Method for synergistically treating Alzheimer's disease by using gene-modified human interdental stem cells

Abstract

The invention discloses a method for synergistically treating Alzheimer's disease through gene-modified human interdental stem cells, and belongs to the field of drug therapy. According to the method, healthy adult dental pulp mesenchymal stem cells (hDPSCs) are extracted, BDNF and NRF2 genes are introduced through lentiviral vectors, the BDNF and NRF2 genes have neurotrophic, antioxidant and anti-inflammatory capacities, and then the hDPSCs are combined with curcumin nanoparticles and injected to the hippocampus of an AD mouse through stereotactic injection. The BDNF can promote nerve regeneration, the NRF2 can improve oxidative stress and inflammation environment, and the curcumin nanoparticles regulate A beta metabolism and activate a BDNF signal channel. Experiments show that the combined therapy can significantly enhance hippocampal neurogenesis of AD mice and improve expression of neuron markers, and it is proved through Morse water maze tests that the combined therapy can improve the spatial learning and memory ability of the mice. The method breaks through the limitation of traditional single-target treatment, multi-dimensional pathological intervention is achieved, the stem cells are rich in source and high in safety, and a new strategy is provided for treatment of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of drug therapy, and in particular relates to a method for synergistically treating Alzheimer's disease using gene-modified human dental pulp mesenchymal stem cells. Background Art

[0002] Alzheimer's disease (AD), a typical neurodegenerative disease, is characterized by the deposition of β-amyloid protein (Aβ) to form plaques, abnormal phosphorylation of tau protein leading to neurofibrillary tangles, and progressive neuronal loss in the hippocampus. Current clinical treatments primarily rely on cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists, which can only temporarily alleviate cognitive decline but cannot halt disease progression. While monoclonal antibody therapies targeting Aβ have shown potential for plaque clearance in clinical trials, they have limitations such as low blood-brain barrier permeability, strong immunogenicity, and the potential for inducing cerebral edema, and have minimal effect on repairing already damaged neurons. Traditional drugs struggle to simultaneously intervene in concomitant pathological processes such as oxidative stress and inflammatory responses, resulting in limited efficacy of single-target therapies. This has become a key bottleneck in the field of AD treatment that urgently needs to be overcome.

[0003] In the development of AD treatment technologies, multiple technical challenges hinder clinical translation. First, the complexity of the neural regenerative microenvironment makes single interventions ineffective. While traditional stem cell therapies can promote neural regeneration through paracrine neurotrophic factors, they lack the ability to regulate oxidative stress and the inflammatory environment. High levels of reactive oxygen species and persistent inflammation in the AD brain significantly inhibit stem cell survival and differentiation. Second, the physiological barrier of the blood-brain barrier (BBB) ​​hinders drug delivery efficiency. While natural active ingredients such as curcumin have the potential to regulate Aβ metabolism and fight inflammation, they are poorly water-soluble and rapidly metabolized in vivo. Conventional formulations have a bioavailability of less than 5%, and only approximately 0.1% crosses the BBB, making it impossible to achieve effective therapeutic concentrations in the lesion. Third, single-target therapies fail to address the multidimensional pathological network of AD: neither strategies targeting Aβ clearance nor interventions promoting neural regeneration can simultaneously address the deterioration of the neuronal survival microenvironment and impaired synaptic remodeling. Clinical data show that therapies targeting Aβ alone have achieved cognitive improvement rates of less than 20% in Phase III clinical trials, highlighting the significant limitations of single-target therapies.

[0004] This patent solves the problems of complex neural environment that is difficult to intervene, low drug delivery efficiency due to the blood-brain barrier, and the inability of a single target to cover multi-dimensional pathological networks. Summary of the Invention

[0005] To solve the problems that the neural environment is complex and difficult to intervene, the blood-brain barrier makes drug delivery inefficient, and a single target cannot cover the multi-dimensional pathological network.

[0006] In order to solve the above problems, the present invention provides the following technical solutions: The method for synergistically treating Alzheimer's disease with gene-modified human dental pulp mesenchymal stem cells comprises the following steps: S1: Complete third molars from healthy adults were extracted and disinfected. The tissue was then rinsed with phosphate buffered saline and minced into pieces. The dental pulp was then digested with 2 mg / mL dispase and 1 mg / mL type I collagenase to obtain mesenchymal stem cells. The cell suspension was then inoculated into a culture flask and incubated with α-modified minimum essential medium. The medium was changed every two days after five days, and the cells were continuously cultured and passaged to passage P3. S2: Construct a lentiviral vector carrying the BDNF and NRF2 genes and package it according to the instructions to obtain a high-titer recombinant lentivirus. P3 human dental pulp mesenchymal stem cells are seeded into well plates. When the cells become confluent, replace the medium with the lentivirus-containing medium and add 8 μg / mL polybrene. Replace the medium with fresh α-MEM medium and continue culturing to obtain gene-modified hDPSCs stably expressing BDNF and NRF2 through resistance screening. S3: 3xTg-AD mice were anesthetized with sodium pentobarbital, and hDPSCs were injected into the bilateral hippocampal DG of the mice at the coordinates of -2.06 mm anteroposterior, ±1.5 mm medial, and -2.1 mm dorsoventral from the bregma. After injection, the needle was left in place for 5 minutes while curcumin nanoparticle solution was injected intraperitoneally until the mice were fully awake. S4: Mice were tested one month after transplantation of gene-modified hDPSCs and concurrent curcumin nanoparticle synergistic treatment. Mice were randomly placed in a quadrant of the Morris water maze filled with milky water and asked to search for a hidden platform within 60 seconds. Each attempt was repeated 15 minutes apart. On the seventh day, an exploratory test without the platform was conducted to observe the exploratory behavior of the mice in the water maze and assess their spatial learning and memory abilities.

[0007] Preferably, the hDPSCs in S2 have all the characteristics of MSCs, including high proliferation capacity, the ability to be induced to differentiate into osteocytes, adipocytes and chondrocytes, and the expression of surface markers of MSCs.

[0008] Preferably, the concentration of hDPSCs in S2 is 2×10 5 cells / μL, which enhances the ability of adult hippocampal neurogenesis in the mouse brain, and the effect of neurogenesis in the hippocampus can be observed by immunofluorescence staining analysis and immunoblotting analysis.

[0009] Preferably, the protein expression of neuronal markers DCX and NeuN in the hippocampus of mice in the hDPSCs-administered group in S2 is increased, and the hDPSCs-administered group also induces an increase in BDNF, which is an essential neurotrophic factor for hippocampal neurogenesis and maintaining cognitive integrity in AD mouse models.

[0010] Preferably, hDPSCs in S2 differentiate into neuron-like cells in the AD brain microenvironment and form functional synaptic connections with host neurons to promote synaptic repair.

[0011] Preferably, in S2, the BDNF gene and the NRF2 gene are introduced into hDPSCs by a lentiviral vector, the viral titer is ≥1×108 transduction units / ml (TU / mL), and the concentration of purified stem cells is >90%. The BDNF therein can promote neuronal survival, differentiation and synapse formation, and NRF2 has antioxidant and anti-inflammatory effects, thereby improving the oxidative stress and inflammatory environment in the patient's brain.

[0012] Preferably, curcumin in S3 is a natural polyphenol compound extracted from ginger plants, which has multiple biological activities such as anti-inflammatory, antioxidant, and regulation of β-amyloid protein (Aβ) metabolism.

[0013] Preferably, the curcumin nanoparticles in S3 are prepared by emulsification-solvent evaporation, with a particle size distribution of 50-200 nm and an encapsulation efficiency of >80%.

[0014] Preferably, in S3, transgenic AD mice (3xTg-AD mice) comprising three mutations associated with familial AD (APP Swedish, MAPT P301L, and PSEN1 M146V) are used, and human dental pulp mesenchymal stem cell preparations are injected into the brain stereotaxically.

[0015] Preferably, BDNF is a core factor of neurogenesis, which can promote the proliferation and differentiation of neural stem cells in the DG region of the hippocampus. Curcumin enhances the survival and maturation of neural precursor cells by activating the BDNF-TrkB signaling pathway, forming a dual drive with the BDNF secreted by gene-modified hDPSCs to promote adult hippocampal neurogenesis.

[0016] Effects and advantages of the method of the present invention for the synergistic treatment of Alzheimer's disease using genetically modified human dental pulp mesenchymal stem cells: 1. This patent uses a lentiviral vector to introduce BDNF and NRF2 genes, which enables hDPSCs to have neurotrophic, antioxidant and anti-inflammatory capabilities, breaking through the limitations of traditional stem cell single-target therapy. The viral titer is ≥1×10 8 TU / mL, stem cell purity>90%, ensuring gene modification efficiency and cell activity.

[0017] 2. This patent uses genetically modified hDPSCs in combination with curcumin nanoparticles to achieve multi-target intervention of neural regeneration + microenvironment repair + Aβ metabolic regulation, which is more comprehensive than single therapy. Curcumin improves bioavailability and blood-brain barrier permeability through nano-sizing.

[0018] 3. This patent uses stereotactic injection technology to precisely deliver hDPSCs to the DG region of the hippocampus with a coordinate error of <0.1 mm, ensuring that the cells are implanted in the key area of ​​neurogenesis. The cell concentration is 2×10 5 cells / μL, which can significantly promote the expression of neuronal markers (DCX, NeuN) and the increase of BDNF levels in the hippocampus.

[0019] 4. This patent uses dental pulp stem cells from a rich source, with minimal trauma to obtain and low immunogenicity, which reduces the risk of allogeneic transplant rejection. The gene modification technology is mature, the lentiviral vector is highly safe, and curcumin is a natural compound with low toxicity and side effects, making it suitable for long-term synergistic treatment.

[0020] 5. Compared with unmodified stem cells, this patent shows that gene-modified hDPSCs can stably express therapeutic factors, avoiding the short-term effect and immunogenicity of exogenous protein injection. The mechanism of action of curcumin and gene-modified cells is complementary, reducing the dose dependence of a single component and lowering potential toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the escape route of mice undergoing the Morris water maze test in the present invention; Figure 2 This is the morphological identification of hDPSCs of the present invention, which are P0 and P3 generation hDPSCs respectively. DETAILED DESCRIPTION

[0022] 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 presence of other identical elements in the process, method, article or apparatus that includes the elements.

[0023] Example 1 This embodiment provides a method for the synergistic treatment of Alzheimer's disease using genetically modified human dental pulp mesenchymal stem cells, which is applicable to the field of medical treatment. The implementation details are as follows: Purpose of the experiment: Gene-modified human dental pulp mesenchymal stem cells as synergistic therapy for Alzheimer's disease.

[0024] Experimental materials: 75% ethanol, phosphate buffered saline (PBS), dispase, collagenase type I, polybrene, 1% sodium pentobarbital, and curcumin nanoparticle solution.

[0025] Experimental steps: S1: Complete third molars from healthy adults were extracted and disinfected. The freshly extracted teeth were then rinsed with phosphate-buffered saline and minced into small pieces. The pulp tissue was then digested with 4 mg / mL dispase and 3 mg / mL type I collagenase for 20 minutes to obtain mesenchymal stem cells. The cell suspension was then seeded into a T-25 culture flask and incubated with α-modified minimum essential medium (α-MEM). The medium was changed on the fifth day and every two days thereafter. The cells were continuously cultured and passaged to passage P3. S2: Construct a lentiviral vector carrying the BDNF and NRF2 genes and package it according to the instructions of the lentiviral packaging kit to obtain high-titer recombinant lentivirus. P3 human dental pulp mesenchymal stem cells (hDPSCs) in the logarithmic growth phase are seeded in 6-well plates. When the cell confluence reaches 60%-70%, the culture medium is replaced with lentivirus-containing culture medium and polybrene at a final concentration of 8 μg / mL is added to promote viral infection. After 6 hours of infection, the culture medium is replaced with fresh α-MEM medium and cultured for another 48 hours. Gene-modified hDPSCs stably expressing BDNF and NRF2 are obtained through resistance screening. S3: 3xTg-AD mice were anesthetized with 1% sodium pentobarbital (0.1 mL / 20 g). The mouse head was fixed in a stereotaxic frame with a mouse adapter. 2.5 µL of hDPSCs were injected into the bilateral hippocampal DG of the mouse at a rate of 100 nL / min. The coordinates were -2.06 mm anteroposterior, ±1.5 mm medial, and -2.1 mm dorsoventral from the bregma. After injection, the needle was left in place for 5 minutes and then slowly withdrawn. At the same time, curcumin nanoparticle solution was injected intraperitoneally. The mouse was then placed on a heating pad until fully awake. S4: All mice were tested one month after transplantation of gene-modified hDPSCs and co-treatment with curcumin nanoparticles. Mice were placed in the testing room in advance. The Morris water maze was filled with milky white water, and the escape platform was fixed 1 cm below the water surface in the northwest quadrant. During the training period, mice were randomly placed in the water in a quadrant and asked to find the hidden platform within 60 seconds. The interval between each training was 15 minutes. If the mouse failed to reach the platform within 60 seconds, it was manually guided to the platform. On the 7th day, an exploratory test without the platform was conducted to observe the exploratory behavior of the mice in the water maze and evaluate their spatial learning and memory abilities.

[0026] Experimental results: See Figure 1 .

[0027] Mice in the hDPSCs plus curcumin nanoparticles group underwent cognitive testing using the Morris water maze, including typical escape trajectories and escape times. The learning curves showed the average daily time mice spent escaping to the hidden platform during the first six days of training, the time mice spent in the target quadrant without the platform within one minute on the seventh day, and the average number of times mice crossed over to the original platform location without the platform on the seventh day. The results showed that during the six days of training prior to testing, the hDPSCs plus curcumin nanoparticles group showed a clear direction in terms of typical escape latencies. Furthermore, mice in this treatment group spent more time exploring the target quadrant and crossed over to the original platform location more times within 60 seconds.

[0028] Example 2 This embodiment provides the use of genetically modified human dental pulp mesenchymal stem cells to treat Alzheimer's disease, wherein the synergistic effect of curcumin nanoparticles is lacking, and the following implementation contents are provided: Purpose of the experiment: Effects of single hDPSCs on Alzheimer's disease.

[0029] Experimental materials: 75% ethanol, phosphate buffered saline (PBS), dispase, collagenase type I, polybrene, 1% sodium pentobarbital.

[0030] Experimental steps: S1: Complete third molars from healthy adults were extracted and disinfected. The freshly extracted teeth were then rinsed with phosphate-buffered saline and minced into small pieces. The pulp tissue was then digested with 4 mg / mL dispase and 3 mg / mL type I collagenase for 20 minutes to obtain mesenchymal stem cells. The cell suspension was then seeded into a T-25 culture flask and incubated with α-modified minimum essential medium (α-MEM). The medium was changed on the fifth day and every two days thereafter. The cells were continuously cultured and passaged to passage P3. S2: Construct a lentiviral vector carrying the BDNF and NRF2 genes and package it according to the instructions of the lentiviral packaging kit to obtain high-titer recombinant lentivirus. P3 human dental pulp mesenchymal stem cells (hDPSCs) in the logarithmic growth phase are seeded in 6-well plates. When the cell confluence reaches 60%-70%, the culture medium is replaced with lentivirus-containing culture medium and polybrene at a final concentration of 8 μg / mL is added to promote viral infection. After 6 hours of infection, the culture medium is replaced with fresh α-MEM medium and cultured for another 48 hours. Gene-modified hDPSCs stably expressing BDNF and NRF2 are obtained through resistance screening. S3: 3xTg-AD mice were anesthetized with 1% sodium pentobarbital (0.1 mL / 20 g). The mouse head was fixed in a stereotaxic frame with a mouse adapter. 2.5 µL of hDPSCs were injected into the bilateral hippocampal DG of the mouse at a rate of 100 nL / min. The coordinates were -2.06 mm anteroposterior, ±1.5 mm medial, and -2.1 mm dorsoventral from the bregma. After injection, the needle was left in place for 5 minutes and then slowly withdrawn. At the same time, curcumin nanoparticle solution was injected intraperitoneally. The mouse was then placed on a heating pad until fully awake. S4: All mice were tested one month after transplantation of gene-modified hDPSCs. Mice were placed in the testing room in advance. The Morris water maze was filled with milky white water, and the escape platform was fixed 1 cm below the water surface in the northwest quadrant. During the training period, mice were randomly placed in the water in one quadrant and given 60 seconds to find the hidden platform. Each training interval was 15 minutes. If the mouse failed to reach the platform within 60 seconds, it was manually guided to the platform. On the 7th day, an exploratory test without the platform was conducted to observe the mice's exploratory behavior in the water maze and assess their spatial learning and memory abilities.

[0031] Experimental results: See Figure 1 .

[0032] The same test as in Example 1 was performed. The results showed that, during the six days of training prior to testing, the hDPSC-treated mice exhibited a typical escape latency, lacking a clear direction. Furthermore, mice in this treatment group spent less time exploring the target quadrant, with significantly fewer crossings of the original platform within 60 seconds.

[0033] Comparative Example 1 3xTg-AD mice were provided for the Morris water maze test, and the following implementation details were performed: Purpose of the experiment: A control group is provided for comparison of Examples 1 and 2.

[0034] Experimental steps: S1: 3xTg-AD mice were anesthetized with 1% sodium pentobarbital. The mouse head was fixed in a stereotaxic frame with a mouse adapter. 2.5 µL of normal saline was injected into the bilateral hippocampal DG of the mouse at the coordinates of -2.06 mm anteroposterior, ±1.5 mm medial, and -2.1 mm dorsoventral from bregma. After injection, the needle was left in place for 5 minutes and then slowly withdrawn. The mouse was then placed on a heating pad until fully awake. S2: All mice were tested one month after injection of normal saline. The mice were placed in the testing room in advance. The Morris water maze was filled with milky white water, and the escape platform was fixed 1 cm below the water surface in the northwest quadrant. During the training period, the mice were randomly placed in the water in a quadrant and asked to find the hidden platform within 60 seconds. The interval between each training was 15 minutes. If the mice failed to reach the platform within 60 seconds, they were manually guided to the platform. On the 7th day, an exploratory test without the platform was carried out to observe the exploratory behavior of the mice in the water maze and evaluate their spatial learning and memory abilities.

[0035] Experimental results: See Figure 1 The same test as in Example 1 was performed, and the results showed that during the 6-day training before the test, the mice in the control group showed a typical disorganized escape route in terms of escape latency. In addition, the mice in this treatment group spent less time exploring the target quadrant, and the number of times they crossed the original platform position within 60 seconds was significantly lower.

[0036] refer to Figure 2 , the cells in Example 1 are iterated 3 times, and the obtained Figure 2 The morphology shown in the figure depicts the morphology of human dental pulp mesenchymal stem cells (hDPSCs) at different passage stages. In the left image, "Passage 0," cells are sparsely distributed and have diverse morphologies. In the right image, "Passage 3," cells have increased density and are uniformly elongated and spindle-shaped, more closely resembling mature stem cells.

[0037] In Example 1, BDNF is a brain-derived neurotrophic factor that can promote neuronal survival, differentiation, and synapse formation, directly stimulate the proliferation and differentiation of neural stem cells in the DG region of the hippocampus, and maintain basic cognitive functions. Genetically modified hDPSCs can continuously secrete BDNF to enhance hippocampal neurogenesis. NRF2 activates the antioxidant response pathway, thereby reducing oxidative stress damage in the brain, while inhibiting the release of inflammatory factors, improving the inflammatory environment in AD patients, and providing an environment for repairing neurons. Genetically modified hDPSCs can differentiate into neuron-like cells in the AD brain microenvironment and form functional synaptic connections with host neurons, directly promoting neural network repair.

[0038] The curcumin in Example 1 can inhibit the aggregation of β-amyloid protein (Aβ) and promote its clearance, thereby reducing the toxic effects of Aβ plaques on neurons. At the same time, it activates the BDNF-TrkB signaling pathway, enhances the survival and maturation of neural precursor cells, and forms a dual drive with BDN secreted by hDPSCs, synergistically promoting adult hippocampal neurogenesis. At the same time, as a natural polyphenol compound, curcumin can supplement the anti-inflammatory oxidative effect of NRF2 and further alleviate the inflammatory response in the brain.

[0039] In Example 1, the co-localization of the neural stem cell markers SOX2 and GFAP in the hippocampus of mice in the hDPSCs plus curcumin treatment group increased, indicating that the transplantation of hDPSCs plus curcumin can promote the increase in the number of quiescent neural stem cells in the DG, thereby activating the activation of neural stem cells and the differentiation process into neurons. The protein expression of neuronal markers DCX and NeuN in the hippocampus of mice in the hDPSCs plus curcumin treatment group increased, and the hDPSCs plus curcumin treatment group also induced an increase in BDNF, a neurotrophic factor that is essential for hippocampal neurogenesis and maintaining cognitive integrity in AD mouse models. Therefore, the results show that the transplantation of hDPSCs can promote adult hippocampal neurogenesis.

[0040] The results of cognitive testing performed on mice in the hDPSCs plus curcumin nanoparticles group in Example 1, including typical escape trajectories and escape times, were analyzed using a learning curve showing the average daily time the mice spent escaping to the hidden platform during the first six days of training, the time the mice spent in the target quadrant without the platform within one minute on the seventh day, and the average number of times the mice crossed the original platform position without the platform on the seventh day. The results showed that during the six days of training prior to testing, the mice in the hDPSCs plus curcumin nanoparticles group exhibited typical escape trajectories with clear direction in terms of escape latency. Furthermore, mice in this treatment group spent more time exploring the target quadrant and crossed the original platform position more times within 60 seconds.

[0041] Example 2 is a single gene-modified hDPSCs treatment, in which the effect is the same as that of Example 1, but lacks curcumin nanoparticles, lacks the synergistic effect of curcumin nanoparticles, lacks the BDNF and curcumin to jointly promote neural stem cell proliferation, differentiation and synapse formation, and restore the function of hippocampal neural circuits, lacks NRF2 and curcumin to synergize and reduce oxidative stress and inflammation levels, alleviate Aβ-induced neuronal damage, and provide a suitable environment for stem cell survival and neural regeneration.

[0042] In Example 2, during the six days of training prior to testing, the mice in the hDPSC-treated group exhibited typical escape latencies, with no clear direction. Mice in this treatment group also spent less time exploring the target quadrant, with significantly fewer crossings of the original platform within 60 seconds.

[0043] refer to Figure 1 Comparing the Morris water maze tests of Example 1 and Example 2, it can be found that the average escape latency of the mice in Example 1 is much shorter than that in Example 2. Compared with Example 2, the swimming time in the target quadrant and the number of times crossing the original platform position within one minute in Example 1 are significantly increased.

[0044] As a control, it is proved that the therapeutic effects of Examples 1 and 2 are not accidental, but are directly caused by the gene-modified hDPSCs and curcumin. This provides a quantitative benchmark to make the conclusion that the combination treatment is superior to the single treatment and the treatment group is superior to the untreated group more convincing. It follows the principle of controlled experiments to exclude the interference of irrelevant variables, meets the methodological requirements of basic medical research, and provides a credible experimental basis for subsequent clinical translation.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 protection of the present invention.

Claims

1. A method for synergistically treating Alzheimer's disease using genetically modified human dental pulp mesenchymal stem cells, characterized in that: The following steps are involved: S1: Complete third molars from healthy adults were extracted and disinfected. The tissue was then rinsed with phosphate buffered saline and minced into pieces. The dental pulp was then digested with 2 mg / mL dispase and 1 mg / mL type I collagenase to obtain mesenchymal stem cells. The cell suspension was then inoculated into a culture flask and incubated with α-modified minimum essential medium. The medium was changed every two days after five days, and the cells were continuously cultured and passaged to passage P3. S2: Construct a lentiviral vector carrying the BDNF and NRF2 genes and package it according to the instructions to obtain a high-titer recombinant lentivirus. P3 human dental pulp mesenchymal stem cells are seeded into well plates. When the cells become confluent, replace the medium with the lentivirus-containing medium and add 8 μg / mL polybrene. Replace the medium with fresh α-MEM medium and continue culturing to obtain gene-modified hDPSCs stably expressing BDNF and NRF2 through resistance screening. S3: 3xTg-AD mice were anesthetized with sodium pentobarbital, and hDPSCs were injected into the bilateral hippocampal DG of the mice at the coordinates of -2.06 mm anteroposterior, ±1.5 mm medial, and -2.1 mm dorsoventral from the bregma. After injection, the needle was left in place for 5 minutes while curcumin nanoparticle solution was injected intraperitoneally until the mice were fully awake. S4: Mice were tested one month after transplantation of gene-modified hDPSCs and concurrent curcumin nanoparticle synergistic treatment. Mice were randomly placed in a quadrant of the Morris water maze filled with milky water and asked to search for a hidden platform within 60 seconds. Each attempt was repeated 15 minutes apart. On the seventh day, an exploratory test without the platform was conducted to observe the exploratory behavior of the mice in the water maze and assess their spatial learning and memory abilities.

2. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: hDPSCs in S2 have all the characteristics of MSCs, including high proliferation capacity, the ability to be induced to differentiate into osteocytes, adipocytes and chondrocytes, and the expression of MSC surface markers.

3. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: The cell concentration of hDPSCs in S2 was 2×10 5 cells / μL, which enhanced the ability of adult hippocampal neurogenesis in the mouse brain. The effect of neurogenesis in the hippocampus could be observed by immunofluorescence staining and immunoblotting analysis.

4. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, wherein: The hDPSCs-administered group in S2 showed increased protein expression of neuronal markers DCX and NeuN in the hippocampus of mice. The hDPSCs-administered group also induced an increase in BDNF, an essential neurotrophic factor for hippocampal neurogenesis and maintaining cognitive integrity in AD mouse models.

5. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: hDPSCs in S2 differentiate into neuron-like cells in the AD brain microenvironment and form functional synaptic connections with host neurons to promote synaptic repair.

6. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: In S2, BDNF gene and NRF2 gene were introduced into hDPSCs by lentiviral vectors with a virus titer of ≥1×10 8 Transduction units / ml (TU / mL), the concentration of purified stem cells is >90%, the BDNF in them can promote neuronal survival, differentiation and synapse formation, and NRF2 has antioxidant and anti-inflammatory effects, improving the oxidative stress and inflammatory environment in the patient's brain.

7. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: Curcumin in S3 is a natural polyphenol compound extracted from ginger plants. It has multiple biological activities such as anti-inflammatory, antioxidant, and regulation of β-amyloid protein (Aβ) metabolism.

8. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, wherein: The curcumin nanoparticles described in S3 are prepared by emulsification-solvent evaporation, with a particle size distribution of 50-200 nm and an encapsulation efficiency of >80%.

9. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, wherein: In the S3, transgenic AD mice (3xTg-AD mice) were used. The transgenic mice included three mutations associated with familial AD (APP Swedish, MAPT P301L, and PSEN1 M146V), and human dental pulp mesenchymal stem cell preparations were injected into the brain stereotaxically.

10. The method for synergistically treating Alzheimer's disease with genetically modified human dental pulp mesenchymal stem cells according to claim 1, characterized in that: The BDNF is a core factor in neurogenesis and can promote the proliferation and differentiation of neural stem cells in the DG region of the hippocampus. Curcumin activates the BDNF-TrkB signaling pathway, enhances the survival and maturation of neural precursor cells, and forms a dual drive with the BDNF secreted by gene-modified hDPSCs to promote adult hippocampal neurogenesis.

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