Neural stem cell peptide for treating Alzheimer's disease and preparation method and application thereof

Abstract

The invention relates to the technical field of stem cell biology. The invention provides a neural stem cell peptide for treating Alzheimer's disease and a preparation method thereof, and the preparation method comprises the following steps: (1) inoculating an iPS cell to a culture medium A, transferring to a culture medium B after an embryo body is formed, and transferring to a culture medium C for continuous culture after induction culture to the 15th-20th day to obtain a neural stem cell; (2) placing the neural stem cells in a lysis buffer solution, and performing ultrasonic treatment and centrifugation to obtain supernate; and (3) carrying out ultrafiltration and ion exchange chromatography on the supernate to obtain the product. According to the technical scheme, the small-molecule active peptide can be efficiently enriched, the obtained neural stem cell peptide can effectively inhibit beta-amyloid protein deposition, the Alzheimer's disease can be better treated, and the neural stem cell peptide has a good application prospect in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell biology, and particularly relates to a neural stem cell peptide for treating Alzheimer's disease, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's Disease (AD) is a neurodegenerative disease characterized by abnormal deposition of β-amyloid protein (Aβ) and hyperphosphorylation of Tau protein. Currently, clinical treatment drugs (such as cholinesterase inhibitors and NMDA receptor antagonists) can only relieve symptoms and cannot prevent the progression of the disease. In recent years, although antibody drugs targeting the clearance of Aβ plaques or the inhibition of Tau pathology have made some progress, there are problems such as limited efficacy, high cost, and potential immune side effects.

[0003] Neural Stem Cells (NSC) have become a research hotspot for the treatment of AD due to their ability to differentiate into functional neurons and promote synaptic regeneration. However, traditional NSC sources (such as embryonic stem cells and adult brain tissues) have problems such as ethical controversies and low amplification efficiency. The emergence of induced pluripotent stem cell (iPS cell) technology has made it possible to prepare NSC on a large scale, but the differentiation of iPS into NSC still faces the following challenges: insufficient differentiation efficiency; difficulty in maintaining homeostasis, and the differentiated NSC are prone to spontaneous differentiation or apoptosis during passage, resulting in unstable concentrations of neurotrophic factors in the secretions; lack of functional peptide segments, and low-molecular-weight active peptides have not been refined and separated, making it difficult to play an efficient role through the blood-brain barrier.

[0004] Based on the above, it is very necessary to develop an efficient and stable iPS to NSC differentiation system to obtain highly pure and low-molecular-weight functional neural stem cell peptides. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a neural stem cell peptide for treating Alzheimer's disease. This technical solution can efficiently enrich small-molecule active peptides, and the obtained neural stem cell peptide can effectively inhibit the deposition of β-amyloid protein, better treat Alzheimer's disease, and has good application prospects in the biomedical field.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method of a neural stem cell peptide for treating Alzheimer's disease, which is characterized by including the following steps:

[0008] (1) Inoculate iPS cells into Medium A. After embryoid bodies are formed, transfer them to Medium B. After induced culture for 15 - 20 days, transfer them to Medium C for continued culture to obtain neural stem cells.

[0009] (2) Place the neural stem cells in a lysis buffer, and perform ultrasonic treatment and centrifugation to obtain a supernatant.

[0010] (3) Subject the supernatant to ultrafiltration and ion exchange chromatography to obtain the product.

[0011] Preferably, Medium A is based on Neurobasal medium and further includes components with the following final concentrations: 0.8 - 1.02% NEAA, 0.6 - 0.8% GlutaMAX, and 0.07 - 0.11 mM β - mercaptoethanol.

[0012] Preferably, Medium B is based on Neurobasal medium and further includes components with the following final concentrations: 7 - 10 μM SB431542, 450 - 510 nM LDN - 193189, and 2.5 - 2.8 μM CHIR99021.

[0013] Preferably, Medium C is based on NSC medium and further includes components with the following final concentrations: 14 - 17 ng / mL EGF, 15 - 20 ng / mL bFGF, 1 - 10 μg / L BDNF, and 8 - 12 mg / L danshen extract.

[0014] Preferably, the lysis buffer in step (2) uses water as a solvent and includes components with the following final concentrations: 40 - 48 mM Tris - HCl, 130 - 140 mM NaCl, 0.5 - 0.8% NP - 40, 0.3 - 0.5% sodium deoxycholate, and 0.5 - 1 mM PMSF.

[0015] Preferably, the frequency of the ultrasonic treatment in step (2) is 20 - 30 kHz, the time of the ultrasonic treatment is 10 - 15 s / time, and the number of ultrasonic treatment times is 2 - 3 times.

[0016] Preferably, the rotation speed of the centrifugation in step (2) is 2000 - 3000 g, and the centrifugation time is 5 - 10 min.

[0017] Preferably, ultrafiltration is performed using a filter membrane with a molecular weight cut - off of 3 - 8 kDa.

[0018] Preferably, an ion exchange column containing SP Sepharose High Performance is used for the ion exchange chromatography.

[0019] Preferably, the ion exchange chromatography process further includes an elution buffer, which includes 0.1-0.5M NaCl, 15-20mM sodium phosphate, and 480-500mM imidazole; the pH of the elution buffer is 7.2-7.5.

[0020] The present invention also provides a neural stem cell peptide for treating Alzheimer's disease prepared by the above preparation method.

[0021] The present invention also provides the application of the neural stem cell peptide in the preparation of a drug for treating Alzheimer's disease.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects:

[0023] The technical solution of the present invention induces the differentiation of iPS cells into high-purity NSCs in stages, and at the same time can efficiently enrich nanoscale small molecule active peptides. The obtained neural stem cell peptides can effectively inhibit the deposition of β-amyloid protein, and can better treat Alzheimer's disease, and have good application prospects in the field of biomedicine. In addition, the addition of Salvia miltiorrhiza extract in the culture medium also enhances the regulatory ability of small molecule active peptides secreted by NSCs on the key pathology of AD to a certain extent. Detailed implementation mode

[0024] The present invention provides a preparation method of a neural stem cell peptide for treating Alzheimer's disease, including the following steps:

[0025] (1) Inoculate iPS cells into medium A, transfer them to medium B after forming embryoid bodies, and transfer them to medium C for continued culture on the 15th-20th day of induction culture to obtain neural stem cells;

[0026] (2) Place the neural stem cells in a lysis buffer, and perform ultrasonic treatment and centrifugation to obtain a supernatant;

[0027] (3) Ultrafilter and perform ion exchange chromatography on the supernatant to obtain the product.

[0028] In the present invention, medium A is based on Neurobasal medium, and preferably further includes components with the following final concentrations: 0.8-1.02% NEAA, 0.6-0.8% GlutaMAX, and 0.07-0.11mM β-mercaptoethanol. More preferably, it includes 0.9-1.01% NEAA, 0.65-0.75% GlutaMAX, and 0.08-0.1mM β-mercaptoethanol. Even more preferably, it includes 1% NEAA, 0.7% GlutaMAX, and 0.095mM β-mercaptoethanol.

[0029] In the present invention, the medium B is based on the Neurobasal medium, and preferably further includes components with the following final concentrations: 7-10 μM SB431542, 450-510 nM LDN-193189, and 2.5-2.8 μM CHIR99021. More preferably, it includes 8-9.5 μM SB431542, 470-500 nM LDN-193189, and 2.6-2.75 μM CHIR99021. Even more preferably, it includes 9 μM SB431542, 490 nM LDN-193189, and 2.7 μM CHIR99021. The medium B of the present invention can efficiently induce neural precursors.

[0030] In the present invention, the medium C is based on the NSC medium, and preferably further includes components with the following final concentrations: 14-17 ng / mL EGF, 15-20 ng / mL bFGF, 1-10 μg / L BDNF, and 8-12 mg / L salvia extract. More preferably, it includes 15-16.5 ng / mL EGF, 16-19 ng / mL bFGF, 2-8 μg / L BDNF, and 8.5-11 mg / L salvia extract. Even more preferably, it includes 16 ng / mL EGF, 18 ng / mL bFGF, 6 μg / L BDNF, and 10 mg / L salvia extract. The medium C of the present invention can preferably achieve the amplification of neural stem cells.

[0031] In the present invention, neural stem cells are placed in a lysis buffer and subjected to sonication and centrifugation to obtain a supernatant. The lysis buffer of the present invention uses water as a solvent and preferably includes components with the following final concentrations: 40-48 mM Tris-HCl, 130-140 mM NaCl, 0.5-0.8% NP-40, 0.3-0.5% sodium deoxycholate, and 0.5-1 mM PMSF. More preferably, it includes 41-46 mM Tris-HCl, 132-138 mM NaCl, 0.6-0.75% NP-40, 0.35-0.45% sodium deoxycholate, and 0.6-0.9 mM PMSF. Even more preferably, it includes 43 mM Tris-HCl, 135 mM NaCl, 0.65% NP-40, 0.4% sodium deoxycholate, and 0.7 mM PMSF. The frequency of the sonication in the present invention is preferably 20-30 kHz, more preferably 22-28 kHz, and even more preferably 25 kHz; the time of the sonication is preferably 10-15 s / time, more preferably 11-14 s / time, and even more preferably 13 s / time; the number of times of the sonication is preferably 2-3 times, more preferably 3 times. The rotation speed of the centrifugation in the present invention is preferably 2000-3000 g, more preferably 2100-2600 g, and even more preferably 2400 g; the time of the centrifugation is preferably 5-10 min, more preferably 6-9 min, and even more preferably 8 min.

[0032] In the present invention, the supernatant is subjected to ultrafiltration and ion exchange chromatography to obtain the neural stem cell peptide. The ultrafiltration in the present invention preferably uses a filter membrane with a molecular weight cut-off of 3-8 kDa. The packing material contained in the ion exchange column used in the ion exchange chromatography process of the present invention is preferably a strong cation exchange material, more preferably SP Sepharose High Performance. Ultrafiltration and ion exchange chromatography of the supernatant in the present invention can better enrich small peptide segments and improve the purity of small peptide segments.

[0033] In the present invention, the ion exchange chromatography process further includes an elution buffer, which preferably includes 0.1-0.5 M NaCl, 15-20 mM sodium phosphate, and 480-500 mM imidazole. More preferably, it includes 0.15-0.45 M NaCl, 16-19 mM sodium phosphate, and 485-495 mM imidazole. Even more preferably, it includes 0.3 M NaCl, 18 mM sodium phosphate, and 490 mM imidazole. The pH of the elution buffer in the present invention is preferably 7.2-7.5, more preferably 7.3-7.4.

[0034] The present invention also provides a neural stem cell peptide prepared by the above-mentioned preparation method for treating Alzheimer's disease.

[0035] The present invention also provides the application of the neural stem cell peptide in the preparation of a drug for treating Alzheimer's disease.

[0036] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] In the present invention, GlutaMAX and NP-40 are purchased from Thermo Fisher Scientific; SB431542, LDN-193189, and CHIR99021 are purchased from AbMole; NEAA, EGF, bFGF, and BDNF are purchased from Wuhan Punosai Life Science Co., Ltd.; PMSF is purchased from Parker Bio.

[0038] Example 1

[0039] A neural stem cell peptide for treating Alzheimer's disease is prepared as follows:

[0040] (1) Inoculate iPS cells into medium A, transfer them to medium B after embryoid bodies are formed, and transfer them to medium C for continuous culture on the 18th day of induction culture to obtain neural stem cells; medium A is based on Neurobasal medium and further contains 1% NEAA, 0.7% GlutaMAX, and 0.095 mM β-mercaptoethanol; medium B is based on Neurobasal medium and further contains 9 μM SB431542, 490 nM LDN-193189, and 2.7 μM CHIR99021; medium C is based on NSC medium and further contains 16 ng / mL EGF, 18 ng / mL bFGF, 6 μg / L BDNF, and 10 mg / L salvia extract;

[0041] (2) Place the neural stem cells in a lysis buffer, sonicate them 3 times at 25 kHz, with each sonication for 13 s, and then centrifuge at 2400 g for 8 min to obtain a supernatant; the lysis buffer uses water as a solvent and further contains 43 mM Tris-HCl, 135 mM NaCl, 0.65% NP-40, 0.4% sodium deoxycholate, and 0.7 mM PMSF;

[0042] (3) The supernatant was ultrafiltered using a filter membrane with a molecular weight cut-off of 8 kDa, and then ion exchange chromatography was performed using an ion exchange column packed with SP Sepharose High Performance. The elution buffer involved consisted of 0.3 M NaCl, 18 mM sodium phosphate, and 490 mM imidazole, with a pH of 7.4.

[0043] Example 2

[0044] A neural stem cell peptide for treating Alzheimer's disease, the preparation method is as follows:

[0045] (1) The iPS cells were inoculated into Medium A. After embryoid bodies were formed, they were transferred to Medium B. After induced culture for 20 days, they were transferred to Medium C for continued culture to obtain neural stem cells. Medium A was based on Neurobasal medium and further supplemented with 0.8% NEAA, 0.6% GlutaMAX, and 0.11 mM β-mercaptoethanol. Medium B was based on Neurobasal medium and further supplemented with 7 μM SB431542, 450 nM LDN-193189, and 2.8 μM CHIR99021. Medium C was based on NSC medium and further supplemented with 14 ng / mL EGF, 20 ng / mL bFGF, 1 μg / L BDNF, and 12 mg / L salvia extract;

[0046] (2) The neural stem cells were placed in a lysis buffer and sonicated 3 times at 20 kHz, with each sonication for 15 s. Then, they were centrifuged at 2000 g for 10 min to obtain the supernatant. The lysis buffer was based on water and further supplemented with 40 mM Tris-HCl, 140 mM NaCl, 0.8% NP-40, 0.5% sodium deoxycholate, and 0.5 mM PMSF;

[0047] (3) The supernatant was ultrafiltered using a filter membrane with a molecular weight cut-off of 8 kDa, and then ion exchange chromatography was performed using an ion exchange column packed with SP Sepharose High Performance. The elution buffer involved consisted of 0.5 M NaCl, 20 mM sodium phosphate, and 480 mM imidazole, with a pH of 7.2.

[0048] Example 3

[0049] A neural stem cell peptide for treating Alzheimer's disease, the preparation method is as follows:

[0050] (1) Inoculate iPS cells into Medium A. After embryoid bodies are formed, transfer them to Medium B. After inducing and culturing for 20 days, transfer them to Medium C for continuous culture to obtain neural stem cells. Medium A is based on Neurobasal medium and further supplemented with 1.02% NEAA, 0.8% GlutaMAX, and 0.7 mM β-mercaptoethanol. Medium B is based on Neurobasal medium and further supplemented with 10 μM SB431542, 510 nM LDN-193189, and 2.5 μM CHIR99021. Medium C is based on NSC medium and further supplemented with 17 ng / mL EGF, 15 ng / mL bFGF, 10 μg / L BDNF, and 8 mg / L salvia extract;

[0051] (2) Place the neural stem cells in lysis buffer and ultrasonically treat them 2 times at 30 kHz, with each ultrasonic treatment lasting for 10 s. Then centrifuge at 3000 g for 5 min to obtain the supernatant. The lysis buffer uses water as the solvent and is further supplemented with 48 mM Tris-HCl, 130 mM NaCl, 0.5% NP-40, 0.3% sodium deoxycholate, and 1 mM PMSF;

[0052] (3) Ultrafilter the supernatant using a filter membrane with a molecular weight cut-off of 3 kDa, and then perform ion exchange chromatography using an ion exchange column packed with SP Sepharose High Performance. The involved elution buffer consists of 0.1 M NaCl, 15 mM sodium phosphate, and 500 mM imidazole, with a pH of 7.5.

[0053] Comparative Example 1

[0054] Same as Example 1, except that “Medium C is based on NSC medium and further supplemented with 16 ng / mL EGF, 18 ng / mL bFGF, 6 μg / L BDNF, and 10 mg / L salvia extract” is replaced with “Medium C is based on NSC medium and further supplemented with 16 ng / mL EGF, 18 ng / mL bFGF, and 6 μg / L BDNF”.

[0055] Comparative Example 2

[0056] Same as Example 1, except that “Medium A is based on Neurobasal medium and further supplemented with 1% NEAA, 0.7% GlutaMAX, and 0.095 mM β-mercaptoethanol” is replaced with “Medium A is based on Neurobasal medium and further supplemented with 1% NEAA and 0.7% GlutaMAX”.

[0057] Comparative Example 3

[0058] Same as Example 1, only remove the following: "The supernatant was ultrafiltered using a filter membrane with a molecular weight cut-off of 8 kDa, and then ion exchange chromatography was performed using an ion exchange column packed with SP Sepharose High Performance. The elution buffer involved consisted of 0.3 M NaCl, 18 mM sodium phosphate, and 490 mM imidazole, with a pH of 7.4."

[0059] Test Example 1

[0060] (1) Cell culture and proliferation

[0061] iPS cells (purchased from Anhui ZhongSheng Suyuan Biotechnology Co., Ltd.) were inoculated at a cell density of 5×10 4 cells per well, and neural stem cell induction culture was carried out based on the preparation methods of Examples 1-3 and Comparative Examples 1-3. The cell culture conditions were 37 °C and 5% CO2. Samples were taken at 48 h and 72 h respectively to observe the proliferation of neural stem cells in different treatment groups, and the CyQuant Cell Proliferation Assay kit (Invitrogen) was used for determination. The results are shown in Table 1.

[0062] Table 1 Proliferation of neural stem cells induced and cultured in different treatment groups

[0063]

[0064]

[0065] It can be seen from Table 1 that the proliferation culture effect of inducing and culturing neural stem cells using the induction differentiation medium described in the technical solution of the present invention is relatively better.

[0066] (2) Safety determination

[0067] Thirty-five male SD rats, SPF grade, weighing 250±10 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0068] The above rats were randomly divided into 7 groups, namely the control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. The rats in Example 1-3 groups and Comparative Example 1-3 groups were intraperitoneally injected with the corresponding prepared neural stem cell peptide suspension (the neural stem cell peptide suspension was diluted with serum-free DMEM, and the final dosage was equivalent to 1 mg / kg); the control group was not treated. The rats were continuously observed for 7 days, and the spontaneous activities, breathing, pupil size, secretions, etc. of each group of rats were recorded.

[0069] The results showed that the rats in each group had the same autonomous activities, breathing, pupil size, secretions, etc., and no abnormal symptoms, which indicated that the neural stem cell peptide of the present invention had certain safety.

[0070] Test Example 2

[0071] Forty male SD rats were selected and randomly divided into 8 groups, with 5 rats in each group, including a normal control group, a model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group.

[0072] Except for the normal control group, the remaining groups of rats were anesthetized with 10% chloral hydrate intraperitoneal injection (0.3mL / 100g). After anesthesia, they were fixed on a rat brain stereotaxic instrument, and needles were inserted into the bilateral hippocampus. The coordinates were: AP-4mm (behind the anterior bregma), MR-2.8mm (right side of the midline), H-3mm (depth from the meninges). After positioning, the skull was drilled open, and the micro-syringe was vertically inserted 3mm. 2μl Aβ1-42 (purchased from AbMole) was slowly injected at a speed of 0.2μL / min, and the needle was retained for 5min. One week after modeling, the drug was administered by intraperitoneal injection, in which the model group was injected with 5μL of normal saline, and the normal control group was not treated in any way. Example 1-3 and Comparative Example 1-3 were respectively given 5μL of the corresponding neural stem cell peptide suspension prepared.

[0073] (A) Morris water maze test

[0074] The water maze is a circular water tank with a diameter of 132 cm and a depth of 60 cm. It is divided into 4 quadrants and placed in the animal breeding room. The water maze is surrounded by windows, animal breeding cages, walls, fluorescent lights, etc. The location of the water maze and the surrounding environment remain unchanged throughout the experiment. All animals were subjected to the water maze experiment 5 days before being killed. During the experiment, the sink was flushed with water, and 0.8 kg of milk powder was added to the water. The water depth was 42 cm and the water temperature was (23+2)℃. There was a platform with a diameter of 9 cm 2 cm below the water surface. The platform was located in the center of one of the quadrants. On the first day, the rats were allowed to swim in the water maze for 60 seconds and then put back into the cage. The water maze experiment began on the second day. Each time, the rats were randomly placed in the water from different quadrants, and the time it took for the rats to escape to the safe platform was recorded. If the rat found the platform, it was allowed to stay on the platform for 10 seconds. If the rat failed to find the platform within 120 seconds, the experiment was terminated and the rat was placed on the platform and waited for 10 seconds. The experiment was continued for 4 days, 4 times a day, with an interval of 60 seconds between each experiment. Finally, the platform was removed and the rats were allowed to swim continuously in the water maze for 60 seconds. The number of times the rats reached the original platform position was recorded to prove whether the rats had remembered the location of the safety zone. The mean escape latency (MEL) and the average number of times they passed the safety zone in the water maze experiment of each group of rats were observed, as shown in Table 2.

[0075] Table 2 Test results of Morris water maze for each treatment group

[0076] Group Average escape latency (s) Number of times passing the platform Normal control group 20.92 4.86 Model group 58.74 0.92 Example 1 group 20.98 4.51 Example 2 group 21.05 4.34 Example 3 group 21.06 4.27 Comparative example 1 group 29.68 2.85 Comparative example 2 group 27.26 2.94 Comparative example 3 group 33.65 2.42

[0077] It can be seen from Table 2 that, compared with the model group, the average escape latency of the rats in the treatment groups given the neural stem cell peptides prepared in Examples 1-3 of the present invention was relatively shorter, and the number of times passing through the platform was relatively more. That is to say, the neural stem cell peptides prepared as described in the present invention have a certain therapeutic effect on Alzheimer's disease.

[0078] In summary, it can be seen that the technical solution of the present invention induces the differentiation of iPS cells into high-purity NSCs in stages, and at the same time can efficiently enrich nano-scale small molecule active peptides. The obtained neural stem cell peptides can effectively inhibit the deposition of β-amyloid protein and can better treat Alzheimer's disease.

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a neural stem cell peptide for treating Alzheimer's disease, characterized in that, It includes the following steps: (1) Inoculate iPS cells into medium A. After embryoid bodies are formed, transfer them to medium B. After induced culture for 15 - 20 days, transfer them to medium C for continuous culture to obtain neural stem cells; (2) Place the neural stem cells in a lysis buffer, and perform ultrasonic treatment and centrifugation to obtain a supernatant; (3) Subject the supernatant to ultrafiltration and ion exchange chromatography to obtain the product; The medium A is based on Neurobasal medium and further includes components with the following final concentrations: 0.8 - 1.02% NEAA, 0.6 - 0.8% GlutaMAX, and 0.07 - 0.11 mM β-mercaptoethanol; The medium B is based on Neurobasal medium and further includes components with the following final concentrations: 7 - 10 μM SB431542, 450 - 510 nM LDN-193189, and 2.5 - 2.8 μM CHIR99021; The medium C is based on NSC medium and further includes components with the following final concentrations: 14 - 17 ng / mL EGF, 15 - 20 ng / mL bFGF, 1 - 10 μg / L BDNF, and 8 - 12 mg / L salvia extract; 2. The preparation method according to claim 1, wherein The lysis buffer in step (2) uses water as a solvent and includes components with the following final concentrations: 40 - 48 mM Tris-HCl, 130 - 140 mM NaCl, 0.5 - 0.8% NP-40, 0.3 - 0.5% sodium deoxycholate, and 0.5 - 1 mM PMSF; 3. The preparation method according to claim 1, characterized in that, The frequency of the ultrasonic treatment in step (2) is 20 - 30 kHz, the time of the ultrasonic treatment is 10 - 15 s / time, and the number of ultrasonic treatment times is 2 - 3 times; 4. The preparation method according to claim 1, characterized in that, The rotation speed of the centrifugation in step (2) is 2000 - 3000 g, and the time of the centrifugation is 5 - 10 min; 5. The preparation method according to claim 1, wherein Ultrafiltration is carried out using a filter membrane with a cut-off molecular weight of 3 - 8 kDa; 6. The preparation method according to claim 1, characterized in that, An ion exchange column containing SP Sepharose High Performance is used during the ion exchange chromatography; The ion exchange chromatography process further includes an elution buffer, and the elution buffer includes 0.1 - 0.5 M NaCl, 15 - 20 mM sodium phosphate, and 480 - 500 mM imidazole; The pH of the elution buffer is 7.2 - 7.5; 7. The neural stem cell peptide for treating Alzheimer's disease prepared by the preparation method according to any one of claims 1 - 6.

8. The application of the neural stem cell peptide according to claim 7 in the preparation of a drug for treating Alzheimer's disease.

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