanism, treatment and neural repair in central nervous system degenerative diseases. Although circCdyl has been reported in the study of the function and mechanism of cancer and tumor-related diseases, the mechanism of mediating changes in the hippocampal microenvironment and participating in the regulation of hippocampal NSCs proliferation and differentiation in terms of neural regeneration is still unclear and lacks relevant research. Therefore, the discovery of new functions of circCdyl in the proliferation and differentiation of hippocampal NSCs has research and application value in solving the problem of hippocampal neural regeneration in neurodegenerative diseases. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide the application of circCdyl in the treatment of related neurodegenerative diseases. The main purpose of the present invention is to discover the new function of circCdyl in the proliferation and differentiation of hippocampal NSCs, and to provide a corresponding recombinant vector to solve the problem of hippocampal neurogenesis in neurodegenerative diseases.

[0005] To solve the above technical problems, the embodiments of the present invention provide the application of circCdyl in the preparation of drugs for the treatment of related neurodegenerative diseases. The drugs overexpress circCdyl and promote the proliferation and differentiation of hippocampal NSCs.

[0006] Furthermore, it includes the following processes:

[0007] S1. Construct a lentiviral vector overexpressing circCdyl: Construct circCdyl on the pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector.

[0008] S2. Detect the effect of circCdyl on the proliferation of hippocampal NSCs in vitro: Transfect the lentivirus carrying circCdyl constructed in step S1 into hippocampal NSCs to increase the intracellular circCdyl level; Use flow cytometry cycle analysis to detect the mitotic phase of cells, and use CCK8 to detect cell proliferation activity.

[0009] S3. Detect the effect of circCdyl on the differentiation of hippocampal NSCs in vitro: Transfect the lentivirus carrying circCdyl constructed in step S1 into hippocampal NSCs, and then induce differentiation. After induction of differentiation, detect the mRNA and protein expression levels of neuron-related markers by RT-qPCR and Western Blot.

[0010] S4. Detect the improvement of the learning and memory function in a rat model of fimbria-fornix transection injury after overexpressing circCdyl in vivo: Construct a rat model of fimbria-fornix transection injury, inject the lentivirus overexpressing circCdyl into the DG area for infection, and then perform Morris and step-through avoidance behavioral tests to observe whether the learning and memory function after injury is improved.

[0011] Furthermore, the construction of the lentiviral vector overexpressing circCdyl in step S1 includes the following steps:

[0012] Vector digestion: The vector used is the pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector, the restriction enzymes are EcoR I and SalI, the digestion temperature is 37 °C, and the digestion time is 3 hours; agarose gel electrophoresis is used for recovery; Target fragment acquisition: Design primers for PCR of circCdyl; Design primers for PCR of circCdyl:

[0013] Forward primer: 5’-CTTAGCTGTTAACGGGAAA-3’,

[0014] Reverse primer: 5’-CTGTTGAAGTCGTGGATGT-3’;

[0015] Recovery of the PCR product of the target gene; Digestion and recovery of the target gene; Ligation of the target gene and the vector; Preparation of competent cells; Transformation; Small-scale plasmid preparation; Identification of recombinant plasmid clones; Virus packaging.

[0016] Furthermore, the detection of the effect of circCdyl on the proliferation of hippocampal NSCs in the in vitro experiment in step S2 includes the following processes:

[0017] (2-1) In vitro culture of neural stem cells: After the instruments are sterilized by high-temperature high-pressure steam, pregnant SD rats on the 15th day of pregnancy are intraperitoneally injected with a compound anesthetic; after the pregnant rats are anesthetized, the abdomen is disinfected by alcohol spraying and exposed, the fur of the pregnant rats is lifted with hemostatic forceps, cut open with surgical scissors, and the uterus containing the embryos is taken out with ophthalmic forceps and placed in a petri dish containing 75% ethanol; the uterus is removed using toothed forceps on an ice box, the embryos are transferred to a petri dish containing DMEM / F-12, the whole brain of the embryonic suckling rats is taken out in the petri dish and the hippocampal region is separated, and transferred to a new petri dish containing DMEM / F-12; the hippocampal tissue in the petri dish is aspirated and transferred to a new centrifuge tube, and repeatedly pipetted until it presents a milky state, centrifuged at 1200 r / min for 3 min; the supernatant is removed, DMEM / F-12 is added, pipetted and mixed evenly, centrifuged at 1200 r / min for 3 min, and repeated twice; the supernatant is discarded, NSCs proliferation medium is added, the cells are pipetted and resuspended, filtered through a cell filter with a pore size of 40 μm, and then transferred to a culture flask, observed under a microscope, and then placed in an incubator at 37 °C and 5% CO2 for 5-7 days;

[0018] (2-2) Passage: Aspirate the suspension in the culture flask and transfer it to a 15 ml centrifuge tube. Centrifuge at 1200 r / min for 3 min; discard the supernatant. Add 0.25% Trypsin-EDTA to the centrifuge tube for digestion at 37°C for 3 min. Subsequently, add an equal volume of complete medium to the centrifuge tube to terminate digestion. Centrifuge at 1200 r / min for 3 min; discard the supernatant. Add D-PBS to resuspend the cells. Centrifuge at 1200 r / min for 3 min; discard the supernatant. Aspirate an appropriate amount of complete medium to resuspend the cells. After filtering through a 40 μm filter, distribute them equally into 2-3 new culture flasks, and then place them in an incubator at 37°C and 5% CO2 for continued culture;

[0019] (2-3) Lentiviral transfection: After plating NSCs, observe the cell growth status; discard the original medium and add fresh complete medium; add overexpressing lentivirus or negative control empty vector lentivirus; after virus infection, incubate in a cell culture incubator at 37°C and 5% CO2; after 24 h or 48 h, remove the virus, discard the medium, replace it with fresh medium, and continue to culture in an incubator at 37°C and 5% CO2;

[0020] (2-4) Detection of immunofluorescence: After culturing the cells in (2-3) for 10 days, remove the culture medium in the culture plate and wash it 3 times with 1×PBS; fix it with 4% paraformaldehyde at room temperature for 15-20 min, wash it 3 times with 1×PBS, place it on a shaker and shake slowly for 5 min each time; block it with blocking solution at room temperature for 2 h; wash it 3 times with 1×PBS, dilute the primary antibody proportionally with antibody diluent, and incubate it overnight at 4°C; wash it 3 times with 1×PBS, 5 min each time, shake slowly on the shaker, dilute the secondary antibody proportionally with antibody diluent, and incubate it at room temperature for 2 h; wash it 3 times with 1×PBS, 5 min each time, shake slowly on the shaker, incubate it with Hoechst33342 live cell staining solution at room temperature for 10 min; wash it 3 times with 1×PBS, 5 min each time, in the dark, drop mounting medium on the glass slide, take out the cell coverslip from the 24-well culture plate and invert it on the glass slide; observe the cells using a ZEISS Axio Scope A1 microscope and process the images using Photoshop CS6 software;

[0021] (2-5) Western Blot experiment: Protein extraction; electrophoresis; membrane transfer; blocking; incubation with primary antibody; incubation with secondary antibody; development and analysis.

[0022] Furthermore, in the in vivo experiment in step S4, the improvement of the learning and memory function of the rat fimbria-fornix transection injury model after overexpressing circCdyl was detected, including the following process:

[0023] (4-1) Establish a rat model of cutting the fimbria-fornix of the hippocampus: Select adult female SD rats, 10 rats in each group, divided into Normal group, Sham group, Transected group, Transected+LV-NC group, and Transected+LV-circ group; After anesthesia, fix the rats on a stereotaxic apparatus for the brain, remove the hair on the top of the skull, disinfect, incise the skin on the top of the skull to expose the skull, cut the periosteum, and find the bregma; Starting from the bregma, 3.6 mm backward, 1.4 mm and 4 mm to the left, and 1.4 mm and 4 mm to the right, a total of 4 points are marked; Use a high-speed skull drill to make a slit at the marked position on the skull, use a fine needle to advance to a depth of 5 mm, and then make left and right cuts; After cutting and stopping bleeding, suture the skin and apply iodophor to the wound;

[0024] (4-2) Inject the virus: Select adult SD rats with a cut fimbria-fornix of the hippocampus model. After anesthesia, fix the rats on a stereotaxic apparatus for the brain, remove the hair on the top of the skull, disinfect, incise the skin on the top of the skull to expose the skull, cut the periosteum, and find the bregma; Locate the dentate gyrus of the hippocampus: 1.39 mm backward from the bregma, 1.8 mm to the left and right, and mark; Use a high-speed skull drill to make a small hole at the marked position, advance the microinjection pump to a depth of 3.9 mm, and inject 5 μL of negative control virus and circCdyl overexpression virus into the dentate gyrus area of the hippocampus respectively. Leave the needle for 10 min after injection; Pull out the needle. After stopping bleeding, suture the skin and apply iodophor to the wound;

[0025] (4-3) Morris water maze behavioral experiment: After modeling, place the adult SD rats head-down into the water pool one by one along the pool wall. Select four different quadrants of front, back, left, and right as the starting points, and record the time, path length, and time for the rats to find the platform; If the experimental animals exceed 2 min to find the platform during the first day of training, guide them to the platform and let them stay still for 30 s; Continuously train for 5 days. Remove the platform on the 5th day, record the data of the number of times the experimental animals cross the platform, and organize and analyze the escape latency of the time for the rats to find the platform, the path and speed of searching for the platform, the time of staying in the platform quadrant, and the number of times of crossing the platform;

[0026] (4-4) Step-through avoidance behavioral experiment: On the first day of training, place the adult SD rats after modeling in the light box and give light stimulation. Due to the natural dark preference of the animals, they will enter the dark box autonomously. After entering the dark box, close the shuttle channel and give an electric shock. The test time is 5 min; Continuously repeat the training for 3 days. Based on the electric shock punishment on the first day of training, the experimental animals will selectively avoid entering the dark box, and record the latency of the time required to enter the dark box from the light box, which is used as an index for detecting short-term memory;

[0027] (4 - 5) Shuttle behavioral experiment: On the first day of training, the adult SD rats after modeling were placed in the test box to move freely for 5 minutes, and then placed in the shock area of the shuttle experiment box. Light stimulation and sound stimulation were given. If they escaped to the safe area before the end of the light and sound stimulation, it was active avoidance. If they escaped to the safe area after the end of the stimulation time through electric shock, it was passive avoidance. After multiple trainings, the experimental animals could gradually form an active avoidance conditioned response and thus obtain memory. Training was carried out continuously for 3 days, with the same method as the first day of training, the cycle number was 30 times, and the test time was 15 minutes. The number of active avoidance times, avoidance times, and passive avoidance times of different groups were recorded, and the data were statistically analyzed.

[0028] The present invention also provides a pharmaceutical composition for treating related neurodegenerative diseases, and the pharmaceutical composition overexpresses circCdyl to promote the proliferation and differentiation of hippocampal NSCs.

[0029] The beneficial effects of the above technical solutions of the present invention are as follows:

[0030] The present invention mainly relates to the application of circCdyl in treating related neurodegenerative diseases and a pharmaceutical composition for treating related neurodegenerative diseases, applying the new function of circCdyl in the proliferation and differentiation of hippocampal NSCs, and providing a corresponding recombinant vector to solve the problem of hippocampal neurogenesis in neurodegenerative diseases. Brief Description of the Drawings

[0031] Figure 1 It is the influence diagram of circCdyl overexpression promoting the proliferation of NSCs in Example 2: Among them, Figure 1 A is the cell cycle situation and statistical analysis of overexpressing and non - overexpressing circCdyl detected by flow cytometry; Figure 1 B is the cell proliferation situation and statistical analysis of overexpressing and non - overexpressing circCdyl detected by CCK8 experiment; Figure 1 C is the immunofluorescence chemical staining to detect Ki67 + / Nestin + and statistical analysis, scale bar = 200μm, *P < 0.05, **P < 0.01;

[0032] Figure 2 It is the influence diagram of circCdyl overexpression on the differentiation of hippocampal NSCs: Among them, Figure 2 A is the detection of the expression levels of MAP2 and Neun in the differentiation of NSCs after circCdyl overexpression by RT - qPCR and statistical analysis; Figure 2B was used to detect the expression levels of Tuj1 protein and MAP2 protein during the differentiation of NSCs after overexpression of circCdyl by Western Blot, and statistical analysis was performed; Figure 2 C was used to detect Tuj1-positive cells in the circCdyl overexpression group and the control group by immunofluorescence and perform statistical analysis. Scale bar = 100 μm; Figure 2 D was used to detect Nissl-positive cells in the circCdyl overexpression group and the control group by flow cytometry and perform statistical analysis. *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 3 For the construction of the rat fimbria-fornix transection model and the identification of lentivirus infection with overexpressed circCdyl: Among them, Figure 3 A is a schematic diagram of model transection; Figure 3 B is Nissl staining; Figure 3 C was used to detect the expression level of circCdyl in the rat fimbria-fornix transection injury model by RT-qPCR; Figure 3 D is a schematic diagram of the infection efficiency of circCdyl. *P<0.05, **P<0.01, ***P<0.001;

[0034] Figure 4 It is the Morris water maze behavior trajectory map of the Morris water maze behavioral experiment in Example 3; Figure 5 For the Morris water maze behavioral experiment in Example 3, the escape latency, the path length to find the platform, and the movement speed were detected and statistically analyzed;

[0035] Figure 6 For the Morris water maze behavioral experiment in Example 3, after removing the platform on the fifth day, the number of times of crossing the platform, the platform residence time, and the path length were detected and statistically analyzed. *P<0.05, **P<0.01, ***P<0.001;

[0036] Figure 7 It is the influence diagram of the step-through avoidance experiment: Figure 7 A, Figure 7 B was used to detect the step-through latency in the step-through avoidance experiment and perform statistical analysis; Figure 7 C, Figure 7 D was used to detect the active avoidance rate in the shuttle experiment and perform statistical analysis. *P<0.05, **P<0.01, ***P<0.001. Detailed implementation manners

[0037] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0038] Embodiments of the present invention provide an application of circCdyl in the preparation of drugs for treating related neurodegenerative diseases, which is manifested in that the drug overexpresses circCdyl and promotes the proliferation and differentiation of hippocampal NSCs. To verify the application of circCdyl provided by the present invention in the preparation of drugs for treating related neurodegenerative diseases, specific embodiments are provided below for further illustration.

[0039] Example 1. Construction of circCdyl lentiviral vector

[0040] 1. Vector digestion: The vector used is pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector (8.8 kb, the maximum insertable gene is 1.7 kb, with EGFP fluorescence and Puro resistance. If wpre is removed, a 2.3 kb gene can be inserted). The endonucleases are EcoR I and Sal I endonucleases. The digestion temperature is 37 °C and the digestion time is 3 hours.

[0041] Among them, the 20 μl digestion system: 4 μl pCDNA3.1 vector (500 ng / μl), 1 μl Sal I, 1 μl EcoRI, 2 μl 10× buffer, 12 μl H2O.

[0042] 2. Gel recovery of digestion products: Agarose gel electrophoresis method is used for recovery.

[0043] Experimental instruments: Agarose gel electrophoresis system; ultraviolet observation analyzer; centrifuge; single-sided blade; constant temperature water bath.

[0044] Reagents: DNA recovery kit; 50× TAE (electrophoresis buffer Tris-acetate); ddH2O; agarose gel.

[0045] Steps:

[0046] 1) Prepare an agarose gel using TAE buffer, and then perform agarose gel electrophoresis on the digestion products (for separating DNA).

[0047] 2) Cut the agarose block containing DNA under ultraviolet light, and remove as much excess agarose as possible. Put it into a 1.5 ml centrifuge tube.

[0048] 3) Add 500 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0). 1 ml of 1 M Tris-HCl (pH 8.0), 0.2 ml of 0.5 M EDTA (pH 8.0), add ddH2O to 100 ml) to 100 mg of agarose in a ratio of 300 - 600 μl per 100 mg of agarose. Place it in a 65°C water bath for 10 minutes to completely dissolve the agarose block. During this period, invert and mix it every 2 minutes to promote dissolution.

[0049] 4) Transfer the melted agarose solution into the adsorption column, centrifuge at 12,000 rpm at room temperature for 1 minute, and pour out the liquid in the collection tube. Then place the adsorption column back into the same collection tube.

[0050] 5) Add 500 μl of wash buffer to the adsorption column, let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm at room temperature for 30 seconds, pour out the liquid in the collection tube, and place the adsorption column back into the same collection tube.

[0051] 6) Add another 500 μl of wash buffer to the adsorption column, centrifuge at 12,000 rpm at room temperature for 15 seconds, pour out the liquid in the collection tube, and place the adsorption column back into the same collection tube.

[0052] 7) Centrifuge at 12,000 rpm at room temperature for 1 minute without sample.

[0053] 8) Place the adsorption column into a clean 1.5 ml centrifuge tube, add 30 μl of elution buffer to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm at room temperature for 1 minute (for higher recovery efficiency, it can be eluted again), and store the 1.5 ml centrifuge tube (DNA) at -20°C.

[0054] 3. Acquisition of target fragment: Design primers for PCR of circCdyl:

[0055] Forward primer: 5’-CTTAGCTGTTAACGGGAAA-3’,

[0056] Reverse primer: 5’-CTGTTGAAGTCGTGGATGT-3’;

[0057] PCR reaction system: ddH2O 12.4 μl, 5×Taq buffer 4 μl, dNTPs (2.5 mM) 1.6 μl, Primer(+)(10 μM) 0.4 μl, Primer(-)(10 μM) 0.4 μl, Template(10 ng / μL) 1 μl, Taq polymerase 0.2 μl

[0058] PCR reaction conditions: 94°C for 5 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 2 min. The above three steps are cycled 35 times, and then 72°C for 10 min.

[0059] 4. Recovery of the PCR product of the target gene: The same as 2 above.

[0060] 5. Digestion and recovery of the target gene: The same as 1 and 2 above.

[0061] 6. Ligation of the target gene and the vector:

[0062] (1) Set the temperature of the dry bath incubator (or the water in the ice box) at 25°C and 42°C in advance;

[0063] (2) Take 4 sterilized 200 μl microcentrifuge tubes and add: dd H2O 10.5 μl, 10×In-Fusion swap enzyme buffer 2 μl, In-Fusion swap enzyme 0.5 μl, linearized vector DNA 1 μl (about 50 ng); purified PCR product fragment 6 μl (about 300 ng)

[0064] (3) After gently shaking the above mixture and then briefly centrifuging, place it in a 25°C dry bath incubator (or 25°C water) for 30 minutes, and then transfer it to 42°C for 15 minutes;

[0065] (4) The ligated product can be immediately used to transform competent cells or stored at 4°C in the refrigerator for later use.

[0066] 7. Preparation of competent cells:

[0067] (1) Prepare the following solutions:

[0068] (1-1) 0.1 M CaCl2 solution, filtered and sterilized with a 0.22 μm filter;

[0069] (1-2) 250 mM KCl2 solution;

[0070] (1-3) 2 M MgCl2 solution, autoclaved;

[0071] (1-4) SOB: Add 1 ml of 250 mM KCl2 solution to 100 ml of LB, adjust the pH value to 7.0 with 5 M NaOH, autoclave, and add 0.5 ml of 2 M MgCl2 solution before use;

[0072] (2) Prepare fresh E. coli competent cells using calcium chloride

[0073] (2-1) Pick a single colony from a fresh plate cultured at 37°C for 16 hours and transfer it to a 1 L flask containing 100 ml of LB medium. Incubate with vigorous shaking at 37°C for 3 hours (rotary shaker, 300 rpm);

[0074] (2-2) Transfer the bacteria to a sterile, disposable, ice-precooled 50 ml polypropylene tube under sterile conditions and place it on ice for 10 minutes to cool the culture to 0°C;

[0075] (2-3) Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells;

[0076] (2-4) Pour out the culture medium, invert the tube for 1 minute to allow the last trace of culture medium to drain;

[0077] (2-5) Resuspend each pellet in 10 ml of ice-precooled 0.1 mol / L CaCl2 and place it in an ice bath;

[0078] (2-6) Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells;

[0079] (2-7) Pour out the culture medium, invert the tube for 1 minute to allow the last trace of culture medium to drain;

[0080] (2-8) Resuspend each cell pellet in 2 ml of ice-precooled 0.1 mol / L CaCl2 per 50 ml of the initial culture;

[0081] (2-9) Aliquot the cells and store them at -70°C.

[0082] 8. Transformation:

[0083] 1) Using a cooled sterile pipette tip, transfer 200 μL from each competent cell suspension to a sterile microcentrifuge tube. Add 10 μL of the ligation solution to each tube and gently rotate to mix the contents. Place on ice for 30 minutes;

[0084] 2) Place the tubes on an EP tube rack placed in a circulating water bath pre-warmed to 42°C and leave for exactly 90 seconds without shaking the EP tube rack;

[0085] 3) Quickly transfer the tubes to an ice bath to cool the cells for 1 - 2 minutes;

[0086] 4) Add 800 μL of LB medium to each tube, warm the medium to 37°C using a water bath, then transfer the tubes to a 37°C shaker and incubate for 45 minutes to allow the bacteria to recover;

[0087] 5) Transfer 150 μL of the transformed competent cells to an LB agar medium with AMP resistance (100 μg / ml);

[0088] 6) Place the plate at room temperature until the liquid is absorbed.

[0089] 7) Invert the petri dish and incubate at 37 °C for 16 hours.

[0090] 9. Small-scale plasmid preparation (alkaline lysis method for plasmid DNA extraction)

[0091] 1) Pick a single colony and add it to 3 ml of LB medium containing the corresponding antibiotic, and culture it with shaking overnight.

[0092] 2) Centrifuge 1.5 ml of the bacterial solution at 12,000 rpm for 30 s in an EP tube to collect the bacteria. Add 100 μl of Solution I containing RNase and mix by shaking. Add 200 μl of Solution II and gently invert to mix. Incubate on ice for 2 min. Then add 200 μl of Solution III, mix quickly to denature, and incubate on ice for 10 min.

[0093] 3) After centrifuging at 12,000 rpm for 10 min, transfer the supernatant to a DNA purification column.

[0094] 4) After centrifuging at 12,000 rpm for 1 min, wash twice with the washing solution.

[0095] 5) After centrifuging at 12,000 rpm, air-dry and add 30 μl of the elution buffer, and let it stand for 1 min.

[0096] 6) Centrifuge again at 12,000 rpm for 1 min to collect the plasmid.

[0097] Note: Solution I: 50 mM (mmol / L) glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0); 12.5 ml of 1 M Tris-HCl (pH 8.0), 10 ml of 0.5 M EDTA (pH 8.0), 4.730 g of glucose, add ddH2O to 500 ml; autoclave at 10 lbf / in2 for 15 min and store at 4 °C. Solution II: 0.2 N NaOH, 1% SDS. 1 ml of 2 N NaOH, 1 ml of 10% SDS, add ddH2O to 10 ml. Prepare it freshly before use (the role of NaOH is to lyse the cells); Solution III: potassium acetate (KAc) buffer, pH 4.8; 300 ml of 5 M KAc, 57.5 ml of glacial acetic acid, add ddH2O to 500 ml.

[0098] 10. Identification of recombinant plasmid clones

[0099] 1) Identification by PCR method: Using the recombinant plasmid as a template, perform PCR amplification with specific primers for the PCR product or universal primers for the vector, and then identify by electrophoresis.

[0100] 2) Restriction enzyme digestion identification: the same as 1 and 2 above.

[0101] 11. Virus packaging

[0102] (1) Cultivation of 293T cells

[0103] Label the cell name, cell passage number, time, and operator on a 10 cm cell culture dish. Aspirate approximately 2.5×10 6 293FT cells into a 15 ml centrifuge tube, then add complete medium to 10 ml and mix well. Then transfer the mixed 293FT cells into a 10 cm culture dish. Place it in an incubator and culture for 48 h.

[0104] (2) Cell passage

[0105] (2-1) Discard the old culture medium, add 5 ml of sterilized PBS solution, gently shake, wash the cell growth surface, and then discard the PBS solution.

[0106] (2-2) Add 2 ml of trypsin digestion solution and digest for 1 - 2 min until the cells are completely digested.

[0107] (2-3) Add 5 ml of DMEM medium containing 10% fetal bovine serum and 100 U / ml double antibody, pipette several times with a graduated pipette to wash down the cells on the bottle wall.

[0108] (2-4) After mixing the cells, divide them into two new culture bottles and continue culturing.

[0109] (3) Cell transfection

[0110] (3-1) 24 h before transfection, digest 293T cells in the logarithmic growth phase with trypsin and adjust the cell density to 1.2x10 7 cells / 20 ml with medium containing 10% serum. Re-seed them into a 15 cm cell culture dish and culture in an incubator at 37℃ and 5% CO2. When the cell density reaches 70% - 80% after 24 h, it can be used for transfection;

[0111] (3-2) Replace the cell medium with serum-free medium 2 h before transfection;

[0112] (3-3) Take out two 1.5 ml centrifuge tubes. In one tube, add 142 ul of serum-free medium and 58 ul of TRL transfection reagent, and pipette to mix well; in the other tube, add 18 ul of packaging plasmid, 10 ul of target plasmid, and serum-free medium to a total volume of 200 ul and mix well. Pipette the liquids in the two centrifuge tubes to mix well and let it stand at room temperature for 20 minutes;

[0113] (3-4) After 20 minutes, take out the 293FT cells from the incubator. Gently drip the mixture into the culture dish with a 200 μl pipette tip to avoid cell floating during transfection. Then gently mix in a cross pattern and place it in the incubator for 8 h;

[0114] (3-5) After 8 h of cell culture, aspirate the original culture medium and slowly add 10 ml of complete culture medium along the wall of the dish. Continue to culture in a 37 °C, 5% CO2 incubator for 48 hours;

[0115] (4) Harvest and concentration of virus

[0116] (4-1) Collect the supernatant of 293T cells 48 hours after transfection (counting from 0 hour of transfection). Centrifuge at 4 °C, 4000 g for 10 min to remove cell debris;

[0117] (4-2) Filter the supernatant through a 0.45 μm filter into a 40 ml ultracentrifuge tube;

[0118] (4-3) Add the crude virus extract sample to the filter cup (up to 19 ml) and cover the lid. Insert the filter cup into the filtrate collection tube;

[0119] (4-4) Centrifuge at 4000 g until the required virus concentration volume is reached. Usually, the required time is 10 - 15 minutes;

[0120] (4-5) After centrifugation, take out the centrifugation device and separate the filter cup from the lower filtrate collection cup;

[0121] (4-6) Invert the filter cup on the sample collection cup. Centrifuge at a centrifugal force not exceeding 1000 g for 2 minutes. The virus concentrate is in the sample collection cup;

[0122] (4-7) Transfer the virus concentrate, aliquot it and store it in a virus tube at -80 °C for long-term storage. Take one of them for virus biological titer determination;

[0123] (5) Lentivirus titer determination

[0124] (5-1) One day before determination, seed 293T cells into a 96-well plate, add 4 × 104 cells per well with a volume of 100 μl;

[0125] (5-2) Prepare 7 - 10 sterile Ep tubes according to the expected titer of the virus. Add 90 μl of serum-free culture medium to each tube.

[0126] (5-3) Take 10 μl of the virus stock solution to be determined and add it to the first tube. After mixing, take 10 μl and add it to the second tube. Continue the same operation until the last tube.

[0127] Description:

[0128] Add 10 μl of the original virus solution into the first Eppendorf tube, denoted as 1E+1 μl;

[0129] Perform the first ten-fold dilution in the second Eppendorf tube. The resulting original virus solution is 1 / 10 of that in the first Eppendorf tube, denoted as 1E+0 μl;

[0130] Perform the second ten-fold dilution in the third Eppendorf tube. The resulting original virus solution is 1 / 10 of that in the second Eppendorf tube, denoted as 1E-1 μl;

[0131] And so on...

[0132] Perform the sixth ten-fold dilution in the seventh Eppendorf tube. The resulting original virus solution is 1 / 10 of that in the sixth Eppendorf tube, denoted as 1E-5 μl;

[0133] Perform the seventh ten-fold dilution in the eighth Eppendorf tube. The resulting original virus solution is 1 / 10 of that in the seventh Eppendorf tube, denoted as 1E-6 μl;

[0134] (5-4) Select the required cell wells, aspirate 90 μl of the culture medium, discard it, and add 90 μl of the diluted virus solution. Place it in the incubator for culture;

[0135] (5-5) After 24 hours, add 100 μl of the complete culture medium;

[0136] (5-6) After 4 days, observe the fluorescence expression. The number of fluorescent cells decreases with the increase of the dilution factor;

[0137] (6) Titer calculation:

[0138] According to the GFP expression, for example, 2 fluorescent cells are observed in the well with 1E-6 μl of the original virus solution added, indicating that at least 2 virus particles have infected the cells in this well. Then the titer of this virus is equal to the number of fluorescent cells divided by the amount of the original virus solution, that is, 2 / (1E-6) = 2E+6, with the unit of TU / μl, which is also equal to 2E+9 TU / ml.

[0139] Example 2: Gene overexpression experiment

[0140] (1) In vitro culture of neural stem cells (NSCs)

[0141] (1-1) After autoclaving the instruments at high temperature and high pressure, intraperitoneally inject a compound anesthetic into pregnant SD rats on the 15th day of pregnancy;

[0142] (1-2) After the pregnant rats are anesthetized, spray and disinfect with alcohol and expose the abdominal cavity. Use hemostatic forceps to lift the fur of the pregnant rats, cut it open with surgical scissors, and use ophthalmic forceps to take out the uterus containing the embryos and place it in a culture dish containing 75% ethanol;

[0143] (1-3) Use forceps with teeth to remove the uterus, transfer the embryo to a culture dish containing DMEM / F-12, and perform the whole process on an ice box;

[0144] (1-4) Take out the whole brain of the embryonic mouse in the culture dish and isolate the hippocampal region, then transfer it to a new culture dish containing DMEM / F-12. This operation is also carried out on an ice box throughout the process;

[0145] (1-5) Aspirate the hippocampal tissue in the culture dish and transfer it to a new centrifuge tube, pipette repeatedly until it shows a milky state, centrifuge at 1200 r / min for 3 min; discard the supernatant, add DMEM / F-12, pipette and mix well, centrifuge at 1200 r / min for 3 min, and repeat twice;

[0146] (1-6) Discard the supernatant, add NSCs proliferation medium, pipette to resuspend the cells, filter through a cell strainer with a pore size of 40 μm, then transfer to a culture flask, observe under a microscope, and then place it in an incubator (37 °C, 5% CO2) for 5-7 d;

[0147] (2) Passage

[0148] (2-1) Aspirate the suspension in the culture flask and place it in a 15 ml centrifuge tube, centrifuge at 1200 r / min for 3 min;

[0149] (2-2) Discard the supernatant, add 0.25% Trypsin-EDTA to the centrifuge tube for digestion at 37 °C for 3 min, then add an equal volume of complete medium to the centrifuge tube to terminate digestion, centrifuge at 1200 r / min for 3 min; discard the supernatant, resuspend the cells with D-PBS, centrifuge at 1200 r / min for 3 min;

[0150] (2-3) Discard the supernatant, aspirate an appropriate amount of complete medium to resuspend, filter through a 40 μm filter, and equally distribute it into 2-3 new culture flasks, then place it in an incubator (37 °C, 5% CO2) for continued culture.

[0151] (3) Lentivirus transfection

[0152] (3-1) After plating NSCs, observe the cell growth status;

[0153] (3-2) Discard the original medium and add fresh complete medium;

[0154] (3-3) Add overexpressing lentivirus or negative control empty vector lentivirus;

[0155] (3-4) After virus infection, incubate in a cell culture incubator (37 °C, 5% CO2);

[0156] After (3 - 5) 24 h or 48 h, remove the virus, discard the culture medium, replace it with fresh culture medium, and continue culturing in an incubator (37 °C, 5% CO2).

[0157] (4) Immunofluorescence assay

[0158] (4 - 1) After culturing the cells in (3) for 10 days, remove the culture solution from the culture plate and wash 3 times with 1×PBS.

[0159] (4 - 2) Fix with 4% paraformaldehyde at room temperature for 15 - 20 min, wash 3 times with 1×PBS, place on a shaker and shake slowly, 5 min / time.

[0160] (4 - 3) Block with the blocking solution at room temperature for 2 h.

[0161] (4 - 4) Wash 3 times with 1×PBS, dilute the primary antibody proportionally with the antibody diluent, and incubate overnight at 4 °C.

[0162] (4 - 5) Wash 3 times with 1×PBS, 5 min / time, shake slowly on a shaker, dilute the secondary antibody proportionally with the antibody diluent, and incubate at room temperature for 2 h (protected from light).

[0163] (4 - 6) Wash 3 times with 1×PBS, 5 min / time, shake slowly on a shaker, incubate with Hoechst 33342 live cell staining solution at room temperature for 10 min (protected from light).

[0164] (4 - 7) Wash 3 times with 1×PBS, 5 min / time, protected from light, drop the mounting medium on the glass slide, take out the cell coverslip from the 24 - well culture plate, and invert it onto the glass slide.

[0165] (4 - 8) Observe the cells using a ZEISS Axio Scope A1 microscope and process the images using Photoshop CS6 software.

[0166] (5) Western Blot experiment

[0167] (5 - 1) Protein extraction:

[0168] ① Aspirate the culture solution in the 6 - well culture plate, rinse 2 times with 1×PBS, and avoid cell floating.

[0169] ② Add 150 μl / well of cell lysis buffer, place in a 1.5 - ml centrifuge tube, and put on ice.

[0170] ③ Centrifuge at 13000 g / m at 4 °C for 30 min, and aspirate the supernatant.

[0171] ④ Protein concentration determination: Use a BCA protein concentration assay kit for protein quantification, and use a multifunctional microplate reader to measure the absorbance and calculate the protein concentration;

[0172] ⑤ Add 5× protein loading buffer in proportion, boil at 100 °C for 8 min, and after cooling, it can be aliquoted and stored at -80 °C for later use.

[0173] (5-2) Electrophoresis:

[0174] ① Prepare the gel: Pour the prepared separating gel into the gap between the glass plates, add isopropanol on the upper layer of the gel to seal, let it stand for 30 min, and discard the upper solution after the gel solidifies; Add the prepared stacking gel, insert the comb, and the stacking gel will solidify in about 40 min;

[0175] ② Place the gel plate into the electrophoresis tank (if running one gel, a plastic plate should be placed on the other side of the electrophoresis tank);

[0176] ③ Add enough electrophoresis buffer into the electrophoresis tank, and add the same amount of each group of proteins and pre-stained protein Marker to each lane;

[0177] ③ Adjust the voltage, the voltage of the stacking gel is 80 V. After the bromophenol blue indicator in the sample migrates to the junction of the stacking gel and the separating gel, change to 120 V and continue electrophoresis until the Marker just runs out of the gel to terminate electrophoresis.

[0178] (5-3) Transfer membrane:

[0179] Activate the cut PVDF membrane with anhydrous methanol, and place the filter paper, gel, and PVDF membrane in sequence, 200 mA, 60 min;

[0180] (5-4) Blocking:

[0181] After the transfer membrane is completed, cut the PVDF membrane into the required size according to the protein molecular weight indicated by the Marker, wash it 3 times in TBST solution, 6 min each time; Place it in 5% skim milk and gently shake and block it at room temperature for 2 h;

[0182] (5-5) Incubate with primary antibody

[0183] ① Place the PVDF membrane in TBST solution and shake it for 3 times, 5 min each time;

[0184] ② Dilute the primary antibody with the blocking solution, add it to the front of the membrane, and incubate overnight at 4 °C;

[0185] (5-6) Incubate with secondary antibody

[0186] ① Take out the membrane, shake it in TBST for 3 times, 5 min each time;

[0187] ② Prepare the secondary antibody with the blocking solution and incubate it at room temperature for 2 h;

[0188] (5-7) Development and analysis

[0189] ① Aspirate the secondary antibody, wash it 3 times with TBST for 5 min each time, mix it in proportion with the ECL kit, and mix well.

[0190] ② Cover the PVDF membrane with the developer, collect the image with the Chemidoc XPS chemiluminescence imaging system, analyze the optical density using Quantity One software, and perform statistical analysis with GraphPad Prism 6.0 software.

[0191] The experimental results are as Figure 2 shown. CircCdyl was overexpressed in cultured hippocampal NSCs by lentiviral transfection and induced to differentiate. The results of RT-qPCR and Western Blot detection showed that compared with the control group, the mRNA and protein expressions of neuron-related markers in the circCdyl overexpression group were significantly up-regulated ( Figure 2 A and Figure 2 B). Immunofluorescence staining experiments found that the proportion of Tuj1-positive cells in the circCdyl overexpression group increased significantly ( Figure 2 C). Flow cytometry detection results showed that the number of Nissl-positive cells in the circCdyl overexpression group increased ( Figure 2 D).

[0192] Example 3. In vivo experiment to detect the improvement of learning and memory function in a rat model of fimbria-fornix transection after overexpression of circCdyl

[0193] (1) Establish a rat model of fimbria-fornix transection

[0194] Take adult female SD rats, 10 rats in each group, divided into Normal group, Sham group, Transected group, Transected + LV-NC group, Transected + LV-circ group; after anesthesia, fix the rats on the stereotaxic apparatus, remove the hair on the skull top, disinfect, incise the skin on the skull top, expose the skull, cut the periosteum, and find the bregma; starting from the bregma, go back 3.6 mm, 1.4 mm and 4 mm to the left, and 1.4 mm and 4 mm to the right, a total of 4 points, mark them; use a high-speed skull drill to make a fine slit at the marked part of the skull, use a fine needle to advance 5 mm in depth, and then make left and right cuts; after cutting and stopping bleeding, suture the skin and apply iodophor to the wound.

[0195] (2) Injecting virus: Select adult SD rats with a transected fimbria-fornix hippocampal model. After anesthesia, fix the rats on a stereotaxic apparatus for the brain, remove the hair on the cranial vertex, disinfect, incise the skin on the cranial vertex to expose the skull, cut the periosteum, and locate the bregma. Locate the dentate gyrus of the hippocampus: 1.39 mm posterior to the bregma, 1.8 mm lateral to the midline on both sides, and mark it. Use a high-speed cranial drill to make a small hole at the marked position, insert a micro-injection pump to a depth of 3.9 mm, and inject 5 μL of negative control virus and circCdyl overexpression virus into the dentate gyrus region of the hippocampus respectively. After injection, keep the needle in place for 10 minutes. Pull out the needle, wait for hemostasis, suture the skin, and apply iodophor to the wound.

[0196] The construction of a rat denervated hippocampal injury model and the identification of circCdyl lentiviral infection are as Figure 3 shown. To study the effect of circCdyl on the hippocampal microenvironment in vivo and its potential role in nerve regeneration therapy, we constructed a rat denervated hippocampal injury model by cutting the fimbria-fornix hippocampal Figure 3 (A). Nissl staining showed that Nissl cells in the transected hippocampal region were absent, indicating successful model construction Figure 3 (B). RT-qPCR analysis showed that the expression level of circCdyl in the hippocampus of rats decreased after cutting the fimbria-fornix hippocampal Figure 3 (C). Then, we injected circCdyl overexpression lentivirus into the dentate gyrus of the hippocampus of model animals Figure 3 (D).

[0197] (3) Morris water maze behavioral experiment: Experiment preparation: Fill a black circular pool with water (pool diameter 200 cm, height 60 cm, platform diameter 10 cm, placed 3 cm below the water surface, water depth 50 cm, water temperature 25 °C, camera located directly above the center of the pool). Divide the above five groups of adult SD rats after modeling (10 rats in each group) into batches, place them head-down along the pool wall one by one, select four different quadrants of front, back, left, and right as the starting points, and record the time, path length, and time for the rats to find the platform. If the experimental animals take more than 2 minutes to find the platform on the first day of training, guide them to the platform and let them stay still for 30 seconds. Continuously train for 5 days, remove the platform on the 5th day, record data such as the number of times the experimental animals cross the platform, and organize and analyze the escape latency of the time for the rats to find the platform, the path, speed, time of staying in the platform quadrant, and the number of times of crossing the platform.

[0198] In the Morris water maze behavioral test, the latency, path length, and movement speed of the rats to find the platform were detected in the first 4 days. The results showed that compared with the LV-NC group, the escape latency and path length of the LV-circ group decreased over time, while there was basically no difference in the movement speed Figure 4 andFigure 5 ), there were significant differences between the two groups statistically; on the 5th day, the platform was removed, and the number of times of crossing the platform, the residence time on the platform and the path length of each group were detected respectively. The results showed that compared with LV-NC, these parameters were significantly up-regulated in LV-circ( Figure 6 ).

[0199] (4) Step-through passive avoidance test: Experimental preparation, ScienCell step-through passive avoidance apparatus, temperature 23 °C, adult SD rats after modeling, divided into 5 groups, Normal group, Sham group, Transected group, Transected + LV-NC group, Transected + LV-circ group, 10 rats in each group; on the first day of training, the adult SD rats after modeling were placed in the light box and given light stimulation. Due to the natural tendency of animals to avoid light, they would enter the dark box autonomously. After being in the dark box, the shuttle channel was closed and electric stimulation was given, and the test time was 5 min; the training was repeated continuously for 3 days. Based on the electric stimulation punishment on the first day of training, the experimental animals would selectively avoid entering the dark box, and the latency from entering the light box to the dark box was recorded as the detection index of short-term memory. The step-through passive avoidance test detected the step-through latency of rats, and the results showed that the step-through latency in the LV-circ group was significantly longer than that in the LV-NC group( Figure 7 A and Figure 7 B)

[0200] (5) Shuttle box test: Experimental preparation, ScienCell shuttle box apparatus, temperature 23 °C, adult SD rats after modeling, divided into 5 groups, Normal group, Sham group, Transected group, Transected + LV-NC group, Transected + LV-circ group, 10 rats in each group; on the first day of training, the adult SD rats after modeling were allowed to move freely in the test box for 5 min, and then placed in the shock area of the shuttle box, and given light stimulation and sound stimulation. If they escaped to the safe area before the end of the light stimulation and sound stimulation time, it was considered active avoidance. If they escaped to the safe area after the end of the stimulation time through electric shock, it was considered passive avoidance. After multiple trainings, the experimental animals could gradually form an active avoidance conditioned response and thus obtain memory; the training was continued for 3 days, the method was the same as that on the first day of training, the number of cycles was 30 times, and the test time was 15 min; the number of active avoidance times, avoidance times and passive avoidance times of different groups were recorded, and the data were statistically analyzed. The shuttle box test detected the active avoidance rate of rats, and it was found that the active avoidance rate in the LV-NC group was significantly lower than that in LV-circ( Figure 7 C and Figure 7 D). Therefore, the experimental results of the above Morris water maze test and step-through passive avoidance test showed that circCdyl affected the hippocampal microenvironment in vivo and had a certain effect on nerve regeneration therapy.

[0201] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. The use of circCdyl in the preparation of drugs for treating related neurodegenerative diseases, characterized in that, The drug overexpresses circCdyl, promoting the proliferation and differentiation of hippocampal NSCs.

2. Use of circCdyl according to claim 1 in the preparation of a medicament for treating related neurodegenerative diseases, characterized in that, It includes the following processes: S1. Construct a lentiviral vector overexpressing circCdyl: Construct circCdyl on the pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector. S2. Detect the effect of circCdyl on the proliferation of hippocampal NSCs in vitro: Transfect the lentivirus carrying circCdyl constructed in step S1 into hippocampal NSCs to increase the intracellular circCdyl level; Use flow cytometry to analyze the mitotic phase of cells and CCK8 to detect cell proliferation activity. S3. Detect the effect of circCdyl on the differentiation of hippocampal NSCs in vitro: Transfect the lentivirus carrying circCdyl constructed in step S1 into hippocampal NSCs, and then induce differentiation. After induction of differentiation, detect the mRNA and protein expression levels of neuron-related markers by RT-qPCR and Western Blot. S4. Detect the improvement of learning and memory function in a rat fimbria-fornix hippocampal lesion model after overexpressing circCdyl in vivo: Construct a rat fimbria-fornix hippocampal lesion model, inject the lentivirus overexpressing circCdyl into the DG area for infection, and then perform Morris and step-through avoidance behavioral tests to observe whether the learning and memory function after injury is improved.

3. Use of circCdyl according to claim 2 in the preparation of a medicament for treating related neurodegenerative diseases, characterized in that, The construction of the lentiviral vector overexpressing circCdyl in step S1 includes the following steps: Vector digestion: The vector used is the pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector, the endonucleases are EcoR I and Sal I endonucleases, the digestion temperature is 37 °C, and the digestion time is 3 hours; Use agarose gel electrophoresis for recovery. Acquisition of the target fragment: Design primers for PCR of circCdyl: Forward primer: 5’-CTTAGCTGTTAACGGGAAA-3’, Reverse primer: 5’-CTGTTGAAGTCGTGGATGT-3’; Recover the PCR product of the target gene; Digest and recover the target gene; Ligate the target gene with the vector; Prepare competent cells; Transformation; Small-scale plasmid preparation; Identification of recombinant plasmid clones; Virus packaging.

4. Use of circCdyl according to claim 2 in the preparation of a medicament for treating related neurodegenerative diseases, characterized in that The detection of the effect of circCdyl on the proliferation of hippocampal NSCs in vitro in step S2 includes the following processes: (2-1) In vitro culture of neural stem cells: After high-temperature and high-pressure steam sterilization of the instruments, pregnant Sprague-Dawley rats on the 15th day of pregnancy were intraperitoneally injected with a compound anesthetic; after the pregnant rats were anesthetized, the abdominal cavity was disinfected by alcohol spraying and exposed. The pregnant rat's fur was lifted with hemostatic forceps, cut open with surgical scissors, and the uterus containing the embryo was removed with ophthalmic forceps and placed in a petri dish containing 75% ethanol; on an ice box, the uterus was dissected with toothed forceps, and the embryo was transferred to a petri dish containing DMEM / F-12. The whole brain of the embryonic mouse was removed in the petri dish and the hippocampal region was isolated and transferred to a new petri dish containing DMEM / F-12; the hippocampal tissue in the petri dish was aspirated and transferred to a new centrifuge tube, and repeatedly pipetted until it presented a milky state, centrifuged at 1200 r / min for 3 min; the supernatant was removed, DMEM / F-12 was added, pipetted and mixed evenly, centrifuged at 1200 r / min for 3 min, and repeated twice; the supernatant was discarded, NSCs proliferation medium was added, the cells were pipetted and resuspended, filtered through a cell strainer with a pore size of 40 μm, and then transferred to a culture flask. Observed under a microscope and then cultured in an incubator at 37 °C and 5% CO2 for 5-7 d; (2-2) Passage: The suspension in the culture flask was aspirated and placed in a 15 ml centrifuge tube, centrifuged at 1200 r / min for 3 min; the supernatant was discarded, 0.25% Trypsin-EDTA was added to the centrifuge tube for digestion at 37 °C for 3 min, and then an equal volume of complete medium was added to the centrifuge tube to terminate digestion, centrifuged at 1200 r / min for 3 min; the supernatant was discarded, D-PBS was added to resuspend the cells, centrifuged at 1200 r / min for 3 min; the supernatant was removed, an appropriate amount of complete medium was aspirated for resuspension, filtered through a 40 μm filter screen and equally distributed into 2-3 new culture flasks, and then cultured in an incubator at 37 °C and 5% CO2; (2-3) Lentiviral transfection: After plating the NSCs, observe the cell growth status; discard the original medium and add fresh complete medium; add overexpressing lentivirus or negative control empty lentivirus; after virus infection, incubate in a cell culture incubator at 37 °C and 5% CO2; after 24 h or 48 h, remove the virus, discard the medium, replace it with fresh medium, and continue to culture in an incubator at 37 °C and 5% CO2; (2-4)Immunofluorescence detection: After culturing the cells in (2-3) for 10 days, remove the culture medium in the culture plate and wash it 3 times with 1×PBS; fix it with 4% paraformaldehyde at room temperature for 15-20 min, wash it 3 times with 1×PBS, place it on a shaker and shake it slowly, 5 min / time; block it with the blocking solution at room temperature for 2 h; wash it 3 times with 1×PBS, dilute the primary antibody proportionally with the antibody diluent, and incubate it overnight at 4°C; wash it 3 times with 1×PBS, 5 min / time, shake it slowly on the shaker, dilute the secondary antibody proportionally with the antibody diluent, and incubate it at room temperature for 2 h; wash it 3 times with 1×PBS, 5 min / time, shake it slowly on the shaker, incubate it with Hoechst 33342 live cell staining solution at room temperature for 10 min; wash it 3 times with 1×PBS, 5 min / time, protect from light, drop the mounting medium on the glass slide, take out the cell coverslip from the 24-well culture plate and invert it on the glass slide; observe the cells with a ZEISS Axio Scope A1 microscope and use Photoshop CS6 software to process the images; (2-5)Western Blot experiment: Protein extraction; Electrophoresis; Transfer membrane; Blocking; Incubation with primary antibody; Incubation with secondary antibody; Development and analysis.

5. Use of circCdyl according to claim 2 in the preparation of a medicament for treating related neurodegenerative diseases, characterized in that, (2-6)In the step S4, the improvement of the learning and memory function of the rat fornix hippocampal fimbria injury model after overexpressing circCdyl was detected by in vivo experiments, including the following processes: (4-1)Constructing a rat model with transected hippocampal fimbria: Take adult female SD rats, 10 rats in each group, divided into Normal group, Sham group, Transected group, Transected+LV-NC group, Transected+LV-circ group; After anesthesia, fix the rats on a stereotaxic apparatus for the brain, remove the hair on the cranial vertex, disinfect, incise the skin on the cranial vertex to expose the skull, cut the periosteum, and find the bregma; Starting from the bregma, 3.6 mm backward, 1.4 mm and 4 mm to the left, 1.4 mm and 4 mm to the right, a total of 4 points, mark them; Use a high-speed skull drill to make a fine slit at the marked position on the skull, use a fine needle to advance to a depth of 5 mm, and then cut left and right; After cutting and stopping bleeding, suture the skin and apply iodophor to the wound; (4-2)Injecting virus: Take adult female SD rats with transected hippocampal fimbria model. After anesthesia, fix the rats on a stereotaxic apparatus for the brain, remove the hair on the cranial vertex, disinfect, incise the skin on the cranial vertex to expose the skull, cut the periosteum, and find the bregma; Locate the hippocampal dentate gyrus: 1.39 mm backward from the bregma, 1.8 mm to the left and right, mark it; Use a high-speed skull drill to make a small hole at the marked position, advance the microinjection pump to a depth of 3.9 mm, and inject 5 μL of negative control virus and circCdyl overexpression virus into the hippocampal dentate gyrus region respectively. After the injection, keep the needle for 10 min; Pull out the needle. After stopping bleeding, suture the skin and apply iodophor to the wound; (4-3) Morris water maze behavioral experiment: After modeling, adult SD rats were placed head-down underwater one by one along the pool wall. Four different quadrants, namely the front, rear, left, and right, were selected as the starting points, and the time, path length, and time for the rats to find the platform were recorded. If the experimental animals took more than 2 minutes to find the platform during the first day of training, they were guided to the platform and left there for 30 seconds. Training was conducted continuously for 5 days. On the 5th day, the platform was removed, and the number of times the experimental animals crossed the platform was recorded. The escape latency, the path and speed of searching for the platform, the time staying in the platform quadrant, and the number of times crossing the platform of the rats were sorted out and analyzed. (4-4) Step-through avoidance behavioral experiment: On the first day of training, the adult SD rats after modeling were placed in the light box and given light stimulation. Due to the natural tendency of the animals to avoid light, they would enter the dark box on their own. After entering the dark box, the shuttle channel was closed, and an electric shock was given. The test time was 5 minutes. Training was repeated continuously for 3 days. Based on the electric shock punishment on the first day of training, the experimental animals would selectively avoid entering the dark box, and the latency of the time required to enter the dark box from the light box was recorded, which was used as an index for detecting short-term memory. (4-5) Shuttle box behavioral experiment: On the first day of training, the adult SD rats after modeling were allowed to move freely in the test box for 5 minutes, and then placed in the shock area of the shuttle box. Light stimulation and sound stimulation were given. If the rats escaped to the safe area before the end of the light and sound stimulation, it was considered active avoidance. If the rats escaped to the safe area after the end of the stimulation time through electric shock, it was considered passive avoidance. After multiple trainings, the experimental animals could gradually form an active avoidance conditioned response and thus obtain memory. Training was conducted continuously for 3 days, with the same method as on the first day of training. The number of cycles was 30 times, and the test time was 15 minutes. The number of active avoidance times, avoidance times, and passive avoidance times of different groups were recorded, and the data were statistically analyzed.

6. A pharmaceutical composition for treating related neurodegenerative diseases, characterized in that, The said pharmaceutical composition overexpresses circCdyl, promoting the proliferation and differentiation of hippocampal NSCs.

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