Neural stem cell-parathymosin compound cell preparation and application thereof in promoting neurogenesis and treating nerve injury

Abstract

The invention relates to a neural stem cell-parathymosin composite cell preparation and application thereof in treatment of nerve injury diseases. According to the invention, it is found for the first time that neural stem cells overexpressing parathymosin (PTMS) can effectively promote neuron neogenesis and axon growth in in-vitro and in-vivo experiments; animal experiment results show that the traditional Chinese medicine composition can also be used as a treatment means for promoting nerve regeneration to resist dyskinesia related to nerve injury diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel treatment regimen for repairing nerve damage by preparing and applying a neural stem cell-parathymidine complex cell preparation. Research Background

[0002] Neurological injury is caused by the apoptosis or necrosis of neurons or glial cells due to trauma or non-traumatic reasons, resulting in functional impairments such as motor or sensory loss at and below the site of injury. This severely impacts a patient's ability to live independently and participate in social activities, commonly occurring in the brain (brain injury), spinal cord (spinal cord injury), and peripheral nerve injuries outside the brain and spinal cord. Taking spinal cord injury as an example, as of 2021, the number of people with spinal cord injury in my country exceeded 3.7 million, with approximately 66,374 new cases of traumatic spinal cord injury each year. Due to the complexity of the disease, patients with spinal cord injury often require lifelong rehabilitation treatment, incurring substantial costs. Spinal cord injury places a heavy economic and psychological burden on patients, their families, and society. However, to date, there are still no effective treatments for neurological injury in clinical practice.

[0003] Neural stem cells are a type of pluripotent stem cell capable of self-renewal and differentiation into various nerve cell types, including neurons, oligodendrocytes, and astrocytes. In recent years, they have been widely used in neuroscience research and clinical treatment studies, and are considered a potentially effective cell type for transplantation to treat nerve injuries. Transplanted neural stem cells can differentiate into specific nerve cells to exert repair functions, and can also secrete cytokines or exosomes to exert immunomodulatory, nerve regeneration-promoting, and myelin regeneration-promoting effects, playing a crucial role in the repair of nerve injuries.

[0004] Although neural stem cells hold great promise for treating nerve injuries, particularly spinal cord injuries, transplanting neural stem cells for spinal cord injury treatment still faces many challenges. For example, after spinal cord injury, the hypoxic and ischemic spinal cord environment, along with various inflammatory responses and oxidative stress, creates an unfavorable metabolic microenvironment that severely restricts nerve and axonal regeneration, resulting in limited therapeutic effects from transplantation.

[0005] Previous research in our laboratory identified a neuroprotective protein, PTMS, from secreted proteins of hypothalamic neural stem cells. Upon release, PTMS can rapidly translocate to the nuclei of various cell types, including neurons and different peripheral cells; and in vivo, it effectively translocates to neuronal nuclei in brain regions, exhibiting neuroprotective functions. Summary of the Invention

[0006] In order to overcome the problems that are difficult to solve in the treatment of nerve injury by neural stem cell transplantation in the existing technology, the present invention provides a novel treatment scheme for repairing nerve injury by preparing and applying neural stem cell-PTMS composite cell preparation.

[0007] The first objective of this invention is to provide a neural stem cell-PTMS composite cell preparation. As a preferred embodiment of this invention, the neural stem cell-PTMS composite cell preparation is prepared by introducing PTMS protein or DNA or RNA encoding PTMS into neural stem cells through electrotransduction, chemical transduction, or biological vector infection.

[0008] As another preferred embodiment of the present invention, the neural stem cells include primary cultured neural stem cells from the hippocampus, olfactory bulb, cortex, subventricular zone (SVZ), and hypothalamus, as well as induced neural stem cells generated by in vitro differentiation of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0009] The second objective of this invention is to provide an application of overexpressing PTMS to promote the differentiation of neural stem cells into neurons. As a preferred example of this invention, PTMS protein or DNA or RNA encoding PTMS is introduced into neural stem cells via electrotransduction, chemical transduction, or biological vector infection to express PTMS and promote the differentiation of neural stem cells into neurons.

[0010] The third objective of this invention is to provide an application of neural stem cell-PTMS composite cell preparations in the treatment of nerve injury. As a preferred example of this invention, transplantation of neural stem cells overexpressing PTMS can significantly improve motor function in mice with spinal cord injury and promote nerve regeneration and axonal regeneration.

[0011] In a preferred embodiment of the present invention, the neural stem cell-PTMS composite cell preparation is administered via intravenous, arterial, oral, parenteral, oral, vaginal, rectal, inhalation, blowing, sublingual, intramuscular, subcutaneous, local, intranasal, intraperitoneal, or intracranial routes.

[0012] The advantages of this invention are:

[0013] 1. For the first time, it was discovered that overexpression of PTMS via a viral vector can effectively promote the differentiation of neural stem cells into neurons, improve differentiation efficiency, and promote neuronal axon development, while knockdown of PTMS expression inhibits the differentiation of neural stem cells into neurons.

[0014] 2. It was discovered for the first time that conditioned medium containing PTMS can effectively promote the differentiation of neural stem cells into long axon neurons.

[0015] 3. For the first time, it was discovered that overexpression of PTMS in neural stem cells / neurons can significantly enhance cellular glucose metabolism (oxidative phosphorylation and glycolysis), while knockdown of PTMS inhibits the levels of oxidative phosphorylation and glycolysis in cells.

[0016] 4. For the first time, PKM2 was discovered to be an intracellular target of PTMS. Inhibition of PKM2 affects the differentiation of neural stem cells into neurons and glucose metabolism by PTMS.

[0017] 5. For the first time, it was discovered that PTMS exerts an effect similar to that of a PKM2 agonist, affecting PKM2 tetramer and thus PK enzyme activity.

[0018] 6. For the first time, it was discovered that transplantation of neural stem cells overexpressing PTMS can promote nerve and axon regeneration in mice with spinal cord injury and improve motor function in mice with spinal cord injury.

[0019] 7. The therapeutic mechanism of neural stem cell-PTMS composite cell preparation in repairing spinal cord injury was revealed. Neural stem cells overexpressing PTMS maintain PKM2 tetramer by paracrine PTMS, thereby maintaining PK enzyme activity, enhancing glycolysis after spinal cord injury, improving the metabolic microenvironment, promoting nerve regeneration and axon regeneration after injury, and improving motor function recovery in mice with spinal cord injury. Attached Figure Description

[0020] Figure 1 PTMS is a neuroprotective protein associated with neuronal loss following spinal cord injury.

[0021] Figure 2 One of the preferred examples for preparing neural stem cell-PTMS composite cell preparations is the construction of neural stem cells overexpressing PTMS via viral vectors.

[0022] Figure 3 To investigate the effect of PTMS overexpression / knockdown on neural stem cell differentiation into neurons using immunofluorescence staining.

[0023] Figure 4 To investigate the effect of PTMS overexpression on glucose metabolism in neural stem cells using Seahorse.

[0024] Figure 5 This is a diagram showing the interaction between PKM2 and PTMS.

[0025] Figure 6 To investigate the effects of PTMS overexpression / knockdown on PKM2 tetramer and enzyme activity.

[0026] Figure 7Behavioral testing of mice with spinal cord injury treated with transplanted neural stem cell-PTMS composite cell preparation.

[0027] Figure 8 To detect the proportion of MAP2-positive and NeuN-positive neurons at the site of spinal cord injury in each group using immunofluorescence.

[0028] Figure 9 The expression of PKM in the spinal cord of each group was detected by RT-qPCR and Western Blot. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0030] Example 1: Preparation of neural stem cell-PTMS composite cell preparation

[0031] (1) Extraction and purification of neural stem cells

[0032] Newborn C57BL / 6 mice were sterilized with 75% alcohol, and the hypothalamus was dissected and minced in a sterile laminar flow hood. The cells were then digested with TrypLE Express enzyme in a 37°C CO2 incubator for 15 min. After digestion, the cells were resuspended in an appropriate amount of neural stem cell culture medium, passed through a 100-mesh sieve, and single-cell suspensions were collected. These suspensions were then cultured in low-absorption 6-well plates using neural stem cell culture medium containing EGF and bFGF. After one week, neurospheres were collected by centrifugation at 700 rpm for 5 min and digested into single cells with TrypLE Express enzyme. The cells were passaged, and the collected cells were identified as hypothalamic neural stem cells (htNSCs). Finally, immunofluorescence staining was used to identify the expression of neural stem cell-specific markers (Nestin and SOX2).

[0033] (2) Constructing a neural stem cell-PTMS composite cell preparation using a viral vector

[0034] First, using Plenti-CMV-Control-GFP and Plenti-CMV-PTMS-HA plasmids, a third-generation lentiviral packaging system, and HEK293T cells, CMV-GFP control virus (CMV-GFP) and PTMS overexpression virus (PTMS-OE) were constructed. Viral supernatants were collected, concentrated by ultracentrifugation, and then aliquoted and stored at -80°C.

[0035] Then, htNSCs single-cell suspensions were infected with neural stem cell culture medium containing appropriate concentrations of lentivirus to construct CMV-GFP htNSCs and PTMS-OE htNSCs, respectively. The infected cells were screened and maintained using neural stem cell culture medium containing 0.2 μg / ml blisterdin. Finally, the effect of PTMS overexpression by PTMS-OE htNSCs was verified by RT-qPCR or Western Blot. The htNSCs that can stably overexpress PTMS obtained by screening are one of the neural stem cell-PTMS composite cell preparations.

[0036] Example 2: Application of PTMS in regulating the differentiation of neural stem cells into neurons

[0037] (1) Constructing neural stem cells with knockdown of PTMS expression using a viral vector

[0038] First, using Control ShRNA and PTMS ShRNA plasmids, a second-generation lentiviral packaging system, and HEK293T cells, Control ShRNA virus (Ctrl ShRNA) and PTMS ShRNA virus (PTMS ShRNA) were constructed. Viral supernatants were collected, concentrated by ultracentrifugation, and then aliquoted and stored at -80°C.

[0039] Then, htNSCs single-cell suspensions were infected with neural stem cell culture medium containing appropriate concentrations of lentivirus to construct Ctrl ShRNA htNSCs and PTMS ShRNA htNSCs, respectively. The infected cells were screened and maintained using neural stem cell culture medium containing 0.2 μg / ml Puromycin. Finally, the effect of PTMSShRNA knocking down PTMS was verified by RT-qPCR or Western Blot.

[0040] (2) Inducing neural stem cells to differentiate into neurons

[0041] First, CMV-GFP htNSCs, PTMS-OE htNSCs, Ctrl ShRNA htNSCs, and PTMS ShRNA htNSCs neurospheres were collected. After enzymatic digestion, single-cell suspensions were seeded at 1×10⁵ cells / well onto poly-L-lysine (PDL)-coated coverslips placed in 24-well plates. The next day, the neural stem cell culture medium was replaced with differentiation medium (Neurobasal-A, 2% B27, 1% fetal bovine serum, and 1 μM retinoic acid). The differentiation medium was replaced with half the amount every other day for 5 days to induce differentiation. Immunofluorescence staining was then performed to calculate the proportion of newly generated TUJ1 neurons and the length of neuronal axons.

[0042] Example 3: Investigating the effect of PTMS on the regulation of glucose metabolism in neural stem cells

[0043] Using the Seahorse XF Real-Time ATP Generation Rate Assay Kit and an Agilent Seahorse XFe96 instrument, the ATP generation rates of CMV-GFP htNSCs, PTMS-OE htNSCs, Ctrl ShRNA htNSCs, and PTMSShRNA htNSCs were measured according to the kit instructions to investigate the effect of PTMS on glycolysis and oxidative phosphorylation of htNSCs.

[0044] The results showed that, compared to CMV-GFP htNSCs, overexpression of PTMS significantly enhanced the rate of ATP production from glycolysis and oxidative phosphorylation in neural stem cells; compared to Ctrl ShRNA, knockdown of PTMS inhibited the rate of ATP production from glycolysis and oxidative phosphorylation in neural stem cells. This indicates that overexpression of PTMS in htNSCs enhances cellular glycolysis and oxidative phosphorylation, while knockdown of PTMS inhibits these processes.

[0045] Example 4: Identification of intracellular regulatory targets of PTMS for glycolysis

[0046] (1) Screening intracellular targets of PTMS using proteomics

[0047] We enriched proteins interacting with PTMS using HA-tagged magnetic beads. PTMS-OE cells were collected, lysed, and magnetic beads were added at a ratio of 20 μl of HA-tagged magnetic bead suspension per 500 μl of protein sample. The cells were placed on a side-shake or rotary mixer and incubated at room temperature for 2 hours. After magnetic separation and denaturation, the resulting proteins were subjected to SDS-PAGE electrophoresis. After Coomassie brilliant blue staining, the gel strips were cut according to protein molecular weight and then sent to Shanghai Jingneng Biotechnology Co., Ltd. for qualitative proteomic analysis of the gel dots and strips. Pyruvate kinase (PKM) was identified as one of the proteins.

[0048] (2) Verify the interaction between PKM2 and PTMS using immunoprecipitation.

[0049] Collect cells to be tested, wash twice with pre-chilled PBS, and resuspend the cell pellet in cell lysis buffer (1×10⁷ cells in 1 ml of lysis buffer). Incubate on ice for 10 minutes. Then, centrifuge at 13000g for 10 minutes at 4°C and collect the supernatant for later use. Prepare magnetic bead antibody complexes in advance using an immunoprecipitation kit (Protein A+G magnetic bead method). After the antibody binds to Protein A+G magnetic beads, wash and resuspend with 1×TBS. Resuspend Protein A+G magnetic beads with TBS according to the initial volume for later use. Then, add 20 μl of antibody-bound Protein A+G magnetic beads to 500 μl of cell lysate and mix overnight at 4°C using a rotary mixer to form immune complexes. The next day, use a magnetic rack to wash the magnetic beads three times with lysis buffer. After removing the buffer, use 100 μl of the solution to prepare the immunoprecipitate. Resuspend the precipitate in 1×SDS loading buffer and mix thoroughly; boil the sample for 5 minutes, use a magnetic rack to adsorb magnetic beads, and collect the supernatant for later use.

[0050] (3) Detection of intracellular colocalization of PKM2 and PTMS using cell immunofluorescence.

[0051] ① Fixation: After discarding the culture medium, add an appropriate amount of pre-cooled PBS to each well, wash three times for 3 minutes each time, then add an appropriate amount of 4% paraformaldehyde and fix at room temperature for 15 minutes; ② Blocking: After fixation, wash three times with PBS for 3 minutes each time, add an appropriate amount of blocking buffer containing 0.3% Triton X 100 and 2% BSA, and block at room temperature for 30 minutes; ③ Applying primary antibody: Remove the blocking buffer, add the diluted primary antibody, and incubate overnight at 4°C; ④ Applying secondary antibody: On the second day, recover the primary antibody, wash three times with PBS, add the corresponding fluorescent secondary antibody according to the primary antibody properties, and incubate at room temperature in the dark for 60 minutes; ⑤ DPAI staining and photography: After the secondary antibody incubation is complete, remove the secondary antibody, wash three times with PBS for 3 minutes each time, then add an appropriate amount of fluorescent antiquencher containing DAPI, and mount the slide with transparent nail polish. After drying, observe and photograph under a confocal microscope.

[0052] Immunoprecipitation experiments confirmed a physically interactive relationship between PTMS and PKM2. Immunofluorescence staining showed that PTMS and PKM2 primarily interact within the intracellular mitochondria.

[0053] Example 4: Treatment of spinal cord injury with transplantation of neural stem cell-PTMS composite cell preparation

[0054] (1) Spinal cord injury modeling

[0055] Eight-week-old female C57BL / 6 mice were anesthetized with 2% sodium pentobarbital. The T10 segment of the spinal cord was exposed, and a precision spinal cord impactor (model: 68099Ⅱ-SM) with a 1mm diameter impact head was used at a speed of 1.5m / s, a depth of 0.8mm, and a dwell time of 0.5s. After the impact, the skin was sutured layer by layer, the wound was disinfected with iodine, and physiological saline was administered. Postoperatively, penicillin sodium was injected to prevent infection, and the animals were artificially urinated three times daily until they could urinate independently. The sham-operated group (Sham group) only had the spinal cord exposed without spinal cord impact.

[0056] (2) Treatment of spinal cord injury in mice with transplantation of neural stem cells-PTMS composite cell preparation

[0057] Mice with spinal cord injury received PTMS-OE htNSCs or Vehicle (saline) transplantation on day 7 post-injury. Under deep anesthesia, the backs of the mice were reopened to expose the dura mater above the injury site. Then, using a microsyringe, 5 μL of cell suspension (containing 1 × 10⁵ cells) was slowly injected at multiple sites into the injury site. After injection, the microsyringe was left in place at the injection site for 1 minute before being slowly withdrawn to prevent leakage of the cell suspension. The skin was sutured after injection, and the mice were returned to their cages for continued rearing after recovery.

[0058] (3) BMS score

[0059] On days 1 and 5 post-spinal cord injury, mice were allowed to walk freely in a closed circular enclosure for 4 minutes. Hind limb movement was observed (joint range of motion, hind limb weight-bearing capacity, forelimb and hind limb coordination, etc.) and assessed using a double-blind scoring system. Functional scores ranged from 0 (no hind limb joint movement) to 9 (normal hind limb movement). All mice underwent daily acclimatization training for the first 3 days preoperatively. Following cell transplantation treatment, BMS scores were assessed every 7 days until week 7 post-surgery.

[0060] (4) Footprint analysis

[0061] Non-toxic black ink was applied to the plantar surfaces and dorsal surfaces of the hind limbs of mice. The mice were then allowed to walk across a wooden board covered with white paper. The black marks left on the paper served as the mice's footprints. Analysis of the footprints (stride length of the left and right hind limbs) was used to assess the recovery of motor function. Similarly, all mice underwent daily acclimatization training for the first three days before surgery. After cell transplantation treatment, footprint analysis was performed every seven days until the seventh week post-surgery.

[0062] (5) Spinal cord frozen sections

[0063] To prepare frozen sections, experimental mice were anesthetized with 2% sodium pentobarbital. After perfusion of the heart with 30 mL of PBS and 10 mL of 4% PFA, the brain tissue was dissected and fixed in 4% PFA overnight. After dehydration with 20% and 30% sucrose respectively, the tissue was embedded in OCT and frozen at -20°C. The sections were then sectioned using a cryostat to a thickness of 10 μm, labeled, and stored at -80°C.

[0064] (6) Tissue immunofluorescence

[0065] ① Equilibration of frozen sections: Equilibrate at room temperature for 30 min, bake at 55-60℃ for 15 min, and soak in PBS for 5 min; ② Antigen retrieval: Place sections in boiling citric acid retrieval solution, cool naturally for 1-2 hours, soak in PBS for 5 min, and repeat once; ③ Blocking: Shake off the water from the sections, add immunofluorescence blocking solution to cover the tissue, and block at 37℃ for 1 h; ④ Incubation with primary antibody: Discard the blocking solution, add the proportionally diluted primary antibody, and incubate at 4℃ overnight; ⑤ Incubation with secondary antibody: The next day, after equilibrating the sections and primary antibody at room temperature for 30 min, recover the primary antibody, wash with PBS for 3×5 min, then add the proportionally diluted secondary antibody, and incubate at room temperature for 1 h; ⑥ Nuclear staining: Discard the secondary antibody, wash with PBS for 3×10 min, shake off the water from the sections, add DAPI staining solution for 10 min, discard the staining solution, and wash with PBS for 3×5 min; ⑦ Mounting: Shake off the water, add anti-fluorescence mounting medium, cover with a coverslip, and take pictures under a confocal microscope.

[0066] (7) The expression of glycolysis-related genes in the spinal cord of different groups of mice was detected by real-time quantitative PCR (RT-qPCR).

[0067] (8) The expression of glycolysis-related proteins in the spinal cord of mice in different groups was detected by Western Blot.

[0068] The above embodiments are merely preferred embodiments of the present invention. It should be understood that those skilled in the art can make other modifications or optimizations without departing from the method of the present invention, and these modifications and optimizations should also be considered within the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A cell preparation derived from a neural stem cell-PTMS complex, characterized in that... The neural stem cells were prepared by expressing or loading PTMS protein.

2. The neural stem cell-PTMS composite cell preparation according to claim 1, characterized in that, PTMS protein or DNA or RNA encoding PTMS can be introduced into neural stem cells via electrotransduction, chemical transduction, or biological vector infection.

3. The neural stem cell-PTMS composite cell preparation according to claim 1, characterized in that... Neural stem cells include primary cultured neural stem cells from the hippocampus, olfactory bulb, cortex, subventricular zone (SVZ), and hypothalamus, as well as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) differentiated in vitro.

4. The method for preparing the neural stem cell-PTMS composite cell preparation according to claim 1, characterized in that, The preparation method includes the step of expressing or loading PTMS into neural stem cells.

5. The preparation method according to claim 4, characterized in that, The preparation method involves introducing PTMS protein or DNA or RNA encoding PTMS into neural stem cells via electrotransduction, chemical transduction, or biological vector infection.

6. The preparation method according to claim 5, characterized in that, The biological vectors include biological gene editing vectors such as viral vectors and bacteriophage vectors.

7. The use of the neural stem cell-PTMS composite cell preparation according to claim 1 in the preparation of a medicament for treating nerve injury diseases.

8. Application of PTMS protein in the preparation of drugs for treating nerve damage diseases.

9. Application of PTMS gene and / or PTMS protein agonists in the preparation of drugs for treating neurological disorders.

10. The application as described in any one of claims 7 to 9, characterized in that, The aforementioned neurological injury disease is: Neurological disorders characterized by damage, loss, degeneration, functional decline, morphological abnormalities, or death of nerve cells caused by trauma (such as falls, blows, etc.) or non-traumatic causes (such as tumors, infections, toxins, genetic defects, poor nutritional support, etc.); or brain nerve injury diseases characterized by damage to brain nerve cells; or spinal cord nerve injury diseases characterized by damage to spinal cord nerve cells; or peripheral nerve injury diseases characterized by damage to peripheral nerve cells.

11. The application as described in claim 9, characterized in that, The agonist is a biological or chemical vector that overexpresses PTMS, including plasmids, viral vectors, phage vectors, liposomes, and nanoparticle vectors.

12. The application according to any one of claims 7 to 9, characterized in that, The drug is available in the form of an injection, an oral preparation, or a skin / mucous membrane penetration preparation.

13. The application according to any one of claims 7 to 9, characterized in that, The drug is administered via intravenous, arterial, oral, parenteral, oral, vaginal, rectal, inhalation, blowing, sublingual, intramuscular, skin, subcutaneous, local, intranasal, intraperitoneal, or intracranial routes.

14. A pharmaceutical composition for treating nerve damage, characterized in that, The drug combination contains the neural stem cell-PTMS complex cell preparation or PTMS as described in claim 1 or 7-9, and a pharmaceutically acceptable carrier.

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