Application of PBX1 or TAT-PBX1 in preparation of product for preventing and treating degenerative neuropathy

Abstract

The invention is applicable to the technical fields of biotechnology and medicine, and provides application of PBX1 or TAT-PBX1 in preparation of products for preventing and treating degenerative neuropathy. The application specifically comprises application of the transcription factor PBX1 or TAT-PBX1 fusion protein in preparation of drugs for preventing and treating degenerative neuropathy / interventional medical instruments, and the nucleotide sequence of the overexpression transcription factor PBX1 is shown as SEQUENCE LISTING. For example, the Alzheimer's disease, which is one of degenerative neuropathy, is improved by improving the expression level of PBX1, so that the learning and spatial memory capabilities of mice are improved. Due to the utilization of the TAT-PBX1 fusion protein, the medicine can break through the blood brain barrier and directly act on cells, and the characteristic solves the major challenge of medicine delivery. A new treatment strategy is provided for treatment of the degenerative neuropathy, the treatment effect is expected to be improved, and the life quality of a patient is improved.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and medical technology, and in particular relates to the application of PBX1 or TAT-PBX1 in preparing products for preventing and treating degenerative neuropathy. Background Art

[0002] Degenerative neuropathy is a progressive disease that affects the central nervous system (CNS) or peripheral nervous system (PNS). The collapse of the structure and function of the neural network and the loss of neurons will lead to the collapse of the core communication circuit, and ultimately lead to impaired memory, cognition, behavior, sensation and motor function. This type of disease is divided into two categories according to the phenotype: one is the disease spectrum dominated by cognitive impairment, such as various types of dementia represented by Alzheimer's disease (AD); the other is the disease spectrum dominated by movement disorders, such as various types of movement disorders represented by Parkinson's disease and Parkinson's syndrome.

[0003] With the acceleration of the aging process, the development of drugs that can reduce neuronal apoptosis has become a major challenge. In degenerative neuropathies, abnormal apoptosis of neurons is the core problem. Taking AD as an example, existing treatments mostly focus on symptom control and are difficult to prevent or reverse the underlying pathological changes of the disease. This is because existing drugs are difficult to break through the blood-brain barrier (BBB) ​​and cannot prevent or reverse the underlying pathological changes of AD, such as amyloid plaques, Tau tangles, or neuronal degeneration and loss. Even if the patient receives treatment, the disease will continue to progress.

[0004] In order to reduce neuronal apoptosis and alleviate degenerative neuropathies to a certain extent, a variety of drugs and therapeutic agents have been developed. However, their effectiveness is often limited by drug delivery challenges, side effects, and the complexity of underlying disease mechanisms. For example, acetylcholinesterase inhibitors and non-competitive NMDA receptor blockers represented by donepezil (Aricept), rivastigmine (Exelon), galantamine (Razadyne), and memantine (Nemenda), although they can treat cognitive symptoms such as memory loss and confusion, cannot change the progression of the disease or resolve the underlying neurodegenerative process. While some new drugs such as Aducanumab and Lecanemab can clear β-amyloid plaques in the brains of AD patients, the efficacy of Aducanumab was only shown to be effective in the high-dose group in two phase III clinical trials; Lecanemab is accompanied by side effects that cannot be ignored, such as inducing amyloid-related imaging abnormalities (ARIA), manifested as adverse reactions such as cerebral edema (ARIA-E) and cerebral hemorrhage (ARIA-H). In addition to drug treatment, non-drug interventions such as diet, music, electroconvulsive therapy, and pet therapy also provide important auxiliary treatment options for AD patients and their families, but they can only alleviate AD symptoms to a certain extent.

[0005] In addition, when developing drugs to treat central nervous system (CNS) diseases, achieving drug delivery across the blood-brain barrier is also a major challenge. Almost all macromolecular drugs, including peptides, recombinant monoclonal antibodies, and drugs based on RNA interference technology, as well as more than 98% of small molecule drugs, cannot pass through the blood-brain barrier, which seriously hinders the effective clinical treatment of CNS diseases. Therefore, while CNS drugs are safe and effective, they must also be able to overcome the blood-brain barrier and achieve adequate exposure in the CNS, which is the key to the successful development of drugs for CNS-related diseases.

[0006] In view of the above challenges, the present invention proposes the use of PBX1 or TAT-PBX1 in the preparation of products for the prevention and treatment of degenerative neuropathy. Pre-B cell leukemia transcription factor 1 (PBX1), as a class of homeobox transcription factors called TALE (ThreeAmino acidLoop Extension), is a core regulator of neurogenesis in the subventricular zone of adults and actively participates in the fate of neural progenitor cells or cell reprogramming. Summary of the invention

[0007] The purpose of the present invention is to provide the use of PBX1 or TAT-PBX1 in the preparation of products for preventing and treating degenerative neuropathy, aiming to solve the problems raised in the above background technology.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The use of transcription factor PBX1 or TAT-PBX1 fusion protein in the preparation of a drug for preventing and treating degenerative neuropathy (such as Alzheimer's disease), the nucleotide sequence of overexpressed transcription factor PBX1 is shown in SEQUENCE LISTING.

[0010] Further, the administration of the drug containing the transcription factor PBX1 includes subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, arterial, spinal cavity injection, nasal or eye instillation, respiratory inhalation, oral administration and brain stereotaxic injection. The administration of the drug containing the TAT-PBX1 fusion protein includes intraperitoneal injection or oral administration.

[0011] Further, PBX1 or TAT-PBX1 is mixed with other excipients to prepare a drug (preparation) for preventing and treating Alzheimer's disease. These excipients can be conventionally used in various preparations, such as isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they can also be selected to be compatible with the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the organism, and then enhancing the drug administration effect. In aqueous injections, excipients generally include isotonic agents and buffers, as well as necessary emulsifiers (such as Tweeen-80, Pluronic and Poloxamer, etc.), solubilizers and antibacterial agents, etc. In addition, other pharmaceutically acceptable pharmaceutical excipients are also included, such as antioxidants, pH regulators and analgesics, etc. Excipients used to prepare liquid preparations generally include solvents (such as water, oils, such as fatty acids), emulsifiers, and necessary preservatives, etc. Various excipients can be combined with drugs to make dosage forms that are conducive to drug delivery, such as aqueous solution injections, nasal drops, eye drops, powder injections, powders, patches, suppositories, emulsions, creams, gels, aerosols, sprays, powder sprays, sustained-release agents, and controlled-release agents, etc.

[0012] In addition, in order to achieve specific drug delivery purposes or methods (such as sustained-release drug delivery, controlled-release drug delivery and pulse drug delivery, etc.), specific excipients can also be used, such as gelatin, albumin, chitosan, polyether and polyester polymer materials (such as polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc.). These excipients help to achieve "favorable drug delivery", mainly in terms of improving therapeutic effects, increasing bioavailability, reducing toxic side effects and improving patient compliance.

[0013] The application of transcription factor PBX1 or TAT-PBX1 fusion protein in the preparation of interventional medical devices for the prevention and treatment of degenerative neuropathy (such as Alzheimer's disease). For example, PBX1 or TAT-PBX1 can be loaded or coated on a scaffold material (such as PLA, PLGA, GelMA and metal, etc.) as an active ingredient to make an interventional medical device for the prevention and treatment of Alzheimer's disease. In addition, it can also be mixed with biocompatible degradable materials to make microneedles and microneedle arrays thereof, or loaded in metal microneedles to make microneedle chips. When the microneedle pierces the skin, PBX1 or TAT-PBX1 will be released in the epithelial tissue and enter the blood, and enter the cell through the blood-brain barrier.

[0014] Furthermore, by increasing the expression of transcription factor PBX1 or injecting TAT-PBX1 fusion protein, Aβ accumulation was inhibited, DNA damage and neuronal apoptosis were reduced, and the learning and spatial memory abilities of mice were improved.

[0015] Furthermore, the overexpression vector of the transcription factor PBX1 or TAT-PBX1 fusion protein is a lentivirus, adenovirus, adeno-associated virus, or retrovirus, wherein the TAT-PBX1 fusion protein is packaged with a lentivirus, adenovirus, adeno-associated virus, or retrovirus, and then transduced into cells (such as mesenchymal stem cells, neural crest stem cells, pigment stem cells, and fibroblasts).

[0016] Furthermore, the overexpression of TAT-PBX1 cells or culture supernatant is achieved by infecting cells with lentivirus to overexpress TAT-PBX1 protein. TAT transmembrane peptide enhances the absorption of proteins by cells such as neurons and mesenchymal stem cells, so that PBX1 contained in the cell culture supernatant can pass through the blood-brain barrier and enter the cells. TAT-PBX1 fusion protein is an exogenous protein expressed by Escherichia coli, yeast or CHO cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention takes Alzheimer's disease, one of the degenerative neuropathy, as an example, and improves the learning and spatial memory ability of mice by increasing the expression level of PBX1. The use of TAT-PBX1 fusion protein enables drugs to break through the blood-brain barrier and act directly on cells. This feature solves the major challenge of drug delivery. The present invention provides a new treatment strategy for the treatment of degenerative neuropathy, which is expected to improve the treatment effect and improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The overexpression plasmid pLVX-hSyn-RFP was designed and synthesized based on the sequence of NM_002585.4-NP_002576.1 of PBX1.

[0020] Figure 2 The overexpression plasmid pLVX-IRES-mCherry was designed and synthesized based on the sequence of NM_002585.4-NP_002576.1 of PBX1.

[0021] Figure 3 The fluorescent spots after bilateral cerebroventricular injection of PBX1 lentivirus were detected by small animal in vivo imaging (Tanon ABL-X6 animal in vivo imaging system).

[0022] Figure 4 Western Blot was used to detect the expression of PBX1 in the hippocampus of mice after bilateral hippocampal injection; the test and analysis method was one way ANOVA, and Dunnett's post hoc test, *: P<0.05.

[0023] Figure 5 The effect of overexpression of PBX1 in AD model neuronal cells on the accumulation of two soluble Aβ fragments in cells (intra-cellular) and supernatant (cell culture supernatant); CTRL: control group (without any treatment); WT_pLVX_NC: empty plasmid overexpressing PBX1, i.e. negative control; WT-pLVX_PBX1: PBX1 overexpression group; SWE-CTRL: overexpression of APPswe; SWE-pLVX_NC: empty plasmid overexpressing APPswe; SWE-pLVX_PBX1: simultaneous overexpression of APPswe and PBX1; detection method was Elisa method; test analysis method was one way ANOVA, after Dunnett's post hoc test, *: P<0.05.

[0024] Figure 6 The results of Western Blot; APP: overexpression of APPswe; pLVX-NC: empty plasmid overexpressing PBX1; pLVX-PBX1: overexpression of PBX1.

[0025] Figure 7 The results of APC / 7AAD double staining flow cytometry; SH-WT: blank control group; SH-NC: empty group overexpressing PBX1; SH-PBX1: PBX1 overexpression group; SWE-WT: APPswe overexpression group; SWE-NC: empty group overexpressing APPswe; SWE-PBX1: overexpression of APPswe and PBX1 at the same time. The test analysis method was one way ANOVA, and Dunnett's post hoc test was performed. *: P<0.05.

[0026] Figure 8 Comet assay was used to detect cell DNA damage. The analysis method was one way ANOVA with Dunnett's post hoc test. *: P<0.05.

[0027] Fig. 9 These are the results of small animal behavior experiments.

[0028] Fig.10 The immunofluorescence results of the mouse brain hippocampus; Note: The box is a representative hippocampal area. The more fluorescent areas, the larger the AβO positive area.

[0029] Fig.11 This is the plasmid map of TAT-PBX1.

[0030] Fig.12This is the construction of the TAT-PBX1 prokaryotic expression system and the denaturation, purification and renaturation results of the recombinant protein.

[0031] Fig.13 In vitro experiments were used to detect the protein levels of DNA damage and apoptosis-related indicators; the test and analysis method was oneway ANOVA, and Dunnett's post hoc test, *: P<0.05; **: P<0.01; ***: P<0.001.

[0032] Fig.14 Two hours after intraperitoneal injection of TAT-PBX1 (0.5 mg / Kg), His tag was detected in the hippocampus of mice.

[0033] Fig.15 Tunel staining results of mouse brain sections (representative areas are shown in the box); the test and analysis method was one way ANOVA, and Dunnett's post hoc test, *: P < 0.05; **: P < 0.01; ***: P < 0.001. TAT-PBX1 Low: TAT-PBX1 low dose (1 mg / kg / d); TAT-PBX1 Mid: TAT-PBX1 medium dose (1.5 mg / kg / d); TAT-PBX1Hi: TAT-PBX1 high dose (2 mg / kg / d).

[0034] Fig.16 Dot Blot was used to detect Aβ deposition in the hippocampus of APP / PS1 mice after intraperitoneal injection of TAT-PBX1, where TAT-PBX1 Low: low dose of TAT-PBX1 (1 mg / kg / d); TAT-PBX1 Mid: medium dose of TAT-PBX1 (1.5 mg / kg / d); TAT-PBX1 Hi: high dose of TAT-PBX1 (2 mg / kg / d); the test and analysis method was one way ANOVA, and Dunnett's post hoc test, *: P<0.05; **: P<0.01; ***: P<0.001.

[0035] Fig.17 These are the results of behavioral experiments, including TAT-PBX1 Low: low dose of TAT-PBX1 (1 mg / kg / d); TAT-PBX1 Mid: medium dose of TAT-PBX1 (1.5 mg / kg / d); TAT-PBX1 Hi: high dose of TAT-PBX1 (2 mg / kg / d); the test and analysis method was one way ANOVA, with Dunnett's post hoc test, *: P<0.05; **: P<0.01; ***: P<0.001. DETAILED DESCRIPTION

[0036] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0038] Example 1: Preparation of PBX1 gene recombinant virus biological preparation, and application in in vivo or in vitro experiments;

[0039] According to the sequence of PBX1 NM_002585.4-NP_002576.1, two overexpression plasmids were designed and synthesized: pLVX-IRES-mCherry ( Figure 2 ) and pLVX-hSyn-RFP( Figure 1 ), these two plasmids were co-transfected with the virus packaging plasmid into 293T cells to prepare the virus. Subsequently, the cell supernatant containing the virus was collected, the virus titer was detected after concentration, and centrifugal filtration was performed. Among them, the pLVX-IRES-mCherry virus was used to transduce SH-SY5Y cells, and the pLVX-hSyn-RFP virus was injected into the bilateral hippocampus by stereotaxic injection technology.

[0040] 1. The lentiviral packaging method is as follows:

[0041] 1) 6×10 6 293T cells were inoculated in a 10 cm culture dish, and transfection was performed when the cell confluence reached 70% the next day;

[0042] 2) Prepare transfection complex according to the Lip2000 manual, prepare two EP tubes, and add 500μL serum-free culture medium (Opti-MEM, Gibco, USA) to each. Add 10μg of target plasmid, 7.5μg of packaging plasmid PAX2, and 2.5μg of PMD2G to the first EP tube; add 30μL transfection reagent to the second EP tube, let stand at room temperature for 5 minutes, then add 500μL system containing plasmid to the system containing transfection reagent, flick the bottom of the tube to mix, and let stand at room temperature for 15 minutes;

[0043] 3) Drop the above transfection complex into a 293T culture dish and place it in an incubator;

[0044] 4) Collect the culture supernatant after 48 h and 72 h, filter with a 0.45 μm filter, and aliquot the viruses and store at -80°C.

[0045] 2. The cell transduction method is as follows:

[0046] 1) Discard the culture medium of SH-SY5Y cells and perform transduction after the cells adhere to the wall. Add pLVX-IRES-mCherry virus and Polybrene to the cells. The final concentration of Polybrene in SH-SY5Y cells is 5 μg / mL;

[0047] 2) Replace the culture medium with fresh medium 24 hours after transduction and observe the transduction of cells using a fluorescence microscope 72 hours after transduction.

[0048] 3. The method of stereotaxic injection of virus into bilateral hippocampus is as follows:

[0049] 1) Use PBS (without Ca 2+ and Mg 2+ ) The titer of all pLVX-hSyn-RFP viruses was adjusted to 2 × 10 9 IU / ml. Before injection, 2.5 μL of 20% mannitol was dissolved in PBS and mixed with 5 μL of virus stock in a 10 μL syringe;

[0050] 2) AD model mice inhaled oxygen containing isoflurane (flow rate 1 L / min, containing 5% (v / v) isoflurane) and maintained anesthesia until the animal's corneal and toe reflexes disappeared;

[0051] 3) Fix the mouse: insert the ear fixation rods into the bilateral external auditory canals, fix the mouse head on the stereotaxic apparatus in a prone position. Use scissors to cut the hair on the top of the mouse head, and use a scalpel to cut the skin after disinfection with iodine;

[0052] 4) Skull drilling: mark the injection point, drill a hole at the marked point with a dental drill, and slowly penetrate the skull until you feel a hollow hole;

[0053] 5) Virus injection: The micropipette was inserted vertically into the dura mater surface to a depth of 2.0 mm. The prepared virus solution was slowly injected, with an injection volume of 7.5 μL on each side (the control group was given the same volume of sterile saline), and an injection speed of 5 nL / S. After the injection, the needle was retained for 5 min to ensure that the solution was fully diffused into the local hippocampal tissue, and then the needle was slowly withdrawn. The skull hole was then filled with bone wax and the skin was sutured with suture clips.

[0054] Figure 3 The image shows the use of a small animal in vivo imager (TanonABL-X6 animal in vivo imaging system) to detect fluorescent spots after bilateral cerebroventricular injection of the PBX1 lentivirus. The bright red spots in the box in the image are the red fluorescent groups after pLVX-hSyn-RFP injection, indicating that the PBX1 lentivirus was successfully injected into the target area (hippocampus).

[0055] Figure 4Western Blot detection showed that after bilateral hippocampal injection, the expression level of PBX1 in the hippocampus region of APP / PS1 mice was increased compared with WT (wild-type mice, i.e. control group) and pLVX-vector group (PBX1 virus empty vector group), indicating that the PBX1 virus was successfully injected into the hippocampus region.

[0056] Example 2: Overexpression of PBX1 in AD neuronal cell models can reduce Aβ accumulation and cell apoptosis;

[0057] To verify the effect of overexpressing PBX1 in AD neuronal cell models, we performed an alkaline comet assay to detect cell DNA damage. The specific steps are as follows:

[0058] 1) Make slides: prepare ordinary glass slides, UV glue, and 2mm×2mm×2mm acrylic strips. Use glue to surround four acrylic strips into a square and stick them on the glass slide. Each glass slide has two glue slots.

[0059] 2) Collect cells, resuspend with PBS, and keep at 37°C for later use. Take low-melting point agarose and add PBS to a final concentration of 1%, heat in a microwave oven to melt, and keep at 37°C after cooling. Gel preparation: Mix the cell suspension and melted low-melting point agarose in a volume ratio of 1:1, immediately spread in the gel tank (about 100 μL per gel tank), and cool at 4°C for 15 minutes;

[0060] 3) Immerse the slide in the lysis solution and place it in a dark room at 4°C for 2 hours. After 2 hours, take out the slide and soak and rinse it in 4°C distilled water twice, 5 minutes each time.

[0061] 4) Immerse the slide with the film in an alkaline solution at 4°C for electrophoresis for 15 minutes at a voltage of 25 V. After the electrophoresis, use filter paper to absorb the solution and soak it in a neutralizing solution for 15 minutes;

[0062] 5) Add DAPI solution to each film, making sure the solution covers the film. After keeping away from light for 1-5 minutes, take photos and observe under a fluorescence microscope.

[0063] The results showed that overexpression of PBX1 in AD model neurons affects soluble Aβ fragments. Figure 5 Compared with the SWE-CTRL group (overexpressing APPswe, i.e. AD model cells), overexpression of PBX1 (i.e. SWE-pLVX-PBX1 group) significantly reduced the accumulation of two soluble Aβ fragments (Aβ1-42 and Aβ1-40) in cells (intra-cellular) and in the supernatant (cell culture supernatant).

[0064] Western Blot results showed that ( Figure 6 ), compared with the AD model group (lane 4), overexpression of PBX1 (lane 6) can reduce the expression of p-Tau. p-Tau is another important marker in the pathological process of AD, and this finding further supports the potential of PBX1 in combating the progression of AD.

[0065] APC / 7AAD double staining flow cytometry results showed ( Figure 7 ), compared with the SWE-WT group (overexpressing APPswe, i.e., AD model cells), the number of positive cells in the second (upper right) and third (lower right) quadrants in the SWE-PBX1 overexpressing group was significantly reduced, that is, the number of early and late apoptotic cells was reduced, indicating that overexpression of PBX1 can reduce cell apoptosis caused by overexpression of APPswe.

[0066] Comet assay to detect cell DNA damage showed that ( Figure 8 ), compared with the AD model group cells (i.e., SH-SWE, row 2, columns 3 and 4), the nuclear tailing was reduced in cells overexpressing PBX1 (pLVX-PBX1, row 3, columns 3 and 4), indicating that overexpression of PBX1 can reduce DNA damage in AD model cells.

[0067] The results of immunofluorescence in the mouse hippocampus showed that ( Fig.10 ), bilateral intracerebroventricular injection of PBX1 lentivirus (APP / PS1+PBX1) significantly reduced the positive area of ​​AβO in the hippocampus, indicating that PBX1 can effectively reduce Aβ deposition in AD model animals.

[0068] Example 3: PBX1 gene therapy can improve the learning and spatial memory abilities of AD model mice;

[0069] Water maze test: Morris water maze (MWM) test can evaluate the learning and spatial memory ability of mice. The experimental apparatus is a circular pool filled with water containing dissolved titanium dioxide, and the pool is divided into four quadrants of equal area. In the first 3 days of the experiment, a platform was hidden 1.2 cm below the water surface in the third quadrant. The mice started from the middle position of one of the four quadrants facing the pool wall and trained to find the platform for 3 consecutive days. Four training sessions were conducted on the first day, and six training sessions were conducted on the second and third days, with an interval of 5-10 minutes between each training. The platform was removed on the fourth day, and the platform was hidden in the fourth quadrant on the fifth and sixth days. The new platform was trained for 2 consecutive days, with four training sessions per day, and the platform was removed on the seventh day for testing. For the training experiment, the mice were allowed to find the platform within 60 seconds and stay on the platform for another 30 seconds. If the animal failed to find the platform within 60 seconds, the time was recorded as 60 seconds, and then these mice were guided to the platform and stayed for 5-10 seconds. The test experiment was conducted 24 hours after the training experiment. The underwater platform was removed and the mice were placed individually in the quadrant opposite to the original platform position. The time it took for the mice to reach the previously placed platform, the number of times they crossed the platform, the swimming speed, and the swimming distance were automatically recorded by a camera, and the data were finally statistically analyzed.

[0070] The results of small animal behavior experiments show that ( Fig. 9 ), the time that APP / PS1 mice stayed in the target quadrant was significantly lower than that of wild-type (WT) mice, and the number of times they crossed the platform decreased, and the latency of crossing the platform was prolonged, and the difference was statistically significant. After overexpressing PBX1 in hippocampal neurons, compared with VECTOR, the time that mice overexpressing PBX1 stayed in the target quadrant and the number of times they crossed the platform increased significantly, and the latency of crossing the platform was significantly reduced. These results indicate that overexpressing PBX1 in hippocampal neurons of AD mice can significantly improve the learning and spatial memory abilities of mice.

[0071] Example 4: Construction of TAT-PBX1 prokaryotic expression system and denaturation, purification and renaturation of recombinant protein;

[0072] First, the recombinant plasmid was constructed using the E. coli expression vector pTAT-HA. After the plasmid construction was completed, the E. coli Rosetta (DE3) containing the construction vector pTAT-HA-PBX1 and the original vector pTAT-HA was grown overnight at 37°C in ampicillin and chloramphenicol resistant LB agar medium. The positive monoclonal strain was picked from the culture medium for amplification, centrifugation, collection of bacterial precipitation, ultrasonic bacterial lysis, and Ni2+-NTA-agarose column purification of the supernatant. The purified protein was slowly stirred overnight at 4°C in a refolding buffer (10mMTris, 10mM glycine, 1mM EDTA, 0.5M arginine (pH 9.5)) to achieve protein refolding. Subsequently, centrifugation was performed to remove impurities, the supernatant was added to a dialysis bag, and dialyzed overnight at 4°C with 20mM phosphate buffer (pH 6.5), and the dialyzed protein solution was concentrated using an ultrafiltration tube to obtain a denatured recombinant protein.

[0073] Fig.11 For the plasmid map of TAT-PBX1, the DNA sequence of PBX1 was added to the C-terminus of the TAT peptide within the plasmid vector pTAT-HA, which contains an N-terminal His 6 tag for purification and an HA tag for distinguishing TAT from PBX1.

[0074] The construction of TAT-PBX1 prokaryotic expression system and the results of denaturation, purification and renaturation of recombinant protein showed that ( Fig.12 ), according to the Coomassie brilliant blue results of the bacterial solution (starting solution, Start), flowthrough solution (Flowthrough), washing solution (Wash) and elution (Elution), it was proved that the purity of the purified TAT-PBX1 was greater than 90%, the molecular weight was correct, and it could be successfully detected.

[0075] The results of in vitro experiments showed that ( Fig.13 ), the protein expressions of Cleaved Caspase3, Cyto C, PARP1, OGG1 and γ-H2AX in the TAT-PBX1+AβO group were significantly lower than those in the AβO group. Among them, Cleaved Caspas3 and Cyto C are markers of apoptosis; PARP1, OGG1 and γ-H2AX are markers of DNA damage. The results showed that the addition of TAT-PBX1 (50μg / mL) significantly reduced AβO-induced DNA damage and apoptosis in SH-SY5Y cells.

[0076] Example 5: TAT-PBX1 intraperitoneal injection detection and its effect on AD model mice;

[0077] 1. Test whether TAT-PBX1 can pass the blood-brain barrier by intraperitoneal injection. The specific steps include: TAT-PBX1 (0.5 mg / Kg, dissolved in ultrapure water) was intraperitoneally injected into 8-week-old male C57 mice. After 2 hours, the mice were sacrificed, the hippocampus was taken and the total protein of the hippocampus was extracted for Western Blot experiment to detect the expression of His tag in the hippocampus. Fig.14 It can be seen that 2 hours after intraperitoneal injection of TAT-PBX1 (0.5 mg / Kg), His tag can be detected in the hippocampus of mice, indicating that TAT-PBX1 can break through the blood-brain barrier and enter the brain. After confirming that TAT-PBX1 can break through the blood-brain barrier and enter the target area, 10-month-old APP / PS1 mice and WT mice of the same age were selected and randomly divided into 6 groups: WT control group of the same age, APP / PS1 solvent control group (intraperitoneal injection of equal volume of PBS), TAT-PBX1 low-dose intervention group (intraperitoneal injection of 1 mg / kg of TAT-PBX1), TAT-PBX1 medium-dose intervention group (intraperitoneal injection of 1.5 mg / kg of TAT-PBX1) and TAT-PBX1 high-dose intervention group (intraperitoneal injection of 2 mg / kg of TAT-PBX1). Injection once every other day, a total of 10 injections. After 10 injections (i.e., 20 days later), the mice were subjected to behavioral experiments (as described in Example 3) to evaluate the changes in cognitive function and brain tissue structure of the mice after TAT-PBX1 treatment. The results of the behavioral experiments showed that ( Fig.17 ), the time that APP / PS1 mice stayed in the target quadrant was significantly lower than that of the control group mice, and the number of times they crossed the platform decreased, and the latency of crossing the platform was prolonged, with statistically significant differences. After intraperitoneal injection of TAT-PBX1, compared with the APP / PS1 model group mice, the time that mice stayed in the target quadrant and the number of times they crossed the platform increased significantly, and the latency of crossing the platform was significantly reduced, indicating that intraperitoneal injection of TAT-PBX1 can significantly improve learning and spatial memory abilities in AD model APP / PS1 mice.

[0078] 2. Use Tunel staining to detect apoptotic changes in mouse hippocampal neurons. The specific steps include: washing brain slices twice with xylene, washing with gradient ethanol (100, 95, 90, 80, 70%) once each, and rinsing with PBS twice. After rinsing, treat the tissue with Proteinase K working solution at 37°C for 15-30min and rinse twice with PBS. Then mix 50μl TdT+450μl fluorescein-labeled dUTP solution to prepare Tunel reaction mixture, react with slices at 37°C for 60min, and keep in a wet box to avoid light. After the reaction is completed, take out the wet box in a dark place, absorb the TUNEL working solution on the slice, and put the slice into a small dye jar and rinse with PBS. Add DAPI working solution to stain the nucleus and react at room temperature for 10-20min. Finally, recover DAPI, add 5-10μl anti-fluorescence attenuation mounting agent or neutral gum, seal the slice with a clean coverslip, and observe and take pictures under a fluorescence microscope.

[0079] Tunel staining results of mouse brain sections (representative areas are shown in the box) show ( Fig.15 ), three doses of TAT-PBX1 (1.0 mg / Kg, 1.5 mg / Kg and 2.0 mg / Kg) all significantly reduced the Tunel-positive area in the hippocampus of APP / PS1 mice, indicating that TAT-PBX1 can reduce the apoptosis of hippocampal neurons in APP / PS1 mice.

[0080] 3. Use Dot Blot to detect Aβ deposition. The specific steps include: extracting total protein from mouse hippocampus and performing protein leveling. Take 2μL of the leveled protein and evenly spot it on the nitrocellulose membrane, with each spot spaced 0.5cm apart. Place the nitrocellulose membrane at room temperature for 5min until the droplets on the strip are dry. Then incubate the strip with 5% BSA solution at room temperature for 30min, rinse the strip with TBST solution for 5min, and aspirate the TBST solution. Incubate at room temperature for 1h, and incubate the nitrocellulose membrane with the sample with anti-oligomeric antibody A11 solution and anti-Aβ antibody 6E10 solution. After 1h, rinse the test strip with TBST solution 3 times, each time at room temperature for 5min, and aspirate the TBST solution. Incubate the strip with the secondary antibody solution for 40min at room temperature. Rinse the test strip with TBST solution 3 times, each time at room temperature for 5min. Apply the mixed ECL liquid evenly on the surface of the strip. Expose the strip in an automatic chemiluminescence imaging system. Grayscale analysis was performed using ImageJ (National Institutes of Health) software to obtain semi-quantitative results.

[0081] Dot Blot analysis of Aβ deposition in the hippocampus of APP / PS1 mice after intraperitoneal injection of TAT-PBX1 showed that ( Fig.16), all three doses of TAT-PBX1 significantly reduced the concentration of AβO and protofibrils in the hippocampus of APP / PS1 mice. Among them, A11 is an AβO antibody that can specifically recognize the Aβ amino acid-independent oligomer form, namely AβO; 6E10 is an Aβ protofibril antibody that can specifically recognize Aβ protofibrils.

[0082] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Use of transcription factor PBX1 or TAT-PBX1 fusion protein in the preparation of a drug for preventing and treating degenerative neuropathy, characterized in that: The nucleotide sequence of the overexpressed transcription factor PBX1 is shown in SEQUENCE LISTING.

2. Use of transcription factor PBX1 or TAT-PBX1 fusion protein in the preparation of interventional medical devices for preventing and treating degenerative neuropathy, characterized in that: The nucleotide sequence of the overexpressed transcription factor PBX1 is shown in SEQUENCE LISTING.

3. The use according to claim 1 or 2, characterized in that: By increasing the expression of transcription factor PBX1 or injecting TAT-PBX1 fusion protein, Aβ accumulation can be inhibited, DNA damage and neuronal apoptosis can be reduced, and the learning and spatial memory abilities of mice can be improved.

4. The use according to claim 1 or 2, characterized in that: The overexpression vector of the transcription factor PBX1 or TAT-PBX1 fusion protein is a lentivirus, adenovirus, adeno-associated virus or retrovirus.

5. The use according to claim 1 or 2, characterized in that: The TAT-PBX1 fusion protein is an exogenous protein expressed by Escherichia coli, yeast or CHO cells.

6. The use according to claim 1 or 2, characterized in that: The degenerative neuropathy is Alzheimer's disease.

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