A method of inducing reprogramming of astrocytes into neurons

Abstract

The present application belongs to the field of biotechnology medicine, and particularly relates to a method for inducing reprogramming of astrocytes into neurons, wherein mouse brain tissue is cut and added with protease for digestion, filtered, inoculated and then placed in a constant temperature incubator for culture. After the cells reach the standard, they are subcultured, and the third generation of astrocytes is digested and resuspended for virus infection, and the infection conditions and MOI of the well-growing group are recorded. After the cell starvation treatment, the CMV-dcas9-VPR-mCherry virus is added first, and then the U6-gRNA-EGFP virus is added to overexpress Ngn2, Prdx2 and Sod1 genes, and the appropriate neuron culture medium is selected according to the induction time for culture; overexpression of Ngn2 can convert astrocytes into mature neurons with normal electrophysiological function. Additional overexpression of Prdx2 and Sod1 can improve the reprogramming efficiency, speed up the reprogramming and promote the maturation of neurons. The mechanism is that the process is accompanied by ferroptosis, and Prdx2 and Sod1 can reduce ferroptosis, thereby improving the reprogramming efficiency and long-term survival rate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and medicine, and in particular relates to a method for inducing astrocytes to reprogram into neurons. Background Technology

[0002] Irreversible neuronal death is the core pathological feature of nervous system injury. This permanent loss of cells directly leads to severe deficits in corresponding nerve functions. Traditional treatments, such as drug intervention or physical rehabilitation therapy, often fail to truly achieve the regeneration and functional reconstruction of nerve tissue. In recent years, directly reprogramming astrocytes into functional neurons has opened up a promising new avenue for the field of nerve repair.

[0003] However, despite the significant theoretical breakthrough of this strategy, existing reprogramming methods still face many key challenges in practical applications, such as generally low reprogramming efficiency, significant oxidative stress damage during the process, and severely insufficient survival rate of newly generated neurons. These issues greatly limit the feasibility and effectiveness of translating this technology into clinical practice. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a method for inducing astrocytes to reprogram into neurons.

[0005] To address the aforementioned technical problems, this invention provides the following technical solution: a method for inducing astrocytes to reprogram into neurons, comprising the following steps:

[0006] Astrocyte isolation and culture steps: The tissue is minced and placed into a centrifuge tube, and protease is added for digestion. Then, the cells are cultured in a complete culture medium to stop the digestion.

[0007] Astrocyte passage steps: Remove cells from the incubator, digest and resuspend cells once they reach the target size, and then seed them into new culture medium according to the cell count;

[0008] Steps for detecting the optimal virus MOI for astrocytes: Digest healthy third-generation astrocytes, resuspend them after digestion to prepare a cell suspension, and then seed them into a culture plate. Add the virus and continue culturing. After a certain period of virus infection, record the infection conditions and MOI corresponding to the group with good cell growth.

[0009] Viral infection steps: After removing the cells from the incubator and starving them, CMVenhancer-MCS-dcas9-VPR-FT2A-mCherry virus was added. After infecting the cells with this virus for a certain period of time, U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP virus was added to overexpress the Ngn2, Prdx2 and Sod1 genes. Depending on the infection time, different neuronal culture media were used for medium replacement.

[0010] Furthermore, in the astrocyte isolation and culture step, the proteases used are trypsin, collagenase, and DNAse I.

[0011] Furthermore, in the astrocyte isolation and culture step, the complete culture medium is a complete culture medium containing 10% fetal bovine serum.

[0012] Furthermore, the astrocyte passage step includes the following steps:

[0013] S1: Preheat PBS solution, trypsin, and astrocyte culture medium;

[0014] S2: Wash the qualified cells multiple times with PBS solution;

[0015] S3: Add trypsin to the culture flask, shake the flask, and place it in an incubator for digestion;

[0016] S4: When the intercellular spaces in the culture flask become wider and the cells become rounder, immediately add complete culture medium to stop digestion and repeatedly pipette the cells from the flask wall.

[0017] S5: Collect the cell suspension into centrifuge tubes, wash the tubes with PBS solution, and centrifuge them in a centrifuge.

[0018] S6: Discard the supernatant and add astrocyte culture medium to the centrifuge tube. Resuspend the cells, transfer an appropriate amount of cell suspension to a cell culture flask, and incubate in an incubator.

[0019] Furthermore, in S6, the temperature of the incubator is 37°C and the CO2 concentration is 5%.

[0020] Furthermore, in the astrocyte optimal virus MOI detection step, trypsin at a concentration of 0.25% is used for digestion.

[0021] Furthermore, in the step of detecting the optimal viral MOI for astrocytes, the volume of the cell suspension is 1–3 mL, and the density is 3 × 10⁻⁶. 4 ~5×10 4 per mL.

[0022] Furthermore, in the step of detecting the optimal virus MOI for astrocytes, the cells need to be cultured to a confluence of 20% to 30% before adding the virus.

[0023] Furthermore, in the viral infection step, before removing the cells from the incubator, the cells need to be digested and resuspended, and then cultured in the incubator until the cell density reaches 60% to 70%.

[0024] Furthermore, in the viral infection step, after the cells are removed from the incubator, they are washed with PBS solution, added with serum-free culture medium, and then starved for 2 hours.

[0025] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: Overexpression of transcription factor Ngn2 can transform astrocytes into mature induced neurons, and these induced neurons possess normal electrophysiological functions. Additional overexpression of mitochondrial antioxidant proteins Prdx2 and Sod1 can significantly improve the reprogramming efficiency of transcription factor Ngn2, increase the number of reprogrammed cells, accelerate the reprogramming speed, and simultaneously make the reprogrammed neurons morphologically more mature. Ferropysis occurs during reprogramming, and Prdx2 and Sod1 can reduce ferroptosis, thereby improving reprogramming efficiency and long-term survival rate. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a graph showing the expression levels of genes and proteins on day 7 of astrocyte reprogramming in this embodiment of the invention.

[0028] A. Expression levels of Ngn2 mRNA in three groups (real-time quantitative PCR); B. Expression levels of Prdx2 mRNA in three groups (real-time quantitative PCR); C. Expression levels of Sod1 mRNA in three groups (real-time quantitative PCR); D. Expression levels of Gpx4 mRNA in three groups (real-time quantitative PCR); E. Expression levels of Slc7a11 mRNA in three groups (real-time quantitative PCR); FI. Protein levels of Ngn2, Prdx2, and Sod1 in three groups, where F is the band diagram of Western blot, tubulin is the internal control of Western blot, and its core function is "control" and "calibration", while G, H, and I are the corresponding statistical graphs; JL. Protein levels of Gpx4 and Slc7a11 in three groups, where J is the band diagram of Western blot, tubulin is the internal control of Western blot, and its core function is "control" and "calibration", while K and L are the corresponding statistical graphs; Note: , , , There was no statistically significant difference in ns. The Ngn2 group was activated alone (VPR+Ngn2), the Ngn2, Prdx2, and Sod1 group was activated simultaneously (VPR+NPS), and the virus group was not activated (VPR+empty).

[0029] Figure 2 This is a fluorescence image of the generation of astrocyte reprogrammed neurons detected by immunofluorescence staining in the early stage of reprogramming in an embodiment of the present invention.

[0030] A. Representative images of immunofluorescence staining of β-type III tubulin (Tubb3) in three groups of cells on day 7 of reprogramming, scale bar = 20 μm;

[0031] B. Representative immunofluorescence staining images of β-type III tubulin (Tubb3) in three groups of cells on day 14 of reprogramming, scale bar = 20 μm;

[0032] C. On day 14 of reprogramming, the three groups of cells were reprogrammed into a bar chart of neuronal conversion rate;

[0033] Note: Activate the Ngn2 group alone (VPR+Ngn2), and activate the Ngn2, Prdx2, and Sod1 groups simultaneously (VPR+NPS), but do not activate the virus group (VPR+empty).

[0034] Figure 3 This is a fluorescence image of the generation of reprogrammed neurons in astrocytes detected by immunofluorescence staining on day 30 of reprogramming in this embodiment of the invention.

[0035] A. Representative images of three groups of cells stained with immunofluorescence for microtubule-associated protein 2 (Map2). Scale bar = 20 μm;

[0036] B. Representative images of immunofluorescence staining of three groups of cellular neuronuclear proteins (NeuN). Scale bar = 50 μm;

[0037] Note: Activate the Ngn2 group alone (VPR+Ngn2), and activate the Ngn2, Prdx2, and Sod1 groups simultaneously (VPR+NPS), but do not activate the virus group (VPR+empty).

[0038] Figure 4 This is an electrophysiological functional map of the induced neurons obtained by whole-cell patch-clamp detection on day 30 of reprogramming in this embodiment of the invention.

[0039] A. Representative image from whole-cell patch-clamp recording;

[0040] B. In voltage clamp mode, inward and outward currents were recorded when a step voltage stimulus was applied, n=3 cells;

[0041] C. In current-clamp mode, action potentials can be recorded when a step current is applied, with n=3 cells. Detailed Implementation

[0042] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0043] Example 1: This example discloses a method for inducing astrocytes to reprogram into neurons, comprising the following steps:

[0044] Isolation and culture of mouse cortical astrocytes:

[0045] Mice aged 3–6 days were cryo-anesthetized and disinfected by immersion in 75% alcohol for 3 minutes. The mouse skull was cut off with ophthalmic scissors and rinsed twice in a culture dish containing pre-chilled HBSS dissecting solution. Then, it was transferred to a new culture dish containing HBSS dissecting solution. The nose was held in place by curved forceps in the left hand, while the scalp and skull were cut along the midcranial suture in the right hand to fully expose the mouse's brain tissue. Then, the entire brain tissue was carefully removed from the base of the skull by curved forceps in the right hand and placed in another culture dish containing pre-chilled HBSS solution.

[0046] The cerebral cortex of the suckling mouse was separated along the midline using curved forceps. Then, under a stereomicroscope, the vascular membrane on the surface of the cortex was peeled off by holding microscope forceps with both hands, and the hippocampus was cut off. The tissue was transferred to a new container containing HBSS solution and washed 1-2 times.

[0047] Use iris scissors to mince the tissue into a paste, transfer it to a 15 mL centrifuge tube, place it on ice and put it into the cell transfer window;

[0048] After putting on a white coat, mask, and gloves as required, enter the cell culture room. Wipe the laminar flow hood with 75% alcohol swabs. Then, spray the surfaces of the consumables, reagents, and instruments required for the experiment (such as test tube racks, alcohol lamps, culture flasks, tweezers, pipettes, autoclaved micropipette tips, micropipettes, complete culture media, waste cups, etc.) with 75% medical alcohol spray and place them in the laminar flow hood. Turn on the ultraviolet lamp and irradiate for 30 minutes.

[0049] Remove a 15 mL centrifuge tube from the transfer window, add 0.25% trypsin, 2 mg / mL collagenase, and 2 mg / mL DNase I. The volume ratio of trypsin, collagenase, and DNase I is 1:1:0.05 (0.5 mL of trypsin is used for one mouse brain). Mix well by pipetting and place in a 37°C water bath for 10-15 min (remove the centrifuge tube every 5 min to observe the tissue digestion and gently shake to mix).

[0050] The digestion was then terminated using a complete culture medium containing 10% fetal bovine serum, and centrifuged at 1000 rpm for 5 min in a horizontal centrifuge.

[0051] After aspirating the supernatant from the centrifuge tube, add 10% complete culture medium to resuspend the cells. Filter the cell suspension through a 40 μm disposable cell sieve. Then, inoculate the filtered cell suspension into a T25 culture flask, add culture medium to 3 mL, spread the cells evenly using the figure-eight method, and place the culture flask in a constant temperature incubator at 37℃ and 5% CO2 for culture.

[0052] Change the medium on day 3, and then change the astrocyte culture medium every 2-3 days thereafter.

[0053] After about 6-7 days of culture, when the cell confluence reaches about 95%, the cells are placed in a constant temperature shaker at 260 rpm and 37 ℃ for 16 hours to remove microglia and oligodendrocytes, and then the cells are passaged.

[0054] Astrocyte passage: Clean bench preparation is the same as before;

[0055] Remove the cells from the incubator and observe their condition and density under a phase-contrast microscope. If the cells are in good condition and the density reaches more than 90%, they can be passaged.

[0056] Before passage, preheat the culture medium with 1×PBS, 0.25% trypsin, and astrocyte culture medium.

[0057] Use a pasteurized tube to aspirate the old culture medium, and then wash the cells 2-3 times with preheated 1×PBS solution.

[0058] Then add an appropriate amount of 0.25% trypsin to the culture flask, gently shake the flask to spread the trypsin evenly, and then immediately place it in an incubator for digestion for about 2 minutes;

[0059] Then, observe the culture flask under a phase contrast microscope. When the intercellular spaces widen and the overall shape becomes rounded, add complete culture medium immediately to stop digestion. Then, gently and repeatedly pipette the cells on the flask wall. The pipetting process should be carried out sequentially, starting from one side of the bottom of the culture flask and ending at the other side, so that the adherent cells can be detached as completely as possible.

[0060] Collect the cell suspension into a 15 mL centrifuge tube, wash the tube with 1×PBS 1-2 times, observe under a microscope whether the cells in the culture flask have been completely digested and detached, and then centrifuge at 800 rpm for 5 min.

[0061] Discard the supernatant, add 1 mL of astrocyte culture medium to the centrifuge tube, and gently resuspend the cells using a pipette. Based on the cell count, transfer an appropriate amount of cell resuspended solution to a T25 cell culture flask, add culture medium to 3 mL, label the culture flask, and then place the cells in a 37°C, 5% CO2 incubator for culture.

[0062] Cell count:

[0063] Gently place a coverslip over the cell counting chamber, and use a pipette to add a 10 μL drop of cell resuspended solution to the edge of the counting cell. The drop will then be drawn into the counting cell below the coverslip by suction. Allow the counting chamber to stand for a few minutes to allow the cells to diffuse and settle before counting the cells under a microscope.

[0064] Under a microscope, move the counting chamber and align the field of view with the counting area. Use a cell counter to count the total number of cells falling within the four large squares of the counting chamber, and then take the average. The counting principle is: "count the top, not the bottom; count the left, not the right." If multiple cells remain clumped without being dispersed, they should only be counted as one cell. If there are many clumps, it is necessary to re-disperse or even re-sample and digest until the vast majority of cells are single cells. The formula for calculating the cell count is: number of cells per milliliter of sample = average number of cells in each large square × 102 4 .

[0065] Detection of the optimal viral MOI for astrocytes (preliminary experiment):

[0066] MOI: Reinfection Index, refers to the ability of a virus to infect cells. The higher the MOI, the more difficult it is for the cells to be infected. Usually, the ratio of the number of virus particles to the number of cells required to infect 80% of a cell line is taken as the MOI of that cell line. MOI = (Virus titer x Virus volume) / Number of cells.

[0067] Cell seeding: Third-generation astrocytes in good growth condition were digested with 0.25% trypsin, then resuspended in complete culture medium to prepare 2 mL of culture medium with a density of 3-5 × 10⁻⁵ cells / mL. 4 100 μL of cell suspension per mL was added to 15 wells of a 96-well plate, with 3 wells designated as the Control group. The cells were incubated at 37°C for 16-24 hours until the cell confluence reached 20%-30%.

[0068] Viral infection: Remove CMV enhancer-MCS-dcas9-VPR-FT2A-mCherry virus and U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP virus from the refrigerator and thaw them slowly on ice; aspirate the supernatant from each well, change the culture medium, add the virus to 3 wells for each virus (MOI=10, 50, 100 respectively), mix well, and continue culturing. Change the medium with complete culture medium 16 h after infection. Observe the cell morphology during the process. If changes occur, the medium can be changed 8 h in advance to maintain normal cell growth.

[0069] Continue culturing: The medium can be changed midway through the process according to the growth status of the cells to maintain cell viability.

[0070] Confirmation of infection efficacy: Observe under a microscope when the fluorescence expression abundance is high at approximately 72 hours after infection. The infection conditions and MOI corresponding to the group with an infection efficiency of around 80% and good cell growth can be used as the basis for subsequent infection experiments (use as little virus as possible).

[0071] Viral infection experiment (formal experiment):

[0072] Cell seeding: Third-generation cells in good growth condition were digested with 0.25% trypsin and then resuspended in complete culture medium.

[0073] Subsequently, 10 μL of cell suspension was taken for cell counting, and the number of cells seeded in each well of a 24-well plate was 5 × 10⁶. 4 indivual.

[0074] Then, the 24-well plate was placed in a 37°C incubator for further culture. The cell status and density were observed daily. When the cell density reached 60%-70% and the cells were in good condition, the next step could be performed.

[0075] Remove the cells from the incubator, wash them twice with PBS, add pre-prepared serum-free culture medium, and starve the cells for 2 hours.

[0076] The virus was then diluted using astrocyte culture medium. Since each cell group required infection with two viruses, a stepwise infection method was employed. Based on the cell MOI and virus titer, an equal volume of CMV enhancer-MCS-dcas9-VPR-FT2A-mCherry virus was added to each cell group.

[0077] 24 hours after infection, the medium was replaced with astrocyte culture medium. Approximately 72 hours post-infection, the virus was diluted using differentiation medium containing 2.5% serum, based on cell MOI and viral titer. Viral U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP was added to form the NPS group, and U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP simultaneously overexpressed the Ngn2, Prdx2, and Sod1 genes.

[0078] Twenty-four hours after infection with U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP virus, the medium was changed using basal induction medium, and then the induction medium was changed every 3-4 days. On day 7, the medium was changed again using mature neuron culture medium. Observe the changes in cell morphology after viral infection. Select appropriate time points for experiments.

[0079] Comparative Example 1: The difference between this comparative example and Example 1 is that the virus U6-gRNA(Ngn2)-SV40-EGFP was added to form the Ngn2 group U6-gRNA(Ngn2)-SV40-EGFP overexpressing the Ngn2 gene.

[0080] Comparative Example 2: The difference between this comparative example and Example 1 is that a virus was added: U6-gRNA-SV40-EGFP was used to form an empty vector group, and the U6-gRNA-SV40-EGFP empty vector group had no overexpressed gene.

[0081] The following experiments were conducted after viral infection:

[0082] Puromycin working concentration:

[0083] Seed cells into 24-well plates 24 hours in advance, at a density of 5 × 10⁶ cells / well. 4 Cells / well, after 24 hours, the cell density is about 70%;

[0084] After 24 hours of cell growth, different final concentrations of Puromycin (0 / 0.5 / 1 / 1.5 / 2 / 2.5 / 3 / 3.5 / 4 / 4.5 / 5 / 5.5 / 6 / 6.5 / 7 / 7.5 / 8 / 8.5 / 9 / 9.5 / 10 μg / mL) were added.

[0085] After culturing for another 48 hours, the cells were observed under a microscope, and the lowest concentration of Puromycin that killed all cells was taken as the working concentration.

[0086] Screening for stable strains:

[0087] 48-72 hours after infection with Puromycin virus (infection steps as before), the cells were cultured in a medium containing an appropriate concentration of Puromycin. After 48 hours of screening, all empty cells in the Puromycin group died. We considered that all remaining cells in the virus-infected group were positive.

[0088] Reduce the Puromycin concentration to the maintenance concentration (1 / 2 to 1 / 4 of the screening concentration), and continue to screen and expand the infected cells for subsequent experiments.

[0089] Cellular immunofluorescence staining:

[0090] Washing: Remove the 24-well plate with built-in climbing slide from the incubator. After confirming cell adhesion, discard the culture medium, add 500 μL of PBS and wash 3 times (30 seconds each time, then let stand). Discard the PBS.

[0091] Fixation: Add 500 μL of 4% paraformaldehyde, incubate at room temperature for 10 min and then discard. Add an equal volume of PBS and wash 3 times (30 s / wash), then discard the PBS.

[0092] Permeability test: Add 500 μL of 0.2% Triton X-100 (prepared with PBS) to each well, leave at room temperature for 10 min, then discard. Next, add an equal volume of PBS and wash 3 times (30 s / wash), then discard the PBS.

[0093] Sealing: Add 500 μL of 10% goat serum to each well, incubate at room temperature for 30 minutes, then discard. No washing is required.

[0094] Incubate primary antibody: Prepare antibody dilution solution, 50 μL per cell slide; cut a small piece of sealing film and stick it on the well plate cover, then drop 50 μL of antibody dilution solution onto the sealing film; carefully remove the cell slide from the well with tweezers, drain the water on paper, and place it upside down on the antibody dilution solution drop;

[0095] Place the above perforated plate cover in a humidified box and incubate overnight at 4 degrees Celsius;

[0096] Secondary antibody incubation (this step requires protection from light): Place the cell slides back into the 24-well plate, add 500 μL of PBS and wash 3 times (5 min each time), then discard the washes. Add the fluorescent secondary antibody dilution buffer (dilute according to the instructions), incubate at room temperature for 1 h, wash 3 times with PBS (5 min each time), then discard the washes.

[0097] DAPI staining (nucleus staining) and mounting: Drop 8 μL of DAPI-containing anti-fluorescence quencher onto a glass slide, and invert the slide onto the drop;

[0098] Apply nail polish around the perimeter of the applicator to secure it.

[0099] Images were captured and saved using a confocal microscope.

[0100] RNA-related experiments

[0101] (1) RNA extraction using TRIzol reagent

[0102] 1) Preparation: Wipe the clean bench with 75% alcohol. All experimental consumables must be autoclaved. The solvents and solutions used should be RNA-specific, and the water used to prepare the solutions should be RNase-free H2O.

[0103] 2) RNA extraction

[0104] ① Extracting RNA from cells: Remove the culture medium, add an appropriate amount of pre-cooled PBS buffer solution to wash the cells thoroughly twice, and use a 1000μL pipette to remove as much PBS buffer solution as possible; ② Extracting RNA from tissue samples: Take the brain tissue collected in a 1.5mL centrifuge tube from the -80℃ freezer and thaw it on ice;

[0105] 3) Add 1 mL of TRIzol reagent and allow it to fully contact the cell or brain tissue sample, then place it on ice for 10 minutes to lyse.

[0106] 4) Transfer the fully lysed cell lysate to a labeled 1.5 mL sterile enzyme-free centrifuge tube, add 0.2 times the volume of TRIzol reagent in chloroform, mix by inverting, let stand at room temperature for 5 minutes, and centrifuge at 12000 rpm for 15 minutes at 4°C.

[0107] 5) After centrifugation, carefully aspirate the supernatant into a new 1.5mL enzyme-free centrifuge tube, being careful not to aspirate the middle or lower layer. Add 0.5-1 times the volume of TRIzol reagent in isopropanol, invert the centrifuge tube to mix the supernatant and isopropanol thoroughly, let stand at room temperature for 10 minutes, and then centrifuge at 12000 rpm for 10 minutes at 4°C.

[0108] 6) Use a pipette to aspirate and discard the supernatant. Add an equal volume of 75% anhydrous ethanol (equal to the TRIzol reagent) to the centrifuge tube to wash the RNA precipitate. Invert the centrifuge tube several times to mix. Centrifuge at 7500 rpm for 5 minutes at 4°C. This process can be repeated 1-2 times. After discarding the supernatant, open the centrifuge tube and let it air dry at room temperature until the precipitate becomes transparent. Immediately add an appropriate amount of enzyme-free water to dissolve the precipitate.

[0109] 7) Take 1 μL of the dissolved RNA solution and use a NanoDrop™ One spectrophotometer to detect the RNA concentration and A260 / A280 ratio. The A260 / A280 ratio should ideally be between 1.8 and 2.1. The extracted RNA sample can be stored at -80°C for later use or reverse transcribed into cDNA for RT-qPCR experiments.

[0110] (2) RT-qPCR experiment

[0111] 1) Removal of genomic DNA: According to the reaction system in Table 1, add the reaction reagents one by one to a 200μL microcentrifuge tube, mix the mixture gently and centrifuge briefly, and use a conventional PCR instrument to perform the reaction. The program is set to 42℃ for 2 minutes. After the reaction is completed, place the tube on ice.

[0112] Table 1: Reaction system for removing genomic gDNA

[0113] 2) Reverse transcription: According to the reverse transcription kit instructions, prepare the reverse transcription reaction system according to Table 2, and aliquot the mixture into RNA from which genomic DNA has been removed. Use a conventional PCR instrument to perform the reaction. Set the program to 37℃ for 15 minutes and 85℃ for 5 seconds. After the program is completed, store the synthesized cDNA at -80℃ or -20℃ for the next reaction.

[0114] Table 2: Reverse Transcription Reaction System

[0115] 3) Real-time quantitative PCR (RT-qPCR) reaction

[0116] ① The target gene mRNA sequence information was obtained from the National Center for Biotechnology Information (NCBI), and Nanning Kedi Biotechnology Co., Ltd. was commissioned to design and synthesize primers for the target gene. The primer sequences used in this study are shown in Table 3.

[0117] Table 3: RT-qPCR primer sequences

[0118] ② Add the RT-qPCR reaction system to a sterile, enzyme-free eight-tube according to the proportions in Table 4, cap the tubes, and centrifuge briefly to allow the reaction system to collect at the bottom of the tubes;

[0119] Table 4: 20 μL RT-qPCR reaction system

[0120] ③ Set the program according to Table 5 in the 7500 Real-Time PCR System instrument and save it according to the instrument's default melting curve acquisition program;

[0121] Table 5: PCR reaction conditions

[0122] ④ Use 2- ΔΔCT The data obtained from the experiment were analyzed using the method.

[0123] Western blot assay

[0124] (1) Extraction of total cell protein

[0125] 1) Prepare a cell lysis mixture using high-efficiency protein lysis buffer (RIRA) and protease inhibitor (PMSF) at a volume ratio of RIPA:PMSF = 100:1; use 150 μL per T25 cell culture flask and 100 μL per well of a six-well plate, and prepare fresh before use.

[0126] 2) After removing the cells from the incubator, discard the old culture medium, rinse the cells twice with pre-cooled PBS buffer, remove the remaining PBS buffer with a pipette, add the prepared cell lysis mixture, shake left and right to ensure the mixture fully covers the cell surface, and place the culture flask or six-well plate on ice for lysis for 15 minutes.

[0127] 3) After lysis on ice, scrape the cells and transfer them to a pre-labeled 1.5 mL microcentrifuge tube. Place the tube on ice and use an ultrasonic processor to break up the cells. The amplitude is 30-35%, the induction time is 30 seconds, the interval is 10 seconds, and the total induction time is 1 minute.

[0128] 4) After sonication of the cells, centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, transfer the supernatant to a new 1.5 mL microcentrifuge tube, which is the total cell protein. Add 1 / 4 volume of 5×SDS-PAGE protein loading buffer according to the volume of the supernatant. Mix thoroughly and heat in a 100°C water bath to denature the protein. Store the treated protein sample in a -20°C freezer.

[0129] (2) Determination of protein concentration by BCA method

[0130] 1) Preparation of protein standards:

[0131] ① Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), dissolve thoroughly to prepare a 25 mg / mL protein standard solution. It can be used immediately after preparation or stored at -20°C for long-term storage.

[0132] ② Take an appropriate amount of 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL. For example, take 20 μL of 25 mg / mL protein standard and add it to 980 μL of PBS to prepare a 0.5 mg / mL protein standard. The diluted 0.5 mg / mL protein standard can be stored at -20℃ for a long time.

[0133] 2) Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1), and mix thoroughly. For example, add 100 μL of BCA reagent B to 5 mL of BCA reagent A, mix well, and prepare 5.1 mL of BCA working solution. The BCA working solution is stable at room temperature for 24 hours.

[0134] 3) Protein concentration detection

[0135] ① Add the standard to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, and then add PBS to bring the total volume to 20 μL. This corresponds to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively.

[0136] ② Add 1 μL of the protein to be tested to a 96-well plate, and add PBS to make up to 20 μL.

[0137] ③ Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0138] ④ Measure the absorbance at wavelength A562 using an ELISA reader.

[0139] ⑤ Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0140] (3) Western blot electrophoresis

[0141] 1) Before starting the experiment, clean the glass plate used for preparing the glue, let it dry, and then align and place it into the glue preparation frame and clamp it tightly.

[0142] 2) Prepare separating gels of different concentrations according to the molecular weight of the target protein. Table 6 provides the corresponding preparation options.

[0143] Table 6: Selection of different concentrations of polyacrylamide gel (SDS-PAGE)

[0144] 3) According to the required volume of separating gel for the experiment, prepare separating gels of different concentrations according to Table 7. After mixing the liquid by blowing, add it to the glass plate trough, then add anhydrous ethanol to seal (fill it up), let it stand at room temperature for 20 minutes, then pour off the anhydrous ethanol and use cut filter paper strips to absorb the anhydrous ethanol.

[0145] Table 7: Preparation of SDS-PAGE Polyacrylamide Gel Separation Gel

[0146] Prepare the stacking gel according to Table 8 based on the required volume of stacking gel for the experiment. After mixing the liquid by blowing, add it to the glass plate trough. Then insert a comb along one side of the glass plate, trying to avoid air bubbles. Let it stand at room temperature for 20-30 minutes.

[0147] Table 8: Solutions used for preparing 5% SDS-polyacrylamide gel electrophoresis stacking gel

[0148] 5) Western blot electrophoresis: After the stacking gel solidifies, install the gel preparation glass plate into the electrophoresis tank, check the airtightness, and add electrophoresis buffer to the inner tank. Slowly and vertically pull out the comb. According to the experimental design, add 30µg of the prepared protein sample to the lanes of the gel in sequence, and add 2μL of marker to both sides of the lane with the protein sample. After replenishing the electrophoresis buffer in both the inner and outer tanks, connect the power supply and set the instrument to a constant voltage of 60V for protein electrophoresis. Determine the electrophoresis time based on the size of the target protein.

[0149] 6) Transfer: Before transfer, according to the gel size of the 0.22μm PVDF membrane, soak the cut PVDF membrane in methanol for 2-3 minutes and then transfer it to the transfer buffer. Before the end of electrophoresis, soak the sponge pad and filter paper in 1× transfer buffer. After electrophoresis, assemble the transfer clamp in a "sandwich" structure in a tray with transfer buffer (sandwich structure: blackboard-sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad-whiteboard, note that there should be no air bubbles between the gel and the membrane). Place the transfer clamp into the transfer device, pour in the pre-cooled transfer buffer at 4℃, place the transfer tank in an ice box, and transfer the membrane for 90 minutes under a constant current of 200mA in the ice box.

[0150] 7) Blocking: After the transfer is completed, the PVDF membrane is washed three times with 1×TBST on a shaker for 10 minutes each time. After washing, the PVDF membrane is placed in 3% BSA blocking solution for 1 hour.

[0151] 8) Immunological reaction: After blocking, wash the PVDF membrane three times with 1×TBST on a shaker for 10 minutes each time. After washing, add the PVDF membrane to the diluted antibody and incubate overnight at 4°C. The next day, remove the band from the primary antibody and wash it three times with 1×TBST for 10 minutes each time. Use the corresponding secondary antibody and dilute it to an appropriate concentration with TBST. Place the protein band to be measured in an incubation box, add the diluted secondary antibody, and incubate on a shaker at room temperature for 1 hour.

[0152] 9) Chemiluminescence development: Remove the band from the secondary antibody and wash it three times with shaking in 1×TBST for 10 minutes each time. Use horseradish peroxidase HRP-ECL chemiluminescence method to develop the color of the band. First, mix ECL-A and ECL-B at a volume ratio of 1:1 to prepare ECL mixture. After slightly blotting the band with absorbent paper, place it in the ECL mixture and shake it at room temperature for 5 minutes. Then, place it in the scanner for exposure and scanning.

[0153] 10) Calculate protein expression levels: Use ImageJ software to analyze the gray values ​​of the obtained imaging bands, measure the gray values ​​of the target proteins in each sample, and calculate the relative expression levels of each target protein after correction using internal reference bands for statistical analysis.

[0154] Peroxides and Fe 2+ Detection

[0155] (1) Dihydroethidium and RhoNox-1 solution

[0156] 1) Dihydroethidium, also known as DHE, is a peroxide indicator. DHE can penetrate the cell membrane, forming a fluorescent protein complex that emits blue fluorescence. After entering the cell, DHE mainly targets the cell membrane, cytoplasm, and nucleus, with the strongest staining effect in the nucleus. DHE produces inherent blue fluorescence with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm. After dehydrogenation, DHE binds to RNA or DNA to produce red fluorescence with a maximum excitation wavelength of 300 nm and a maximum emission wavelength of 610 nm. In practical observation, 535 nm can be used as the excitation wavelength.

[0157] 2) RhoNox-1 is a fluorescent probe that specifically detects divalent iron ions. When RhoNox-1 reacts with Fe... 2+ Following the reaction, an irreversible orange (red) fluorescent product (Ex / Em: 540 / 575 nm) is generated. RhoNox-1 can penetrate well into cells and is suitable for intracellular Fe production. 2+The detection tends to locate in the Golgi apparatus.

[0158] (2) Preparation of stock solution

[0159] Dissolve 1 mg of DHE in 0.31 mL of DMSO to obtain 10 mM Dihydroethidium. Dissolve 1 mg of RhoNox-1 in 0.2181 mL of DMSO to obtain 10 mM RhoNox-1.

[0160] Note: Store the stock solution at -20°C or -80°C away from light and avoid repeated freeze-thaw cycles.

[0161] (3) Preparation of working solution

[0162] Prepare 1-10 μM DHE / RhoNox-1 working solutions by diluting the stock solution with preheated serum-free cell culture medium or PBS. Adjust the concentration of the working solution according to the actual situation.

[0163] (4) Cell staining

[0164] 1) Plate the cells in confocal microplates. When infecting with the virus, replace VPR-mCherry with VPR-puro (to avoid fluorescence crosstalk). The remaining infection steps are the same as before. On day 7 after infection with the second virus, remove the confocal microplates from the incubator, discard the culture medium, add 500 μL of PBS and wash 3 times (5 min each time, let stand), then discard the PBS.

[0165] 2) Add 100 μL of dye working solution to the bottom of the glass in the center of the confocal dish;

[0166] (5) Incubate at room temperature in the dark for 5-30 minutes.

[0167] (6) Discard the dye working solution, add 500 μL of PBS and wash 3 times (5 min / time) and discard.

[0168] (7) Take and save images using a confocal microscope.

[0169] Whole-cell patch-clamp recording

[0170] (1) Immerse the glass microelectrode in 95% alcohol, place it in an ultrasonic cleaner, sonicate for 5-10 minutes, repeat twice, then replace the alcohol with double distilled water, sonicate for 5-10 minutes, repeat 5 times, and place it in a 50℃ oven to dry for later use.

[0171] (2) A glass blank with an outer diameter of 1.5 mm is drawn into a microelectrode with an outer diameter of 3~5 MΩ using a two-step drawing method with a microelectrode drawing instrument.

[0172] (3) Dip the tip of the glass microelectrode into the electrode liquid, fill the tail end with the corresponding electrode liquid, and remove air bubbles.

[0173] (4) Remove the cells from the incubator, discard the culture medium, add freshly prepared ACSF solution, fix the culture dish with modeling clay, find neurons with intact and shiny cell membranes under a high-power (40x) microscope, and use a micromanipulator to move the electrode to the target position under a low-power (10x) microscope, record the electrode being pressed into the water so that it is close to the cell.

[0174] (5) Adjust the position of the recording electrode under high magnification. After the electrode contacts the cell membrane, remove the negative pressure to form a high-resistance seal. After it stabilizes, use instantaneous negative pressure to burst the membrane. When the cell series impedance stabilizes, the relevant recording can be performed.

[0175] (6) The electrical signal of the electrode is guided by the Ag / AgC1 electrode, then amplified by the patch clamp amplifier (Multiclamp700B, MolecularDevices), and finally converted into a data signal by the digital-to-analog converter (Digidata 1550B, MolecularDevices).

[0176] Image Acquisition

[0177] The Bio-Rad system was used to collect and process Western Blot experimental results;

[0178] The EVOS fluorescence microscope and Zeiss / Leica laser confocal microscopy were used to acquire and process fluorescence and bright-field images of cells and brain tissue. ImageJ was used for image annotation, editing, cropping, and counting analysis. Photoshop and Illustrator were used for image layout and annotation.

[0179] Data statistics

[0180] (1) SPSS 26.0 was used for statistical analysis, and GraphPad Prism 8.0 software was used to draw the graphs;

[0181] (2) For statistical analysis of the data, the t-test (Student's t-test) was used to compare the means of two groups; one-way ANOVA was used to compare the means of multiple groups. All statistical data were expressed in the form of mean ± standard error (Mean ± SEM) or mean ± standard deviation (Mean ± SD), and a two-tailed P < 0.05 was considered statistically significant.

[0182] Through the above experiments, such as Figure 1As shown, on day 7 of reprogramming, the expression levels of target genes (Ngn2, Prdx2, and Sod1) were detected by qRT-PCR and Western blot; the expression levels of ferroptosis-related genes (Gpx4 and Slc7a11) were also detected. Figure 2 As shown, on days 7 and 14 of reprogramming, immunofluorescence staining was used to analyze the expression level of the neuron-specific marker Tubb3 and assess transdifferentiation efficiency; Figure 3 , Figure 4 As shown, on day 30 of reprogramming, immunofluorescence staining was used to analyze the expression levels of Map2 and NeuN, specific markers of mature neurons, to assess the maturity of induced neurons; morphological changes of cells in each group were collected under a microscope on day 30 of reprogramming; and whole-cell patch-clamp assays were performed to detect the electrophysiological function of induced neurons.

[0183] Results: Over 95% of the primary astrocytes cultured in vitro expressed the astrocyte marker S100β. The optimal viral MOI for primary astrocytes was 50. On day 7 of reprogramming, dCas9-VPR upregulated the expression of Ngn2, Prdx2, and Sod1 genes and proteins in primary astrocytes under specific gRNA guidance. On day 7 of reprogramming, the gene expression of ferroptosis-related genes Gpx4 and Slc7a11 decreased by approximately 1-fold and 1.5-fold, respectively. In the NPS group, the gene expression levels of Gpx4 and Slc7a11 were not significantly different from those in the empty vector group. On days 7 and 14 of reprogramming, there were no Tubb3+ cells in the empty vector group, while the Ngn2 and NPS groups had Tubb3+ cells, with a higher number in the NPS group. On day 30 of reprogramming, the empty vector group had no Map2+ or NeuN+ cells, while both the Ngn2 group and the NPS group had Map2+ or NeuN+ cells. Furthermore, the NPS group had a greater number of induced neurons (iNs) with more complex morphology. On day 30 of reprogramming, the original stellate processes of astrocytes disappeared, the cell bodies shrank, and they transformed into mature neurons with single, slender axons. Induced neurons could generate repetitive action potentials and exhibited typical neurophysiological activity.

[0184] Therefore, overexpression of the transcription factor Ngn2 can transform astrocytes into mature induced neurons with normal electrophysiological function. Additional overexpression of the mitochondrial antioxidant proteins Prdx2 and Sod1 significantly improves the reprogramming efficiency of Ngn2, increases the number of reprogrammed cells, accelerates the reprogramming process, and promotes more mature morphology in the reprogrammed neurons. Ferropysis occurs during reprogramming, and Prdx2 and Sod1 can reduce ferroptosis, thereby improving reprogramming efficiency and long-term survival.

[0185] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing astrocytes to reprogram into neurons, characterized in that, Includes the following steps: Astrocyte isolation and culture steps: The tissue is minced and placed into a centrifuge tube, and protease is added for digestion. Then, the cells are cultured in a complete culture medium to stop the digestion. Astrocyte passage steps: Remove cells from the incubator, digest and resuspend cells once they reach the target size, and then seed them into new culture medium according to the cell count; Steps for detecting the optimal virus MOI for astrocytes: Digest healthy third-generation astrocytes, resuspend them after digestion to prepare a cell suspension, and then seed them into a culture plate. Add the virus and continue culturing. After a certain period of virus infection, record the infection conditions and MOI corresponding to the group with good cell growth. Viral infection steps: After removing the cells from the incubator and starving them, CMV enhancer-MCS-dcas9-VPR-FT2A-mCherry virus was added. After infecting the cells with this virus for a certain period of time, U6-gRNA(mPrdx2-mSod1-mNgn2)-SV40-EGFP virus was added to overexpress the Ngn2, Prdx2 and Sod1 genes. The Prdx2 and Sod1 genes can reduce ferroptosis.

2. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the astrocyte isolation and culture step, the proteases used are trypsin, collagenase, and DNAse I.

3. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the astrocyte isolation and culture step, the complete culture medium is a complete culture medium containing 10% fetal bovine serum.

4. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, The astrocyte passage process includes the following steps: S1: Preheat PBS solution, trypsin, and astrocyte culture medium; S2: Wash the qualified cells multiple times with PBS solution; S3: Add trypsin to the culture flask, shake the flask, and place it in an incubator for digestion; S4: When the intercellular spaces in the culture flask become wider and the cells become rounder, immediately add complete culture medium to stop digestion and repeatedly pipette the cells from the flask wall. S5: Collect the cell suspension into centrifuge tubes, wash the tubes with PBS solution, and centrifuge them in a centrifuge. S6: Discard the supernatant, add astrocyte culture medium to the centrifuge tube, resuspend the cells, transfer an appropriate amount of cell suspension to a cell culture flask, and place it in an incubator for culture.

5. The method for inducing astrocyte reprogramming into neurons according to claim 4, characterized in that, In S6, the temperature of the incubator is 37°C and the CO2 concentration is 5%.

6. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the astrocyte optimal virus MOI detection step, trypsin at a concentration of 0.25% is used for digestion.

7. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the astrocyte optimal virus MOI detection step, the cell suspension volume is 1–3 mL, and the density is 3 × 10⁻⁶. 4 ~5×10 4 per mL.

8. The method for inducing astrocytes to reprogram into neurons according to claim 1, characterized in that, In the step of detecting the optimal viral MOI for astrocytes, the cells need to be cultured to a confluence of 20% to 30% before adding the virus.

9. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the viral infection step, before removing the cells from the incubator, the cells need to be digested and resuspended, and then cultured in the incubator until the cell density reaches 60% to 70%.

10. The method for inducing astrocyte reprogramming into neurons according to claim 1, characterized in that, In the viral infection step, after the cells are removed from the incubator, they are washed with PBS solution, serum-free culture medium is added, and then the cells are starved for 2 hours.

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