Application of transcription factor Bhlhe41 in central nervous system injury repair

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a transcription factor Bhlhe41 in a central nervous injury repair process. The invention finds that Bhlhe41 plays a negative regulation role in optic nerve injury, that is, knocking down or knocking out Bhlhe41 in retinal ganglion cells (RGC) can promote the survival of RGC and axon regeneration after central nervous injury, thereby further promoting the functional reconstruction of the injured optic nerve. Therefore, the knockout of the transcription factor bhlhe41 in the RGC or the inhibition of the expression of the transcription factor bhlhe41 is beneficial to promoting the functional repair of the central nervous and promoting the survival and regeneration of the RGC.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of transcription factor Bhlhe41 in the process of central nervous system injury repair. Background Art

[0002] The difficulty in regenerating neuronal axons after nerve injury is a hot topic and difficulty in regenerative medicine. Unlike peripheral nerves that can regenerate spontaneously, neurons in the adult central nervous system (CNS) usually do not have the ability to regenerate after being damaged, which has catastrophic consequences for those who suffer from spinal cord injury, stroke, brain trauma or optic neuropathy.

[0003] The main reasons for the failure of axon regeneration in neurons are usually due to the lack of endogenous regeneration ability and the presence of exogenous environmental inhibitors. At present, multiple endogenous signaling pathways that regulate axon regeneration have been discovered, among which the mitogen-activated protein kinase (MAPK) signaling pathway and the phosphatidylinositol 3-kinase / protein kinase B (PI3K / Akt) signaling pathway are the most representative. The MAPK signaling pathway is involved in the perception of axonal injury, the initiation and maintenance of regeneration, and regulates axon regeneration by regulating protein synthesis and the cytoskeleton; the PI3K / Akt signaling pathway regulates axon regeneration by regulating the transcription and translation of related genes in neurons after injury. The combined action of multiple regeneration-promoting signals can further enhance the regeneration ability of axons. For exogenous environmental inhibitors, such as chondroitin sulfate proteoglycans (CSPGs), which are expressed by glial cells, they send inhibitory signals to growth cones and, combined with the downregulation of intrinsic growth ability, inhibit axon regeneration. Therefore, axonal injury leads to loss of function of the affected neurons and cannot be completely repaired by existing methods.

[0004] Transcription factors (TFs) are the basis for transmitting complex patterns of intrinsic and exogenous signals to dynamic gene expression programs that define cell type identity. Some transcription factors such as Sox11, KLF4, Atf3, CREB, etc. can be regulated to activate elements that promote neuronal survival and regeneration. Studies have shown that targeting these molecules can enhance the growth capacity of mature neuronal axons, but can only promote a certain degree of regeneration of damaged CNS axons, so other unidentified genes may play a key role in controlling neuronal growth and regeneration.

[0005] Therefore, finding the key factors that regulate neuronal survival and regeneration after nerve injury will help promote the repair of central nervous system function and provide a theoretical basis for clinical treatment.

[0006] The present invention clarifies that the transcription factor Bhlhe41 has a regulatory effect on the survival and regeneration of retinal ganglion cells (RGC) during the repair of central nervous system damage. By reducing the expression of the transcription factor Bhlhe41 gene, the survival of retinal ganglion cells (RGC) can be effectively regulated, thereby promoting the repair of central nervous system damage. Summary of the invention

[0007] The present invention demonstrates that Bhlhe41 plays a negative regulatory role in optic nerve injury, that is, both knocking down and knocking out Bhlhe41 can promote the survival and axon regeneration of RGCs after optic nerve injury, thereby further promoting the functional reconstruction of the damaged optic nerve, indicating that Bhlhe41 can become a target for treatment after optic nerve injury.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution provided by the present invention is as follows:

[0009] The invention provides application of transcription factor Bhlhe41 in preparing central nervous system injury therapeutic products.

[0010] In the present invention, the therapeutic product includes a Bhlhe41 inhibitor for inhibiting the expression of the Bhlhe41 gene or the Bhlhe41 protein.

[0011] In the present invention, the Bhlhe41 inhibitor is used to promote the survival or regeneration of neurons.

[0012] In the present invention, the neurons are retinal ganglion cells.

[0013] In the present invention, the Bhlhe41 inhibitor inhibits the transcriptional activity of the Bhlhe41 gene by inhibiting the activity of transcription factors, chromatin remodeling, DNA methylation, and activating or inhibiting cell signaling pathways.

[0014] In the present invention, the Bhlhe41 inhibitor selectively reduces Bhlhe41 translation through RNA splicing regulation, RNA stability regulation, RNA interference, and post-translational modification, thereby controlling the expression level of Bhlhe41 protein.

[0015] In the present invention, the Bhlhe41 inhibitor is a viral vector that inhibits the expression of Bhlhe41.

[0016] In the present invention, the viral vector is an adeno-associated viral vector.

[0017] The present invention provides the use of Bhlhe41 gene or Bhlhe41 protein as a drug target for preparing a drug for treating central nervous system injury.

[0018] In the present invention, the central nervous system injury is optic nerve injury.

[0019] In the present invention, the central nervous system injury is optic nerve crush.

[0020] According to the present invention, the transcription factor bhlhe41 can participate in the repair of central nervous system injury by regulating the survival of retinal ganglion cells (RGCs), which helps to better understand the important role of transcription factors in the process of nerve injury repair, and provides new targets for treatment after nerve injury, which will help promote the functional repair of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Next, in the description of the drawings and embodiments, for convenience, the term "retinal ganglion cell" is abbreviated as "RGC"; the term "adeno-associated virus (Adeno-associated Virus)" is abbreviated as "AAV"; the term "central nervous system" is abbreviated as "CNS"; the term "human embryonic stem cell" is abbreviated as "hESCs".

[0022] Figure 1 is a schematic diagram of the effect of knocking out the Bhlhe41 gene on RGC survival and axon regeneration according to an embodiment of the present invention;

[0023] Figure 2 is a schematic diagram showing the effect of knocking out the Bhlhe41 gene on the survival of RGCs after optic nerve crush according to an embodiment of the present invention;

[0024] Figure 3 is a schematic diagram showing the effect of knocking out the Bhlhe41 gene on the survival of RGCs after optic nerve crush according to another embodiment of the present invention;

[0025] Figure 4 Schematic diagram of the effect of knocking out the Bhlhe41 gene on the phosphorylation level of AKT in RGC according to an embodiment of the present invention;

[0026] Figure 5 Schematic diagram of the effect of knocking down the Bhlhe41 gene on the survival of RGCs differentiated from hESCs according to an embodiment of the present invention;

[0027] Figure 6 It is a schematic diagram of the effect of knocking out the bhlhe41 gene on the thickness of the retinal layer after optic nerve crush according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] Hereinafter, the present invention will be described in detail according to exemplary embodiments, but the present invention is not limited to these embodiments. The present invention is embodied in the following various forms, but should not be construed as being limited to the exemplary embodiments set forth herein. Therefore, the detailed description and embodiments of the present invention will convey the scope of the present invention to those of ordinary skill in the art and are construed as being within the scope of the present invention.

[0029] Example 1 Animal experiments showed that knocking out the bhlhe41 gene based on CRISPR / cas9 technology in vivo promoted the survival and axon regeneration of RGCs.

[0030] 1. Experimental methods

[0031] (1) Construction and identification of Vglut2-Cre:LSL-Cas9 mice

[0032] The genetically engineered mouse strain Vglut2-Cre:LSL-Cas9 for neuroscience research was selected for breeding. When the breeding mice were about 2 weeks old, the tails were collected for identification. The identification method is: add 300ul lysis solution + 5ul proteinase K to each sample and digest overnight at 55℃. The next day, add 600ul pre-cooled anhydrous ethanol, turn it upside down, and know that flocculent precipitation appears. Centrifuge at 12000rpm for 5min. After centrifugation, aspirate the upper liquid, turn it upside down at room temperature to dry until the precipitate becomes transparent. Add 200ul Milli-Q (laboratory pure water) and shake it up and down to fully dissolve the DNA. Take 3ul gDNA template and perform PCR in a 25ul reaction system. Prepare the gel according to the target band, and add an appropriate amount of DNA and DNA maker to the agarose gel. Set the electrophoresis instrument to 120V voltage and run for 20-30min. Then use the gel imaging system to observe and take pictures.

[0033] (2) Optic nerve crush model and anterograde labeling with CTB conjugate

[0034] Based on the successfully bred Vglut2-Cre:LSL-Cas9 genotype mice, an optic nerve crush model was constructed. The construction process is as shown in the attached Figure 1As shown in A: First, five groups of adeno-associated viruses AVV-sgPten, AVV-sgCebpg, AAV-sgJip3, AAV-sgBhlhe41 and control Nontarget sgRNA were injected into the vitreous cavity of Vglut2-Cre:LSL-Cas9 genotype mice, so that the viral vector was directly injected into the vitreous cavity, and the viral suspension was diffused into the retina with the vitreous body, thereby knocking out the target gene. The Jip3, Pten and Cebpg are factors that have been proven to have a certain effect on survival and axon regeneration. After 2 weeks, the mice were anesthetized, the optic nerve was exposed, and the optic nerve was clamped at 2mm behind the ball for 3s. After 2 weeks of feeding, the CTB tracer was injected into the vitreous body with a micropipette to track the regenerated axons anterogradely, and the injection volume was 2μl per mouse. Two days later, the mice were perfused with 4% paraformaldehyde, the eyeballs were removed and post-fixed with 4% formaldehyde, and then replaced with PBS buffer and stored in a 4°C refrigerator one day later. (3) Retinal flat mount and immunofluorescence staining

[0035] The eyeballs of the six groups of mice were taken out, namely, AVV-sgPten group, AVV-sgCebpg group, AAV-sgJip3 group, AAV-sgBhlhe41 group, Nontarget sgRNA-intact group and Nontarget sgRNA group. The Bhlhe41 sgRNA group was the experimental group, the Nontarget sgRNA group was the control group, and the Jip3 sgRNA group, Cebpg sgRNA group and Pten sgRNA group were positive controls.

[0036] Use microtweezers to peel off each group of retinas and put them into 96-well plates. Add 100ul antibody diluent to each well for blocking and membrane permeation for 1-2d. At the same time, add PBS buffer to the blank wells around the retina to prevent evaporation. Dilute the primary antibody multiple splicing RNA binding protein (RBPMS) with antibody diluent. The multiple splicing RNA binding protein is a typical marker of the entire retinal ganglion cells. 100μl per well, seal the 96-well plate with a sealing film, tie a rubber band to prevent leakage, and incubate for 1-2d at 4°C. Then wash three times with PBS buffer, each time with a shaker for 10min. Dilute the secondary antibody with antibody diluent, 100ul per well, seal the 96-well plate with a sealing film, tie a rubber band to prevent leakage, and incubate for 1-2d at 4°C. Then wash three times with PBS buffer, each time with a shaker for 10min. Take a certain distance in the middle of the slide to place the retina, then put two square cover glasses on each side and fix them with 502 glue. Place a layer of blue circular filter paper in the middle to prevent reflection, then soak the retina in PBS buffer, open it like a bowl, cut a cut on each of the top, bottom, left and right sides, and then place the bowl with the bottom facing up (the side containing the RGC on top) on the filter paper, add a small amount of special sealing liquid, and cover with a square coverslip for sealing.

[0037] The images were taken under a fluorescence microscope and the number of surviving RGCs in each group was counted.

[0038] (4) Optic nerve transparency

[0039] The ScaleS method was used to perform transparent treatment on the sciatic nerve tissue, and ScaleS0, ScaleA2, ScaleB4, AbScale and ScaleS4 solutions were used in the transparent treatment. The formulas of the solutions are as follows:

[0040] ScaleS0: 10g sorbitol + 0.065g methyl-β-cyclodextrin + 0.065g γ-cyclodextrin + 0.5g N-acetyl-L-hydroxyproline + 2ml glycerol + 1.5ml DMSO PBS fixed volume 50ml;

[0041] Scale A2: 12g urea + 1ml glycerol + 50μl TritonX-100, ddH 2 OFix volume to 50ml;

[0042] Scale B4: 24 g urea, ddH 2 OFix volume to 50ml;

[0043] AbScale: 1g urea + 100μl TritonX-100, PBS to 50ml;

[0044] Scale S4: 20g sorbitol + 12g urea + 4ml glycerol + 7.5ml DMSO + 100μl TritonX-100ddH 2 OFix volume to 50 ml.

[0045] The collected sciatic nerves were placed in ScaleS0, ScaleA2, ScaleB4(0), and ScaleA2 solutions and incubated in a 37°C water bath overnight in sequence for tissue permeabilization. The permeabilized optic nerves were washed with 1x PBS buffer for 6 hours. The optic nerves were incubated with SCG10 (1:200) primary antibody in a 37°C water bath for 3 days, and then washed twice with AbScale solution at room temperature for 3 hours each. The nerves were incubated with fluorescent secondary antibodies (1:500) in a 37°C water bath for 3 days, and then washed twice in AbScale solution at room temperature for 3 hours. Afterwards, the nerves were re-fixed with 4% PFA for 1 hour and washed twice with 1xPBS for 1 hour. Finally, the nerves were incubated with ScaleS4 solution in a 37°C water bath. Three days later, optical observation was performed using a Leica microscope. Among them, the Bhlhe41 sgRNA group was the experimental group, the Nontarget sgRNA group was the normal control group, the Jip3sgRNA group, the Pten sgRNA group and the Cebpg sgRNA group were positive controls. Jip3, Pten and Cebpg are factors that have been confirmed to have effects on axon regeneration.

[0046] 2. Experimental results

[0047] Attached Figure 1 B shows the immunofluorescence images of retinal flat mounts, which are GFP (green, the viral vector carries the GFP sequence, representing the viral infection efficiency) and RBPMS (red). It can be seen from the figure that the number and intensity of red fluorescence representing RGC in the Bhlhe41 sgRNA are higher than those in the Nontarget sgRNA control group. In the Bhlhe41 sgRNA group, the co-labeled fluorescence of GFP and RBPMS is obvious.

[0048] Attached Figure 1 C shows the magnified immunofluorescence image of the retinal flat mount of the Bhlhe41 sgRNA group, which shows the specific expression and co-labeling of the red fluorescence representing RGC, indicating the survival of RGC after optic nerve injury. Figure 1 F, Quantitative analysis results also showed that the number of surviving RGCs in the Bhlhe41 sgRNA group increased compared with the control group.

[0049] Attached Figure 1 D shows the optic nerve tissue transparency. Figure 1 E shows a statistical graph of the number of regenerated axons after optic nerve crush. The results show that the number and length of regenerated axons in the Bhlhe41 sgRNA group after optic nerve injury were higher than those in the control group, that is, knocking out Bhlhe41 can promote axon regeneration after optic nerve crush.

[0050] Example 2 In vivo knockout of Bhlhe41 promotes RGC survival after optic nerve crush 1. Experimental methods

[0051] (1) Retinal immunofluorescence

[0052] First, immunofluorescence staining was performed on the retinas of Wt mice to observe the expression of Bhlhe41 in normal mice in order to construct subsequent knockout mice. The obtained retinas were fixed and dehydrated, and retinal frozen sections were prepared. They were washed three times with 1xPBS buffer for 10 minutes each time, permeabilized in 0.3% Triton X-100 for 10 minutes, and then blocked with immunohistochemical blocking solution at room temperature for 2 hours. The retinas were incubated with anti-Bhlhe41 and anti-Tuj1 primary antibodies at 4°C overnight. The next day, the primary antibodies were discarded and washed three times with 1xPBS buffer for 10 minutes each time, and then incubated with the corresponding secondary antibodies at room temperature for 2 hours. Finally, they were washed three times with 1xPBS buffer for 10 minutes each time. They were stained with anti-fluorescence quenching sealing solution containing DAPI. The stained retinal tissue was imaged using a Leica microscope.

[0053] (2)Bhlhe41 flox / flox Construction and characterization of mice

[0054] Order Bhlhe41 flox / flox Conditional knockout male and female mice were bred. When the breeding mice were about 2 weeks old, the tails were collected for identification to verify their phenotype. 300ul lysis solution + 5ul proteinase K were added to each sample and digested at 55℃ overnight. The next day, 600ul pre-cooled anhydrous ethanol was added and inverted until flocculent precipitation appeared. Centrifugation was performed at 12000rpm for 5min. After centrifugation, the upper liquid was aspirated and inverted at room temperature to dry until the precipitate became transparent. 200ul laboratory pure water was added and the mixture was shaken up and down to fully dissolve the DNA. 3ul DNA template was taken and PCR was performed in a 25ul reaction system. Gel was prepared according to the target band and appropriate amount of DNA and DNA maker were added to the agarose gel. The electrophoresis instrument was set to 120V voltage and run for 20-30min. Then the gel imaging system was used to observe and take pictures.

[0055] (3) Construction of optic nerve crush model

[0056] Based on the successful breeding of Bhlhe41 flox / flox Mice were used to construct the optic nerve crush model. The experimental procedure is shown in the attached Figure 2 B, first inject 2ul of adeno-associated virus into 4-week-old Bhlhe41 flox / floxThe mice were placed in the vitreous cavity. After 2 weeks, the mice were anesthetized, the optic nerve was exposed, and the optic nerve was injured by clamping for 3 seconds at 2 mm behind the eyeball. After 2 weeks of feeding, the mice were perfused with 4% paraformaldehyde, the eyeballs were removed and post-fixed with 4% formaldehyde, and then replaced with PBS after 1 day and stored in a 4°C refrigerator.

[0057] (4) Retinal flat mount

[0058] Remove the eyeball and use microtweezers to peel off the retina and put it into a 96-well plate. Add 100ul antibody diluent to each well for blocking and membrane permeation for 1-2 days. At the same time, add PBS buffer to the blank wells around the retina to prevent evaporation. Then wash three times with PBS, shaking for 10 minutes each time. Take a certain distance in the middle of the slide to place the retina, and then put two square coverslips on the left and right sides and fix them with 502 glue. Put a layer of blue round filter paper in the middle to prevent reflection, then soak the retina in PBS buffer, open it like a bowl, cut it up and down, left and right, and then put the bottom of the bowl up (the side containing RGC on top) on the filter paper, add a small amount of special sealing liquid, and cover it with a square coverslip for sealing.

[0059] Photographs were taken and the number of surviving RGCs was counted.

[0060] 2. Experimental results

[0061] Attached Figure 2 A shows an immunofluorescence image, and the experimental results demonstrate the expression of Bhlhe41 in normal mice, in which Bhlhe41 is in red and Tuj1 is in green.

[0062] Attached Figure 2 C shows the light microscopic image of the retinal flat mount. Figure 2 D shows the statistical graph of the number of RGC survival, and the results show that Bhlhe41 flox / flox In the mouse optic nerve crush model, AAV-Cre induced in vivo knockout of bhlhe41 promoted the survival of RGCs after optic nerve crush. AAV-GFP was used as a control virus.

[0063] Example 3 In vivo knockout of Bhlhe41 promotes RGC survival after optic nerve crush 1. Experimental methods

[0064] (1) Optic nerve crush model and anterograde labeling with CTB conjugate

[0065] Based on Bhlhe41 in Example 2 flox / flox The optic nerve crush model was constructed using mice of different genotypes. The experimental procedure is as shown in the attached Figure 3 As shown in A: In the experimental group, adeno-associated viruses AAV-Cre and AAV-PLAP were first injected into the vitreous cavity of Bhlhe41 knockout mice, while Bhlhe41 was used in the control group.flox / flox Mouse retinas were used, but the optic nerve was not crushed. After 2 weeks, the mice were anesthetized, the optic nerve was exposed, and the optic nerve was crushed for 3 seconds at 2 mm behind the eyeball. After 2 weeks of feeding, CTB tracer was injected into the vitreous body with a micropipette to trace the regenerated axons anterogradely, 2 μl per mouse. After 2 days, the mice were perfused with 4% paraformaldehyde, the mouse eyeballs were removed and post-fixed with 4% formaldehyde, and PBS was replaced after 1 day and stored in a 4°C refrigerator.

[0066] (2) Retinal immunofluorescence

[0067] The retina was fixed and dehydrated, and retinal frozen sections were prepared. The retina was washed three times with 1×PBS for 10 minutes each time, and permeabilized in 0.3% Triton X-100 for 10 minutes, and then blocked with immunohistochemical blocking solution for 2 hours at room temperature. The retina was incubated with anti-RBPMS primary antibody overnight at 4°C. The next day, the primary antibody was discarded and the retina was washed three times with 1×PBS for 10 minutes each time, and then incubated with the corresponding secondary antibody for 2 hours at room temperature. Finally, it was washed three times with 1XPBS for 10 minutes each time. Staining was performed with anti-fluorescence quenching sealing solution containing DAPI.

[0068] The stained retinal tissue was imaged by a Leica microscope.

[0069] 2. Experimental results

[0070] Attached Figure 3 B shows the retinal immunofluorescence image, in which the RBPMS antibody appears green. The immunofluorescence results show that after knocking out bhlhe41, the number of RBPMS fluorescence representing retinal ganglion cells increased significantly. Figure 3 C shows a statistical graph of RGC survival rate, indicating that knockdown of bhlhe41 in vivo can significantly promote the survival of RGCs in the retina.

[0071] Example 4 Knockout of bhlhe41 in vivo can significantly promote AKT phosphorylation levels in RGCs 1. Experimental methods

[0072] (1) Optic nerve crush model and anterograde labeling with CTB conjugate

[0073] Based on Bhlhe41 in Example 2 flox / flox The optic nerve crush model was constructed using mice of different genotypes. The experimental procedure is as shown in the attached Figure 3 As shown in A: In the experimental group, adeno-associated viruses AAV-Cre and AAV-PLAP were first injected into the vitreous cavity of Bhlhe41 knockout mice, while Bhlhe41 was used in the control group. flox / floxMouse retina, but not optic nerve crush model. After 2 weeks, the mice were anesthetized, the optic nerve was exposed, and the optic nerve was crushed for 3s at 2mm behind the eyeball. After 2 weeks of feeding, CTB tracer (2μl per mouse) was injected into the vitreous body with a micropipette to track the regenerated axons anterogradely. After 2 days, the mice were perfused with 4% paraformaldehyde, the mouse eyeballs were removed and post-fixed with 4% formaldehyde, and PBS was replaced after 1 day and stored in a 4℃ refrigerator.

[0074] (2) Retinal immunofluorescence

[0075] The retina was fixed and dehydrated, and retinal frozen sections were prepared. Washed 3 times with 1X PBS, 10 minutes each time, and permeabilized in 0.3% TritonX-100 for 10 minutes, and then blocked with immunohistochemistry blocking solution at room temperature for 2 hours. The retina was incubated with anti-Tuj1 primary antibody and anti-P-AKT primary antibody at 4°C overnight. The next day, the primary antibody was discarded and washed 3 times with 1xPBS buffer, 10 minutes each time, and then incubated with the corresponding secondary antibody at room temperature for 2 hours. Finally, it was washed 3 times with 1xPBS buffer, 10 minutes each time. Stained with anti-fluorescence quenching sealing solution containing DAPI.

[0076] The stained retinal tissue was imaged by a Leica microscope.

[0077] 2. Experimental results

[0078] Attached Figure 4 B shows the retinal immunofluorescence image, where the Tuj1 group is green and the P-AKT group is red. The immunofluorescence results showed that after knocking out bhlhe41 compared with the control group, the number of fluorescent Tuj1 and P-akt representing axons in the retina increased significantly, indicating that knocking out Bhlhe41 in vivo can significantly promote the phosphorylation level of AKT in the retina.

[0079] Example 5 Knockdown of bhlhe41 in RGCs differentiated from human embryonic stem cells (hESCs) promotes RGC survival

[0080] 1. Experimental methods

[0081] Attached Figure 5 A shows the operation process, which is as follows:

[0082] (1) Human embryonic stem cell (hESCs) culture

[0083] Order hESCs cell lines expressing meCherry and culture them normally in mTeSRTM1 complete medium, which is a feeder-free maintenance medium specifically for human embryonic stem cells ES and human induced pluripotent stem cells iPS. Before culture, Matrigel matrix gel is required to coat the cell culture plate to provide support for the feeder-free culture of human ES and iPS cells, and culture at 37° for 1 hour. After cell inoculation, ROCK inhibitor Y27632 is added to the final concentration of 10nm. The medium needs to be changed every day during the culture process. If needed, use ReleSR digestion solution for subculture. The ReLeSR TM It is an enzyme-free reagent for dissociating human ES cell and human iPS cell colonies into cell aggregates.

[0084] (2) Induction of human embryonic stem cells (hESCs) into RGCs

[0085] After the stem cells grew to 75% confluence, they were dissociated into single cell suspension using Accutase cell digestion solution and then centrifuged at 52.6K / cm 2The cells were inoculated into 24-well plates at a density of 1:1. After 24 hours, the cells were replaced with N2B27 medium, which was prepared by mixing DMEM / F12 with Neurobasal (a culture medium conventionally used for culturing neural cells) medium containing 1x GlutaMAX (a substitute for L-glutamine, a factor necessary for neural cell culture), 1xantibiotic-antimycotic (antibiotic-antimycotic, used to prevent bacterial and fungal contamination during cell culture), 1% N2 Supplement (N-2 supplement is a serum-free, chemically defined neural cell culture supplement), and 2% B27 Supplement (B-27 supplement is a serum-free supplement with defined ingredients, containing antioxidants, proteins, vitamins and fatty acids, which can support the culture of neural cells when mixed in the optimal ratio). The prepared N2B27 medium is a special medium for hES induction differentiation, which can gradually induce hES cells to differentiate into RGCs. Then, small molecule compounds were added. Specifically, on days 1-6, the medium was replaced with N2B27 basal medium containing FSK (Forskolin is an effective adenylate cyclase activator with a working concentration of 25uM), NIC (Nicotinamide is a form of vitamin B3 and niacin with a working concentration of 10mM), DSM (dihydroorotate dehydrogenase DHODH inhibitor with a working concentration of 1uM), and IDE2 (a small molecule inducer of cell permeability that can eventually form endoderm in human ESCs by activating the TGF-β signaling pathway with a working concentration of 2.5uM). On days 7-10, the medium was replaced with N2B27 basal medium containing FSK (25uM) and NIC (10mM). On days 10-18, the medium was replaced with N2B27 basal medium containing FSK (25uM). On days 18-30, the medium was replaced with N2B27 basal medium containing FSK (25uM) and DAPT (DAPT (GMP) is an effective oral active γ-secretase inhibitor used to induce stem cells to develop into functional neurons, with a working concentration of 10UM). The addition of the above small molecule compounds can effectively induce hESC to differentiate into retinal ganglion cells (RGC). The medium was changed every day, and the fluorescence was observed under the microscope. Red fluorescence appeared when RGC began to differentiate.

[0086] (3) Lentiviral transfection of hESCs to induce differentiation of RGCs

[0087] After RGC differentiation was successful, the cells were transfected with a lentiviral vector library targeting Pten sgRNA, Cebpg sgRNA, and bhlhe41 sgRNA. The cells were cultured for 14 days to allow enough time for gene editing. The successfully transfected RGCs showed green fluorescence.

[0088] (4) Retinal immunofluorescence staining

[0089] The eyeball tissue samples obtained from a 64-year-old person were fixed and dehydrated to prepare retinal frozen sections. The retina was washed three times with 1X PBS for 10 minutes each time, permeabilized in 0.3% TritonX-100 for 10 minutes, and then blocked with immunohistochemistry blocking solution at room temperature for 2 hours. The retina was incubated with anti-RBPMS primary antibody, anti-bhlhe41 primary antibody, and anti-jip3 primary antibody at 4°C overnight. The next day, the primary antibody was discarded and washed three times with 1XPBS for 10 minutes each time, and then incubated with the corresponding secondary antibody at room temperature for 2 hours. Finally, it was washed three times with 1xPBS buffer for 10 minutes each time. It was stained with anti-fluorescence quenching sealing solution containing DAPI. The stained retinal tissue was imaged using a Leica microscope.

[0090] (5) RGC resuspension

[0091] After the RGCs grow to 14 days, they are resuspended to interrupt axonal growth. Digested with 1 mg / ml collagenase type II and incubated at 37° for 30 min. Then, serum-containing DMEM medium is added to terminate digestion at 1000 rpm for 3 min. Resuspended with N2B27 medium and inoculated into 24-well plates.

[0092] Imaging was performed using a fluorescence microscope 48 hours later.

[0093] 2. Experimental Results

[0094] Attached Figure 5 A shows the process of inducing human embryonic stem cells (hESCs) to differentiate into RGCs and the process of virus infection and cell resuspension.

[0095] Attached Figure 5 B shows immunofluorescence images of the retina (GCL) of a 64-year-old human eye, with the Bhlhe41 group in green, the JIP3 group in green, and the RBPMS group in red, demonstrating the expression of Bhlhe41 and JIP3 in RGCs in the human retina.

[0096] Attached Figure 5 C shows the fluorescence image of RGC 48 hours after resuspension, GFP (green) indicates the virus transfection situation, MeCherry (red) indicates the number of surviving RGC after resuspension and the statistical graph of RGC survival rate. It can be seen from the figure that the number of surviving RGCs in the Bhlhe41 sgRNA group is significantly higher than that in the Nontarget sgRNA control group.

[0097] Attached Figure 5 D Quantitative analysis results also demonstrated that the survival rate of RGCs in the Bhlhe41 sgRNA group was significantly higher than that in the NontargetsgRNA control group.

[0098] The experimental results showed that knocking down Bhlhe41 in hESCs-differentiated RGCs promoted RGC survival.

[0099] As determined in the above results, the transcription factor Bhlhe41 can participate in the repair of central nervous system injury by regulating the survival of retinal ganglion cells (RGC). Therefore, knocking out the transcription factor bhlhe41 or inhibiting its expression will help promote the functional repair of the central nervous system and promote the survival and regeneration of retinal ganglion cells.

[0100] Example 6 Knockout of bhlhe41 gene inhibits retinal thinning after optic nerve crush 1. Experimental methods

[0101] Spectral domain optical coherence tomography (SD-OCT,) was used before and after ONC. Before imaging, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg / kg). During imaging, the cornea was kept moist with 2.5% hydroxycellulose eye drops. Four images (en face images of scans 1, 26, 51, and 76 at 0°, 45°, 90°, and 135°) were used for retinal thickness measurement. For each selected image, a vertical caliper was placed on each side of the optic nerve head, 500 μm away from the center of the optic nerve head. The thickness of the ganglion cell complex (GCC), which consists of three layers of the innermost retina: the nerve fiber layer (NFL), the ganglion cell layer (GCL), and the inner plexiform layer (IPL), was measured with a caliper. The GCC thickness of each retina was taken as the average of a total of eight measurements.

[0102] 2. Experimental results

[0103] Attached Figure 6 The results of OCT measurement of mouse retina are shown in Figure 1. The results show that knocking out the bhlhe41 gene inhibits the thinning of the retinal layer after optic nerve crush.

[0104] It should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for limiting purposes. Descriptions of features or aspects in each embodiment should generally be considered to be feasible for similar features or aspects in other embodiments.

Claims

1. Application of transcription factor Bhlhe41 in the preparation of therapeutic products for central nervous system injury.

2. The application according to claim 1, characterized in that: The therapeutic products include Bhlhe41 inhibitors for inhibiting the expression of Bhlhe41 gene or Bhlhe41 protein.

3. The application according to claim 2, characterized in that: The Bhlhe41 inhibitor is used to promote the survival or regeneration of neurons.

4. The application according to claim 3, characterized in that: The neurons are retinal ganglion cells.

5. The application according to claim 2, characterized in that: The Bhlhe41 inhibitor inhibits the transcriptional activity of the Bhlhe41 gene by inhibiting the activity of transcription factors, chromatin remodeling, DNA methylation, and activating or inhibiting cell signaling pathways.

6. The application according to claim 2, characterized in that: The Bhlhe41 inhibitor selectively reduces Bhlhe41 translation through RNA splicing regulation, RNA stability regulation, RNA interference, and post-translational modification, thereby controlling the expression level of Bhlhe41 protein.

7. The use according to claim 2, 3 or 4, characterized in that: The Bhlhe41 inhibitor is a viral vector that inhibits the expression of Bhlhe41.

8. The application according to claim 7, characterized in that: The viral vector is an adeno-associated viral vector.

9. Use of Bhlhe41 gene or Bhlhe41 protein as a drug target for preparing drugs for treating central nervous system damage.

10. The use according to claim 9, characterized in that: The central nervous system injury is optic nerve injury.

11. The use according to claim 9, characterized in that: The central nervous system injury is optic nerve crush.

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