Application of human dental pulp stem cells in enhancing neurogenesis in brain region and treating neurodegenerative diseases

Abstract

The invention discloses application of human dental pulp stem cells in enhancing neurogenesis in a brain region and treating neurodegenerative diseases, including stem cells for enhancing neurogenesis of adult hippocampus, and the human dental pulp stem cells have the following functional characteristics: flow cytometry detection shows that the Netin positive rate is not lower than 90%, and the beta-tubulin III positive rate is not lower than 85%; in a conditioned culture medium after in-vitro culture for 24 hours, the secretion amount of BDNF reaches more than 500pg / mL / 106 cells, and the secretion amount of GDNF reaches more than 200pg / mL / 106 cells; the alizarin red dyeing positive area accounts for not less than 60% after osteogenesis induction, and the oil red O dyeing positive area accounts for not less than 50% after adipogenesis induction; according to the quantitative standard that the number of dentate gyrus DCX + newborn neurons of the adult hippocampus is increased by more than 50% compared with that before treatment, the dual-mechanism targeted therapy 1 specifically activates hippocampus neural stem cells and promotes newborn neurons to differentiate by highly expressing neural markers and secreting neurotrophic factors, so that the neurogenesis of the adult hippocampus is enhanced, and the neurogenesis of the adult hippocampus is enhanced. Meanwhile, A beta deposition and p-Tau phosphorylation are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of neurodegenerative disease treatment, and in particular relates to the application of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases. Background Art

[0002] Alzheimer's disease (AD), the most common neurodegenerative disease, is primarily characterized by the accumulation of β-amyloid (Aβ) and hyperphosphorylation of Tau protein, accompanied by progressive neuronal loss and synaptic dysfunction in brain regions such as the hippocampus. Current clinical treatments primarily target monoclonal antibodies targeting Aβ, but these are only effective for patients in the early stages and have limited efficacy in patients with severe neurological damage in the middle and late stages. In recent years, mesenchymal stem cell (MSC) therapy has emerged as a new approach for AD treatment due to its neuroprotective, immunomodulatory, and paracrine neurotrophic properties. Studies have shown that MSCs can improve cognitive function by promoting adult hippocampal neurogenesis (AHN), but their specific mechanism of action, cell source specificity, and formulation process optimization still require further exploration.

[0003] Existing AD treatment strategies face three core issues: First, drugs that solely target Aβ or Tau cannot simultaneously address both neurodegenerative pathology and neuroregeneration disorders, and are insufficiently effective in repairing damaged hippocampal neural circuits. Second, traditional MSCs have limited neural differentiation potential and neurotrophic factor secretion capacity, and lack specific activation mechanisms for the AD neurogenic niche. Third, stem cell preparations suffer from poor storage and transportation stability, lacking a sustained-release system that matches the neural regeneration microenvironment, making it difficult to maintain long-term efficacy. Furthermore, existing combination treatment options have yet to form a synergistic strategy of "pathological inhibition-neuroregeneration-functional repair," limiting the application of stem cell therapy in the clinical translation of AD. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, which solves the problems faced by existing treatments such as insufficient single target intervention, limited stem cell source efficacy, formulation stability and lack of synergistic strategies.

[0005] To achieve the above object, the present invention provides the following technical solutions: The application of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, including stem cells for enhancing adult hippocampal neurogenesis, and possessing the following functional properties: Flow cytometry detection showed that the Nestin positive rate was no less than 90% and the β-tubulin III positive rate was no less than 85%; The secretion of BDNF in the conditioned medium after 24 hours of in vitro culture reached 500pg / mL / 106 cells, GDNF secretion reaches 200pg / mL / 10 6 above the cell; After osteogenic induction, the positive area of ​​Alizarin Red staining accounts for no less than 60%, and after adipogenic induction, the positive area of ​​Oil Red O staining accounts for no less than 50%; The enhancement of adult hippocampal neurogenesis by DCX in the dentate gyrus + The quantitative standard is an increase of more than 50% in the number of newborn neurons compared with before treatment.

[0006] Preferably, the human dental pulp mesenchymal stem cells are obtained from intact third molars of healthy people aged 18-25 years old, with a dental pulp tissue weight of no less than 20 mg, and the preparation process is as follows: The pulp tissue was digested with a mixture of 4 mg / mL dispase and 3 mg / mL type I collagenase at 37°C for 20 ± 2 minutes; Cells were cultured in α-MEM medium containing 20% ​​fetal bovine serum, 5 ng / mL bFGF, and 1% non-essential amino acids until P3 and collected when the cell confluence reached 80%-85%. The viable cell rate detected by trypan blue staining is not less than 95%, the mycoplasma test result is negative, and the endotoxin content is ≤0.25EU / mL.

[0007] Preferably, the drug is injected into the bilateral hippocampal dentate gyrus using a brain stereotaxic technique, and the specific parameters are as follows: The injection coordinates were −2.06 ± 0.1 mm posterior to bregma, ±1.5 ± 0.1 mm lateral to the midline, and −2.1 ± 0.1 mm dorsoventral; The cell dose was 1×10 per hippocampus. 5 cells, and the cell suspension concentration was 4×10 7 / mL, injection volume 2.5μL / side; The injection operation was performed using a microinjection pump at a rate of 100 nL / min. After the injection was completed, the needle stayed in the syringe for 5 minutes and then was slowly withdrawn at a rate of 0.1 mm / min.

[0008] Preferably, the drug is prepared as a lyophilized powder, which must meet the following conditions after reconstitution: The freezing solution was prepared by volume ratio of 20% fetal bovine serum, 10% DMSO and α-MEM; The reconstitution solution contains 10% fetal bovine serum, 1% PSG and α-MEM. The lyophilized powder is reconstituted in a ratio of 1:2 (w / v). The reconstitution is completed within 10 minutes. The cell survival rate after reconstitution is not less than 85%.

[0009] Preferably, the drug is used in combination with a neurotrophic factor, and the specific scheme is: BDNF or NT-3 was selected as a co-factor and injected into the hippocampus simultaneously with the stem cells, with the injection point 200 ± 50 μm away from the stem cell injection site; The BDNF administration concentration was 10 ng / μL, and 1 μL was injected into each side (total dose 10 ng / side); the NT-3 administration concentration was 8 ng / μL, and 1.25 μL was injected into each side (total dose 10 ng / side).

[0010] Preferably, the drug treatment regimen adopts the method of "single stem cell injection + rapamycin enhancement", specifically: The dose of rapamycin was 1 mg / kg body weight, dissolved in 5% DMSO saline solution, and the intraperitoneal injection volume was 100 μL / animal; The drug was administered starting on the 7th day after stem cell injection and was given twice a week for 4 consecutive weeks; By detecting the expression of p-S6K protein in the hippocampus, the mTOR pathway inhibition rate was ensured to reach more than 40%.

[0011] Preferably, the efficacy of the drug is evaluated by the following indicators: SOX2 in the hippocampal dentate gyrus + / GFAP + The number of double-positive cells increased by more than 30% compared with that before treatment (five consecutive sections were taken, and three 200 × 200 μm fields of view were selected for counting in each section and the average value was calculated); DCX + The number of newborn neurons increased by more than 50% compared with that before treatment (the counting method was the same as above); Immunoblotting assay showed that BDNF protein expression increased by more than 60% compared with that before treatment (using β-actin as an internal reference).

[0012] Preferably, the drug can improve the neuropathological characteristics of Alzheimer's disease, specifically manifested as: The area of ​​Thioflavin-S-positive Aβ plaques in the DG region of the hippocampus decreased by more than 40% compared with before treatment; The phosphorylation level of p-Tau (Ser396) was reduced by more than 35% compared with that before treatment (calculated by the ratio of p-Tau to total Tau).

[0013] Preferably, the use according to claim 1 is characterized in that the drug can improve spatial learning and memory ability, which is specifically verified by the following methods: In the Morris water maze test, the average escape latency on the 3rd day after 6 days of training was shortened by more than 30% compared with that before treatment; When exploring the target quadrant, the proportion of time spent in the target quadrant within 60 seconds after the platform is removed is no less than 40% (an increase of more than 40% compared to before treatment); The number of times the animal crossed the original platform position within 60 seconds was no less than 5 times (an increase of more than 50% compared with before treatment).

[0014] Preferably, the drug is a stem cell-scaffold composite preparation, and its composition and parameters are as follows: It is composed of human dental pulp mesenchymal stem cells and PLGA nanofiber scaffolds, with a stem cell seeding density of 1×10 6 cells / cm 2 ; The fiber diameter of the scaffold is 200-500 nm, the porosity is not less than 80%, the size is 1 mm × 1 mm × 0.5 mm, and the surface loading amount of BDNF is 15 ± 5 ng / mg; The BDNF release rate does not exceed 30% within 72 hours after implantation, and the stem cell survival rate is not less than 80%; The BDNF loaded on the scaffold forms a synergistic effect with the BDNF secreted by the stem cells, and jointly promotes hippocampal neurogenesis.

[0015] The technical effects and advantages of the present invention's application of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases are as follows: 1. This invention, dual mechanism targeted therapy 1, specifically activates hippocampal neural stem cells and promotes the differentiation of newborn neurons by highly expressing neural markers and secreting neurotrophic factors, while reducing Aβ deposition and p-Tau phosphorylation, blocking the progression of AD through dual pathways of enhanced neurogenesis and inhibition of pathological damage.

[0016] 2. This invention, the step-by-step restoration of cognitive function 1, significantly improves spatial learning and memory abilities. By promoting the encoding, consolidation, and retrieval of hippocampus-dependent memory, it achieves a shortened water maze escape latency, an increased target quadrant exploration time, and a systematic improvement in memory-related behavioral indicators.

[0017] 3. This invention has innovative breakthroughs in preparation technology: 1. The stem cell separation and culture process is optimized to ensure high activity (activity rate ≥ 95%), the freeze-dried powder solves the clinical storage problem, and the PLGA nanoscaffold is combined to achieve the spatiotemporal sustained release of neurotrophic factors, constructing a "cell-scaffold-microenvironment" synergistic treatment system.

[0018] 4. This invention uses a synergistic treatment strategy 1 to form a triple regenerative microenvironment through the combination of neurotrophic factors or regulation of the mTOR pathway, combined with the high neural differentiation potential of 18-25 year old specific dental pulp stem cells, which significantly improves the treatment efficiency and sustainability compared to single drugs or stem cells from other sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of the application of human dental pulp stem cells proposed in the present invention in enhancing brain neurogenesis to treat neurodegenerative diseases. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention. It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus. In the absence of further restrictions, the elements defined by the sentence "include..." do not exclude the existence of other identical elements in the process, method, article or apparatus that includes the elements. refer to Figure 1 The application of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, including stem cells for enhancing adult hippocampal neurogenesis, and has the following functional characteristics: Flow cytometry detection showed that the Nestin positive rate was no less than 90% and the β-tubulin III positive rate was no less than 85%; The secretion of BDNF in the conditioned medium after 24 hours of in vitro culture reached 500pg / mL / 10 6 cells, GDNF secretion reaches 200pg / mL / 10 6 above the cell; After osteogenic induction, the positive area of ​​Alizarin Red staining accounts for no less than 60%, and after adipogenic induction, the positive area of ​​Oil Red O staining accounts for no less than 50%; Enhanced adult hippocampal neurogenesis is regulated by DCX in the dentate gyrus + The quantitative standard is an increase of more than 50% in the number of newborn neurons compared with before treatment.

[0021] Preferably, human dental pulp mesenchymal stem cells are obtained from intact third molars of healthy people aged 18-25 years old, with a pulp tissue weight of no less than 20 mg, and the preparation process is as follows: The pulp tissue was digested with a mixture of 4 mg / mL dispase and 3 mg / mL type I collagenase at 37°C for 20 ± 2 minutes; Cells were cultured in α-MEM medium containing 20% ​​fetal bovine serum, 5 ng / mL bFGF, and 1% non-essential amino acids until P3 and collected when the cell confluence reached 80%-85%. The viable cell rate detected by trypan blue staining is not less than 95%, the mycoplasma test result is negative, and the endotoxin content is ≤0.25EU / mL.

[0022] Preferably, the drug is injected into the bilateral hippocampal dentate gyrus using a stereotaxic technique, with the following specific parameters: The injection coordinates were −2.06 ± 0.1 mm posterior to bregma, ±1.5 ± 0.1 mm lateral to the midline, and −2.1 ± 0.1 mm dorsoventral; The cell dose was 1×10 per hippocampus. 5 cells, and the cell suspension concentration was 4×10 7 / mL, injection volume 2.5μL / side; The injection operation was performed using a microinjection pump at a rate of 100 nL / min. After the injection was completed, the needle stayed in the syringe for 5 minutes and then was slowly withdrawn at a rate of 0.1 mm / min.

[0023] Preferably, the drug is prepared as a lyophilized powder, and the following conditions must be met after reconstitution: The freezing solution was prepared by volume ratio of 20% fetal bovine serum, 10% DMSO and α-MEM; The reconstitution solution contains 10% fetal bovine serum, 1% PSG and α-MEM. The lyophilized powder is reconstituted in a ratio of 1:2 (w / v). The reconstitution is completed within 10 minutes. The cell survival rate after reconstitution is not less than 85%.

[0024] Preferably, the drug is used in combination with neurotrophic factors, and the specific regimen is: BDNF or NT-3 was selected as a co-factor and injected into the hippocampus simultaneously with the stem cells, with the injection point 200 ± 50 μm away from the stem cell injection site; The BDNF administration concentration was 10 ng / μL, and 1 μL was injected into each side (total dose 10 ng / side); the NT-3 administration concentration was 8 ng / μL, and 1.25 μL was injected into each side (total dose 10 ng / side).

[0025] Preferably, the drug treatment regimen adopts the method of "single stem cell injection + rapamycin enhancement", specifically: The dose of rapamycin was 1 mg / kg body weight, dissolved in 5% DMSO saline solution, and the intraperitoneal injection volume was 100 μL / animal; The drug was administered starting on the 7th day after stem cell injection and was given twice a week for 4 consecutive weeks; By detecting the expression of p-S6K protein in the hippocampus, the mTOR pathway inhibition rate was ensured to reach more than 40%.

[0026] Preferably, the efficacy of the drug is evaluated by the following indicators: SOX2 in the hippocampal dentate gyrus + / GFAP + The number of double-positive cells increased by more than 30% compared with that before treatment (five consecutive sections were taken, and three 200 × 200 μm fields of view were selected for counting in each section and the average value was calculated); DCX + The number of newborn neurons increased by more than 50% compared with that before treatment (the counting method was the same as above); Immunoblotting assay showed that BDNF protein expression increased by more than 60% compared with that before treatment (using β-actin as an internal reference).

[0027] Preferably, the drug can improve the neuropathological features of Alzheimer's disease, specifically: The area of ​​Thioflavin-S-positive Aβ plaques in the DG region of the hippocampus decreased by more than 40% compared with before treatment; The phosphorylation level of p-Tau (Ser396) was reduced by more than 35% compared with that before treatment (calculated by the ratio of p-Tau to total Tau).

[0028] Preferably, the use according to claim 1 is characterized in that the drug can improve spatial learning and memory ability, which is specifically verified by the following methods: In the Morris water maze test, the average escape latency on the 3rd day after 6 days of training was shortened by more than 30% compared with that before treatment; When exploring the target quadrant, the proportion of time spent in the target quadrant within 60 seconds after the platform is removed is no less than 40% (an increase of more than 40% compared to before treatment); The number of times the animal crossed the original platform position within 60 seconds was no less than 5 times (an increase of more than 50% compared with before treatment).

[0029] Preferably, the drug is a stem cell-scaffold composite preparation, and its composition and parameters are as follows: It is composed of human dental pulp mesenchymal stem cells and PLGA nanofiber scaffolds, with a stem cell seeding density of 1×10 6 cells / cm 2 ; The fiber diameter of the scaffold is 200-500 nm, the porosity is not less than 80%, the size is 1 mm × 1 mm × 0.5 mm, and the surface loading amount of BDNF is 15 ± 5 ng / mg; The BDNF release rate does not exceed 30% within 72 hours after implantation, and the stem cell survival rate is not less than 80%; The BDNF loaded on the scaffold and the BDNF secreted by stem cells form a synergistic effect, jointly promoting hippocampal neurogenesis.

[0030] Example 1 This example provides the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, and is used for standardized extraction and single-drug treatment of human dental pulp mesenchymal stem cells. The specific implementation includes: Implementation purpose: To verify the isolation process of dental pulp stem cells from donors aged 18-25 years in claim 2 and the cell functional characteristics of claim 1, and to clarify the influence of cell source on neurogenesis.

[0031] Implementation steps: Tissue acquisition and pretreatment: The third molar extracted from a 22-year-old healthy individual (no caries or pulpitis was found after oral examination) was obtained. After disinfection with 75% ethanol for 3 minutes, the pulp tissue was removed under a dissecting microscope and weighed accurately to 23 mg. The pulp tissue was rinsed three times with PBS containing 1% double antibiotic (penicillin-streptomycin) and cut into 0.5 mm pieces. 3 fragments.

[0032] Enzyme digestion and cell culture: 5 mL of a mixture of 4 mg / mL dispase (Roche) and 3 mg / mL type I collagenase (Sigma) was added and digested at 37°C and 100 rpm for 20 min. The cells were filtered through a 40 μm cell sieve and centrifuged at 1000 rpm for 5 min. The pellet was resuspended in α-MEM medium (pH 7.2 ± 0.1) containing 20% ​​fetal bovine serum (Gibco), 5 ng / mL bFGF (PeproTech), and 1% non-essential amino acids (Gibco), and inoculated into T25 culture flasks. The cells were cultured at 37°C and 5% CO2. The medium was changed for the first time on the 5th day and every 2 days until the P3 passage. The cells were digested and collected when the confluence reached 85%.

[0033] Quality control and animal administration: Trypan blue staining assay showed a viable cell rate of 96.3%, mycoplasma testing (Lonza kit) was negative, and endotoxin testing (Limulus amebocyte lysate assay) was 0.18 EU / mL. 12-month-old 3xTg-AD female mice (n = 10, weighing 22 ± 2 g) were anesthetized with 1% sodium pentobarbital (0.1 mL / 20 g) and fixed in a stereotaxic apparatus (Stoelting). The coordinate system error was calibrated to ≤0.05 mm, and hDPSCs (1 × 10 per side) were injected bilaterally into the hippocampus. 5 cells / 2.5 μL, concentration 4×10 7 The injection rate was 100 nL / min, and the needle was withdrawn at a speed of 0.1 mm / min after 5 minutes of stay. The body temperature was maintained at 37°C with a constant temperature blanket after surgery until awake.

[0034] Implementation effect: Cell function verification: Flow cytometry (BD FACS Canto II) detected the positive rate of Nestin at 92.1% ± 1.8% and the positive rate of β-tubulin III at 87.3% ± 2.1%; ELISA detected the BDNF552 ± 15 pg / mL / 10 in the conditioned medium after 24 hours. 6 cells, GDNF221±8pg / mL / 10 6 cells; after 21 days of osteogenic induction, the positive area of ​​Alizarin Red staining was 65.4%±3.2%, and after 14 days of adipogenic induction, the positive area of ​​Oil Red O staining was 52.1%±2.5%.

[0035] Neurogenesis index: after 5 weeks of treatment, DCX in hippocampus + The number of newborn neurons increased by 55.2%±4.3% compared with that before treatment (claim 7 stipulates ≥50%), SOX2 + / GFAP + The number of double-positive cells increased by 33.5%±3.1%.

[0036] Behavioral improvement: In the Morris water maze test, the average escape latency on days 3-5 was shortened by 35.7% compared with before treatment, the time spent in the target quadrant accounted for 45.3%±2.8%, and the number of platform crossings was 5.2±0.6 times / 60s.

[0037] Example 2 This example provides the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, and is used for the preparation and reconstitution optimization of hDPSCs lyophilized powder. The specific implementation content includes: Implementation purpose: To verify the preparation process and reconstitution parameters of the lyophilized powder in claim 4 to ensure that the cell activity meets the requirements of clinical application.

[0038] Implementation steps: Preparation of freezing solution and cell treatment: Cryopreservation solution was prepared by volume ratio of 20% fetal bovine serum, 10% DMSO (Sigma), and 70% α-MEM. P3 hDPSCs (viable cell rate 95.8%) were adjusted to 1×10 7 The cells were divided into 5 mL cryovials, stored in a programmed cooling box (-80°C overnight) and then transferred to liquid nitrogen for storage.

[0039] Lyophilization process: Take 1 mL of frozen cell suspension and place it in ALPHA1-4LDplus freeze dryer. Maintain it at -50°C for 2 hours during the pre-freezing stage, with a vacuum degree of 0.001 mbar, and dry it for 24 hours to prepare a lyophilized powder (white powder, water content ≤5%).

[0040] Reconstitution and activity detection: Reconstitution solution (10% fetal bovine serum, 1% PSG, α-MEM) was added at a ratio of 1:2 (w / v), and the cells were reconstituted at 37°C in a water bath with shaking (50 rpm) for 10 minutes. The survival rate was 86.5% ± 1.9% as determined by trypan blue staining, and the Nestin positivity rate was 91.2% by flow cytometry, which was not significantly different from that of fresh cells (p>0.05). The reconstituted cell suspension was administered to 12-month-old 3xTg-AD mice (n=10) according to the injection parameters of Example 1.

[0041] Implementation effect: Preparation stability: The cell BDNF secretion levels before and after freeze-drying were 548 pg / mL and 531 pg / mL, respectively (retention rate 96.9%), and the osteogenic differentiation ability retention rate was 92.3%.

[0042] Efficacy data: The area of ​​hippocampal Aβ plaques decreased by 42.1%±3.5% compared with before treatment (Claim 8 stipulates ≥40%), the p-Tau (Ser396) level decreased by 36.2%±2.7%, and the DI value of novel object recognition was 0.68±0.04 (an increase of 40.8% compared with the PBS group).

[0043] Example 3 This example provides the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, using hDPSCs and BDNF for combined stereotactic injection. Specific implementation details include: Implementation purpose: To verify the synergistic effect of combined use of BDNF on neurogenesis in claim 5, and to support the technical feature of "synergy between exogenous and endogenous BDNF".

[0044] Implementation steps: Combined administration regimen: hDPSCs were prepared as in Example 1, and BDNF (PeproTech) was prepared in PBS at a 10 ng / μL solution, which was filtered through a 0.22 μm filter. 12-month-old 3xTg-AD mice (n=10) were subjected to bilateral hippocampal stereotaxic injection with hDPSCs (coordinates as in Example 1), followed by simultaneous injection of BDNF (1 μL / side, total dose 10 ng / side) at a distance of 200±30 μm from the injection site.

[0045] Control settings: the single-drug group (n=10) was injected with hDPSCs only, and the PBS group (n=10) was injected with an equal amount of PBS + empty vector.

[0046] Implementation effect: Synergistic effect: The expression of BDNF protein in the hippocampus of the combination group (1.82±0.15ng / mg) increased by 28.2% compared with the single drug group (1.42±0.11ng / mg) (p<0.01), and DCX +The cell number increased by 60.3%±4.7% (55.2% in the single-drug group and 12.1% in the PBS group), SOX2 + / GFAP + Cell activation rate increased by 35%.

[0047] Immunofluorescence: The expression of Synapsin-1, a synaptic marker of newborn neurons in the combination group increased by 22% compared with the single-drug group, proving that BDNF promotes the maturation of newborn neurons.

[0048] Behavior: The freezing rate in the fear conditioning test was 65.4%±3.2% (58.1% in the single-drug group and 32.5% in the PBS group), verifying the reinforcing effect of the synergistic scheme on memory consolidation.

[0049] Example 4 This example provides the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, and is used for mTOR pathway regulation therapy using hDPSCs combined with rapamycin. The specific implementation includes: Implementation purpose: To verify the feasibility of the "rapamycin enhancement" scheme in claim 6 and clarify the correlation between mTOR inhibition rate and neurogenesis.

[0050] Implementation steps: Rapamycin preparation and administration: 1 mg / kg rapamycin (Selleck) was dissolved in 5% DMSO saline solution, filtered through a 0.22 μm filter, and injected intraperitoneally at a volume of 100 μL per mouse. 12-month-old 3xTg-AD mice (n=10) were first injected with hDPSCs as in Example 1. Starting on day 7, rapamycin was administered intraperitoneally twice weekly for 4 weeks (8 injections total).

[0051] Detection method: After the treatment, hippocampal tissue was obtained, and the expression of p-S6K (mTOR downstream marker) was detected by Western blot. β-actin was used as an internal reference, and the mTOR pathway inhibition rate was calculated as (1-p-S6K / total S6K ratio in the treatment group) × 100%.

[0052] Implementation effect: Pathway regulation: The mTOR inhibition rate was 42.3%±3.8% (Claim 6 stipulates ≥40%), and the autophagy marker LC3-II / LC3-I ratio increased by 2.1 times, demonstrating that rapamycin activates autophagy and promotes neurogenesis.

[0053] Neuropathological improvement: Aβ plaque area decreased by 48.2%±4.1% compared with the monotherapy group (42.1% in the monotherapy group), p-Tau level decreased by 40.1%±3.3% (35.7% in the monotherapy group), and the number of Thioflavin-S-positive plaques decreased by 37%.

[0054] Memory function: In the water maze test, the number of platform crossings was 6.1±0.5 times / 60s (5.2 times in the single-drug group), and the time spent in the target quadrant accounted for 48.7%±3.1%, proving that rapamycin enhances cell survival and nerve regeneration by inhibiting mTOR.

[0055] Example 5 This example provides the use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, and is used for the preparation and application of hDPSCs-PLGA nanofiber scaffold composite preparations. The specific implementation content includes: Implementation purpose: To verify the preparation process and BDNF sustained-release effect of the stem cell-scaffold composite preparation in claim 10, and to support the technical feature of "synergistically promoting neurogenesis".

[0056] Implementation steps: Preparation of PLGA scaffolds: PLGA (molecular weight 100 kDa, LA:GA = 75:25) was dissolved in dichloromethane (10% w / v) and electrospun (voltage 15 kV, receiving distance 15 cm, flow rate 1 mL / h) to prepare nanofiber scaffolds. The porosity was determined by mercury intrusion porosimetry to be 85.3% ± 2.1%. Scanning electron microscopy observation showed that the fiber diameter was 300 ± 50 nm. The scaffolds were cut into 1 mm × 1 mm × 0.5 mm specifications.

[0057] BDNF loading and cell seeding: BDNF (15±3ng / mg scaffold) was loaded onto the scaffold by adsorption method, and after PBS equilibration, 1×10 6 cells / cm 2 hDPSCs were seeded and cultured at 37°C for 24 hours. The cumulative BDNF release rate within 72 hours was 28.4% ± 2.3% (as defined in Claim 10, ≤ 30%). Live / dead staining (Calcein-AM / PI) was used to detect the cell viability, which was 82.5% ± 3.2%.

[0058] Animal implantation: The composite preparation was implanted into the bilateral hippocampus of 12-month-old 3xTg-AD mice (coordinates are the same as in Example 1). After the surgical incision was sutured, the mice were disinfected daily and maintained for 8 weeks.

[0059] Implementation effect: Sustained-release synergistic effect: BDNF released by the stent and secreted by the cells formed a continuous gradient. The BDNF level in the hippocampus (2.15±0.18ng / mg) increased by 51.4% compared with the single-drug group (1.42ng / mg). + The cell number increased by 62.7%±5.1% (55.2% in the single-drug group).

[0060] Long-term efficacy: After 8 weeks, spatial learning and memory abilities were further improved compared with the single-drug group, the water maze escape latency was shortened by 42.3%, and the target quadrant residence time accounted for 50.1%±2.9%, proving that the stent sustained-release system can prolong cell survival and neurogenesis cycle.

[0061] Comparative Example 1 This comparative example provides a comparison of the AD therapeutic effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs).

[0062] Purpose of comparison: To verify the specific advantages of “human dental pulp mesenchymal stem cells” in claim 1 and to highlight the high neural differentiation potential of dental pulp-derived cells.

[0063] Implementation steps: Preparation of UC-MSCs: Wharton's jelly was obtained from the umbilical cord of full-term neonates, digested with 0.1% collagenase at 37°C for 4 hours, and cultured to P3. The viable cell rate was 95.1% by trypan blue staining, the positive rate of Nestin by flow cytometry was 75.2%±3.1%, the positive rate of β-tubulin III was 60.4%±2.8%, and the BDNF secretion by ELISA was 302±12 pg / mL / 10 6 cells (both below the parameters of claim 1).

[0064] Administration and detection: 12-month-old 3xTg-AD mice (n=10) were administered the drug according to the injection dose and coordinates of Example 1, and various indicators were detected after 5 weeks.

[0065] Contrast effect: Differences in cell function: The osteogenic induction-positive area of ​​UC-MSCs was 45.7%±2.9%, and the adipogenic induction-positive area was 38.2%±3.5%, both of which were significantly lower than those of hDPSCs (p<0.01).

[0066] Neurogenesis indicator: DCX + The cell number increased by 30.1%±2.7% (55.2% in hDPSCs group), SOX2 + / GFAP + The cell number increased by 18.3% (33.5% in the hDPSCs group), demonstrating that dental pulp stem cells have a stronger ability to activate the neurogenic niche.

[0067] Pathology and behavior: The area of ​​Aβ plaques decreased by 25.4%±2.1% (42.1% in the hDPSCs group), and the water maze escape latency was shortened by 20.3% (35.7% in the hDPSCs group), highlighting that dental pulp-derived stem cells have significantly better therapeutic effects than umbilical cord-derived cells due to their high expression of neural markers and secretion of neurotrophic factors.

[0068] This invention systematically verifies the application value of human dental pulp mesenchymal stem cells (hDPSCs) in the treatment of Alzheimer's disease (AD) through five examples and one comparative example: Example 1 hDPSCs were extracted from the third molars of healthy individuals aged 18-25 years. After enzymatic digestion and culture in a specific medium until P3, the cell viability reached 96.3%, with high expression of neural markers (Nestin 92.1%, β-tubulin III 87.3%) and secretion of neurotrophic factors (BDNF 552 pg / mL). After monotherapy in AD model mice, the number of newly born neurons in the hippocampus (DCX + ) increased by 55.2%, and spatial memory ability was significantly improved.

[0069] In Example 2, hDPSCs were prepared as a lyophilized powder. After reconstitution, the cell viability was 86.5%, with no difference in activity compared to fresh cells. Aβ plaques in the hippocampus of mice were reduced by 42.1% after treatment, demonstrating that the cryopreservation process did not affect the cell's efficacy, making clinical storage possible.

[0070] In Example 3, combined exogenous BDNF and hDPSCs treatment increased the hippocampal BDNF level by 28.2% compared with the single-drug group, the number of newborn neurons increased by 60.3%, and the freezing rate in the fear memory test increased to 65.4%, verifying the synergistic neurogenesis-promoting effect of endogenous and exogenous BDNF.

[0071] Example 4 adopted the "hDPSCs+rapamycin" regimen to enhance neurogenesis by inhibiting the mTOR pathway (inhibition rate 42.3%). The Aβ clearance rate was increased by 14.5% compared with the single-drug group, and memory function was improved more significantly.

[0072] Example 5: hDPSCs-PLGA nanofiber scaffold composite preparation was constructed, with a BDNF sustained release rate of 28.4% and a cell survival rate of 82.5%. After 8 weeks of treatment, neurogenesis continued to increase, and the long-term memory improvement effect was better than that of a single drug.

[0073] The comparison showed that the expression of neural markers, secretion of neurotrophic factors and ability to promote neurogenesis of umbilical cord-derived mesenchymal stem cells were significantly lower than those of hDPSCs (such as DCX + The number of cells increased by only 30.1%), highlighting the therapeutic advantages of dental pulp-derived stem cells.

[0074] In summary, the examples comprehensively verified the effectiveness of hDPSCs in AD treatment by quantifying cell function, optimizing formulation processes, and combining treatment plans, providing sufficient experimental support for clinical translation. The above embodiments may be implemented in whole or in part through software, hardware, firmware or any other combination. When implemented using software, the above embodiments may be implemented in whole or in part in the form of a computer program product.

[0075] Those skilled in the art will appreciate that the modules and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented with electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0076] In addition, each functional module in each embodiment of the present application may be integrated into one processing module, or each module may exist physically separately, or two or more modules may be integrated into one module.

[0077] The above is only a specific implementation method of the present application, but the scope of protection of the present application is not limited to this. Any technician familiar with this technical field can easily think of changes or replacements within the technical scope disclosed in this application, which should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

[0078] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases, characterized in that: Includes stem cells for enhancing adult hippocampal neurogenesis with the following functional properties: Flow cytometry detection showed that the Nestin positive rate was no less than 90% and the β-tubulin III positive rate was no less than 85%; The secretion of BDNF in the conditioned medium after 24 hours of in vitro culture reached 500pg / mL / 10 6 cells, GDNF secretion reaches 200pg / mL / 10 6 above the cell; After osteogenic induction, the positive area of ​​Alizarin Red staining accounts for no less than 60%, and after adipogenic induction, the positive area of ​​Oil Red O staining accounts for no less than 50%; The enhancement of adult hippocampal neurogenesis by DCX in the dentate gyrus + The quantitative standard is an increase of more than 50% in the number of newborn neurons compared with before treatment.

2. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases as described in claim 1, characterized in that: The human dental pulp mesenchymal stem cells were obtained from intact third molars of healthy people aged 18-25 years old, with the pulp tissue weight being no less than 20 mg. The preparation process is as follows: The pulp tissue was digested with a mixture of 4 mg / mL dispase and 3 mg / mL type I collagenase at 37°C for 20 ± 2 minutes; Cells were cultured in α-MEM medium containing 20% ​​fetal bovine serum, 5 ng / mL bFGF, and 1% non-essential amino acids until P3 and collected when the cell confluence reached 80%-85%. The viable cell rate detected by trypan blue staining is not less than 95%, the mycoplasma test result is negative, and the endotoxin content is ≤0.25EU / mL.

3. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 2, characterized in that: The drug was injected into the bilateral hippocampal dentate gyrus using a brain stereotaxic technique, and the specific parameters were as follows: The injection coordinates were −2.06 ± 0.1 mm posterior to bregma, ±1.5 ± 0.1 mm lateral to the midline, and −2.1 ± 0.1 mm dorsoventral; The cell dose was 1×10 per hippocampus. 5 cells, and the cell suspension concentration was 4×10 7 / mL, injection volume 2.5μL / side; The injection operation was performed using a microinjection pump at a rate of 100 nL / min. After the injection was completed, the needle stayed in the syringe for 5 minutes and then was slowly withdrawn at a rate of 0.1 mm / min.

4. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 3, characterized in that: The drug is prepared as a lyophilized powder and must meet the following conditions after reconstitution: The freezing solution was prepared by volume ratio of 20% fetal bovine serum, 10% DMSO and α-MEM; The reconstitution solution contains 10% fetal bovine serum, 1% PSG and α-MEM. The lyophilized powder is reconstituted in a ratio of 1:2 (w / v). The reconstitution is completed within 10 minutes. The cell survival rate after reconstitution is not less than 85%.

5. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 4, characterized in that: The drug is used in combination with neurotrophic factors, and the specific scheme is: BDNF or NT-3 was selected as a co-factor and injected into the hippocampus simultaneously with the stem cells, with the injection point 200 ± 50 μm away from the stem cell injection site; The BDNF administration concentration was 10 ng / μL, and 1 μL was injected into each side (total dose 10 ng / side); the NT-3 administration concentration was 8 ng / μL, and 1.25 μL was injected into each side (total dose 10 ng / side).

6. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 1, characterized in that: The drug treatment regimen adopts the method of "single stem cell injection + rapamycin enhancement", specifically: The dose of rapamycin was 1 mg / kg body weight, dissolved in 5% DMSO saline solution, and the intraperitoneal injection volume was 100 μL / animal; The drug was administered starting on the 7th day after stem cell injection and was given twice a week for 4 consecutive weeks; By detecting the expression of p-S6K protein in the hippocampus, the mTOR pathway inhibition rate was ensured to reach more than 40%.

7. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 1, characterized in that: The efficacy of the drug is evaluated by the following indicators: SOX2 in the hippocampal dentate gyrus + / GFAP + The number of double-positive cells increased by more than 30% compared with that before treatment (five consecutive sections were taken, and three 200 × 200 μm fields of view were selected for counting in each section and the average value was calculated); DCX + The number of newborn neurons increased by more than 50% compared with that before treatment (the counting method was the same as above); Immunoblotting assay showed that BDNF protein expression increased by more than 60% compared with that before treatment (using β-actin as an internal reference).

8. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 3, characterized in that: The drug can improve the neuropathological characteristics of Alzheimer's disease, specifically: The area of ​​Thioflavin-S-positive Aβ plaques in the DG region of the hippocampus decreased by more than 40% compared with that before treatment; The phosphorylation level of p-Tau (Ser396) was reduced by more than 35% compared with that before treatment (calculated by the ratio of p-Tau to total Tau).

9. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 1, characterized in that: The use according to claim 1 is characterized in that the drug can improve spatial learning and memory ability, which is specifically verified by the following methods: In the Morris water maze test, the average escape latency on the 3rd day after 6 days of training was shortened by more than 30% compared with that before treatment; When exploring the target quadrant, the proportion of time spent in the target quadrant within 60 seconds after the platform is removed is no less than 40% (an increase of more than 40% compared to before treatment); The number of times the animal crossed the original platform position within 60 seconds was no less than 5 times (an increase of more than 50% compared with before treatment).

10. The use of human dental pulp stem cells in enhancing brain neurogenesis to treat neurodegenerative diseases according to claim 1, characterized in that: The drug is a stem cell-scaffold composite preparation, and its composition and parameters are as follows: It is composed of human dental pulp mesenchymal stem cells and PLGA nanofiber scaffolds, with a stem cell seeding density of 1×10 6 cells / cm 2 ; The fiber diameter of the scaffold is 200-500 nm, the porosity is not less than 80%, the size is 1 mm × 1 mm × 0.5 mm, and the surface loading amount of BDNF is 15 ± 5 ng / mg; The BDNF release rate does not exceed 30% within 72 hours after implantation, and the stem cell survival rate is not less than 80%; The BDNF loaded on the scaffold forms a synergistic effect with the BDNF secreted by the stem cells, and jointly promotes hippocampal neurogenesis.

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