Application of protein polymer in preparation of medicine for treating Alzheimer's disease

Abstract

The invention belongs to the field of biological medicines, and discloses an application of a protein polymer in preparation of a medicine for treating Alzheimer's disease, a production process of the protein polymer comprises the following steps: S1) using ultraviolet irradiation to stimulate mesenchymal stem cells and culturing; and S2) splitting the mesenchymal stem cells, separating and purifying to obtain the protein polymer. According to the present invention, the mesenchymal stem cells are stimulated to express the protein with the nerve repair function through the ultraviolet irradiation stress mode, and the protein polymer is obtained through further separation and purification, such that the protein polymer can be adapted to diversified application modes, can improve the abnormal conditions of the Alzheimer's disease related biomarkers, and can provide the significant treatment effect on the Alzheimer's disease.

Description

Technical Field

[0001] This application belongs to the field of medicine, and particularly relates to the use of a protein polymer as a drug for the treatment of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, which mostly occurs in the elderly population and is the main cause of senile dementia. Currently, the prevalent pathogenic mechanism hypotheses of Alzheimer's disease are divided into three types: amyloid hypothesis, p-Tau protein hypothesis, and inflammation hypothesis. Among them, the amyloid hypothesis holds that the abnormal deposition of Aβ molecules is the main cause of AD; the p-Tau protein hypothesis emphasizes that the hyperphosphorylation of Tau protein to form neurofibrillary tangles will lead to AD; the inflammation hypothesis has noticed that inflammatory factors of neuroinflammation and even systemic inflammation are prone to accumulate at amyloid and neurofibrillary tangles, and there is a certain association between inflammation in the organism and AD.

[0003] One existing method for the treatment of Alzheimer's disease is to administer to a subject a preparation of allogeneic mesenchymal stromal cells (MSC or MSCs) derived from healthy young adult donors. The MSC preparation contains mesenchymal stem cells and a vehicle, and is administered to the subject by infusion.

[0004] However, cell products such as the above MSC preparation have relatively strict requirements for production, transportation, and storage conditions, and also require the assistance of professionals in a professional institution during administration, with many restrictions at the application end. Based on the characteristic that mesenchymal stem cells can produce different stress proteins under different stimulation conditions, how to use MSCs to produce a protein polymer that can improve the abnormalities of Alzheimer's disease-related biomarkers and prepare the protein polymer into a drug that can treat Alzheimer's disease is a very challenging task. Summary of the Invention

[0005] Based on this, the inventors of this application have found that after culturing mesenchymal stem cells under appropriate stress conditions, a variety of proteins can be isolated from their lysates. The isolated protein polymer can improve the abnormalities of Alzheimer's disease-related biomarkers and can be prepared into a drug for the treatment of Alzheimer's disease.

[0006] The specific technical solution of this application is: the use of a protein polymer as a drug for the treatment of Alzheimer's disease, and the production process of the protein polymer includes:

[0007] S1) Stimulate mesenchymal stem cells with ultraviolet irradiation and culture;

[0008] S2) Lyse the mesenchymal stem cells and isolate and purify to obtain the protein polymer.

[0009] In some alternative application scenarios, the protein polymer satisfies at least one of the following properties:

[0010] 1) During molecular sieve exclusion chromatography, under the exclusion chromatography conditions where the elution flow rate is 0.2 mL / min - 0.4 mL / min and the eluent is PBS, the elution volume at which the first component elutes is 12 mL - 13.2 mL, the elution volume at which the second component elutes is 15.2 mL - 17 mL, the elution volume at which the third component elutes is 17 mL - 20 mL, the elution volume at which the fourth component elutes is 29 mL - 31 mL, and the elution volume at which the fifth component elutes is 31 mL - 34 mL;

[0011] 2) During SDS - PAGE detection, when using a 4% - 20% precast gel for sample separation and detection, the sample bands are mainly distributed between 11 KD - 100 KD. Among them, from the largest to the smallest molecular weight, the first band is between 75 KD - 100 KD; the second band is between 63 KD - 75 KD;

[0012] 3) During reverse - phase HPLC detection, with a sample injection volume of 25 μL, a column temperature of 30 °C, a flow rate of 0.4 mL / min, a detection wavelength of 280 nm, mobile phase A being 0.1% TFA aqueous solution, mobile phase B being 0.1% TFA acetonitrile solution, and the gradient elution conditions being:

[0013]

[0014] Under the reverse - phase HPLC conditions where..., the peak elution time after sample separation is between 2 min - 20 min. The peak elution time of the first group of sample components is between 12 min - 20 min, where the characteristic peak 1 elutes at approximately 13 min - 17 min, and the peak elution time of the second group of sample components is 2 min - 5 min, and the characteristic peak 2 elutes at approximately 3 min.

[0015] In some alternative application scenarios, the protein polymer at least includes the following proteins:

[0016] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens OX=9606 GN=ALB;

[0017] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens OX=9606 GN=TF; and

[0018] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens OX=9606 GN=LUM;

[0019] Moreover, the mass of the above three proteins accounts for more than 40% of the total mass of the protein polymer.

[0020] In some alternative application scenarios, the protein polymer further comprises at least one of the following proteins:

[0021] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens OX=9606GN=ACTA2 PE=1SV=1;

[0022] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens OX=9606GN=SERPINA1 PE=1SV=3;

[0023] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens OX=9606GN=TPM2 PE=1SV=1;

[0024] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens OX=9606GN=VIM PE=1SV=4;

[0025] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens OX=9606GN=FN1PE=1SV=5;

[0026] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens OX=9606GN=TPM1 PE=1SV=2;

[0027] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens OX=9606GN=FLNA PE=1SV=4;

[0028] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens OX=9606PE=1SV=2;

[0029] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens OX=9606 GN=TNC PE=1 SV=3;

[0030] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens OX=9606 GN=A2M PE=1 SV=3;

[0031] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens OX=9606 GN=ACTB PE=1 SV=1; and

[0032] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens OX=9606 GN=HBG1 PE=1 SV=2.

[0033] In some alternative application scenarios, the operation of the molecular sieve size exclusion chromatography includes:

[0034] After equilibrating a Superdex 150 molecular sieve 8×500 size exclusion chromatography column, loading the sample, eluting with PBS at an elution rate of 0.2 mL / min, starting to collect from a UV absorption value of 4 mAU at 280 nm, and eluting and collecting 5 components with elution volumes of 12 mL - 13.2 mL, 15.2 mL - 17 mL, 17 mL - 20 mL, 29 mL - 31 mL, and 31 mL - 34 mL.

[0035] In some alternative application scenarios, the time for ultraviolet irradiation to stimulate mesenchymal stem cells is 1 h - 30 h; the intensity of ultraviolet irradiation stimulation is 10 μW / cm2 - 60 μW / cm 2 , and the wavelength of the ultraviolet light is 290 nm - 340 nm.

[0036] In some alternative application scenarios, the culture medium used for the mesenchymal stem cells during ultraviolet irradiation stimulation is a serum-free MSCs culture medium.

[0037] In some alternative application scenarios, the mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived human mesenchymal stem cells, and human placenta-derived human mesenchymal stem cells.

[0038] In some alternative application scenarios, the protein polymer can be made into any one of the dosage forms of freeze-dried powder, injection solution, gel, spray, pill, tablet, powder, ointment, and microneedle preparation.

[0039] In some alternative application scenarios, the administration route of the protein polymer during application is selected from one of intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, oral administration, and subcutaneous injection.

[0040] In some alternative application scenarios, the protein polymer is used to reduce the concentrations of β-amyloid Aβ-40 and Aβ-42 in plasma, and / or to increase the concentration ratio of β-amyloid Aβ-42 to Aβ-40 in plasma.

[0041] In some alternative application scenarios, the protein polymer is used to inhibit the abnormal phosphorylation of Tau protein on motor neurons in a neuroinflammatory environment.

[0042] The beneficial effects of this application are as follows:

[0043] In this application, mesenchymal stem cells are stimulated by ultraviolet irradiation, and after stress culture, the stressed mesenchymal stem cells are lysed, and a protein polymer (denoted as Alitor or ALT) is isolated and purified. Alitor can be prepared into different dosage forms such as freeze-dried powder, injection, gel, spray, pill, tablet, powder, ointment, and microneedle preparation, and can be combined with different administration routes such as intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, oral administration, and subcutaneous injection. And through relevant experimental verification, Alitor can improve the abnormalities of Alzheimer's disease-related biomarkers, reduce the concentrations of β-amyloid Aβ-40 and Aβ-42, and the degree of abnormal phosphorylation of Tau protein on motor neurons. On the one hand, the deposition of β-amyloid Aβ-40 and Aβ-42 is prone to aggregation to form a β-sheet structure, and then form amyloid plaques, which directly damage neurons and have strong neurotoxicity. Alitor can reduce the concentrations of Aβ-40 and Aβ-42 in plasma, and / or can increase the concentration ratio of Aβ-42 to Aβ-40 in plasma, which means that Alitor can repair neuron damage to a certain extent, relieve the symptoms of Alzheimer's disease, and achieve a therapeutic effect. On the other hand, hyperphosphorylated Tau protein will aggregate in cells to form neurofibrillary tangles, and directly damage the structure and function of neurons, leading to memory loss and cognitive impairment. Alitor inhibits the abnormal phosphorylation of Tau protein on motor neurons, proving that Alitor can reverse the phosphorylation trend of Tau protein and inhibit the spread of inflammation in the nervous system, and has the potential to treat Alzheimer's disease. Description of the Drawings

[0044] In order to more clearly illustrate the technical solutions of this application, the drawings required for the description of this application will be briefly introduced below.

[0045] Figure 1It is a photo of the growth state of MSCs after 3D culture by Tang Yi in Experiment 1.

[0046] Figures 2 - 6 It is a photo of the cell state after different ultraviolet irradiation times in Experiment 1.

[0047] Figure 7 It is the SDS-PAGE result of Experiment 1.

[0048] Figure 8 It is the elution curve of Experiment 2.

[0049] Figure 9 It is the elution curve of HPLC-SEC.

[0050] Figure 10 It is the SDS-PAGE electrophoresis diagram of Sample No. 12 (240305-pure-12#) after purification in Experiment 3.

[0051] Figure 11 It is the immunofluorescence map of p-Tau protein in damaged motor neurons.

[0052] Figure 12 It is for Figure 11 The statistical chart of the average fluorescence intensity. Detailed implementation manner

[0053] The embodiments of the present implementation manner are described in detail below. The embodiments are only used to explain the present implementation manner and cannot be understood as a limitation to the present implementation manner.

[0054] In the present application, for the convenience of description, mesenchymal stem cells are used as an example in the embodiments. However, in practical applications, it is not limited to mesenchymal stem cells. Other stem cells can also produce similar stress proteins after stress induction. Therefore, other stem cells that can be used include, but are not limited to, at least one of embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.

[0055] In theory, all mesenchymal stem cells can induce the production of corresponding stress proteins after undergoing stress condition treatment. For the convenience of description, ultraviolet irradiation was used as the stress condition in the examples to prepare protein polymers that can be used as drugs for treating Alzheimer's disease. In practical applications, other stress conditions can achieve similar effects. Other stress conditions include but are not limited to at least one of the following stress methods: infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, oxidation (under conditions containing oxidants such as hydrogen peroxide or hypochlorous acid), high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, ray, ray, ray, ion beam, high CO2, low CO2. Various stress treatments can be carried out successively or simultaneously when there is no conflict.

[0056] In the examples, human umbilical cord-derived mesenchymal stem cells (HUC-MSC) were used as an example to illustrate the problem. In practical applications, the sources of mesenchymal stem cells also include but are not limited to bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0057] In theory, any method that can be used to separate proteins from cell lysates can be used to isolate and purify the protein polymers described in this application from stressed cultured stem cells. For the convenience of description, chromatography, electrophoresis, and size exclusion (molecular sieve) were used in some examples of this application. In practical applications, it can also be any method that can separate and extract the target protein from the filtrate, including but not limited to at least one of the following methods: chromatography, spectroscopy, size exclusion, dialysis, salting out, precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, centrifugation, etc.

[0058] Spectroscopy uses the absorbance of the target protein at a specific wavelength of light to screen out the target extract; dialysis uses a dialysis bag to exchange solutions with a buffer to finally obtain a purified and concentrated target extract; organic solvent precipitation method adds an organic solvent to the cell lysate to achieve a precipitation effect similar to that of high-concentration salt, and the precipitate filtered is the target extract; acid extraction method obtains the target extract by adding concentrated hydrochloric acid; alkali extraction method treats the cell lysate by adding sodium hydroxide or potassium hydroxide to obtain the target extract; ultrafiltration method adds the lysate after cell lysis to an ultrafiltration column, continuously centrifuges, and adds a buffer, and performs ultrafiltration filtration multiple times to finally concentrate and obtain the target extract; chromatography includes ion exchange chromatography, gel filtration chromatography, and affinity chromatography, and different chromatography methods are selected according to the characteristics of the target extract; electrophoresis passes an electric current to separate protein molecules in a dispersion matrix to obtain the target extract; centrifugation method filters the target extract by centrifuging the lysate.

[0059] The technical solution of the present application will be further described below in conjunction with experimental embodiments. It can be understood that the Alitou (also known as ALT) defined in the present application is a collection of various stress proteins with specific biological activities expressed by mesenchymal stem cells under stress conditions. The intracellular protein obtained by simply filtering the cell lysate is the protein polymer; and the intracellular protein is further purified to remove impurities of non-target proteins, and the purified product obtained is also a protein polymer.

[0060] It should be noted that the drug prepared from the protein polymer can be one of freeze-dried powder, injection, gel, spray, pill, tablet, powder, ointment and microneedle preparation composed of the protein polymer and the corresponding medically acceptable carrier and / or excipient. The administration method can be one of intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, oral administration and subcutaneous injection.

[0061] Experiment 1: Effects of different treatments on protein expression levels:

[0062] A total of 2 L of HUC-MSC was cultured in the HK-G050(PRF)3D medium of Tangyi Huike Biotechnology. The total number of cells was about 5×10^8, and they were divided and placed in 4 T225 bottles, batch number: TYHK24011701. The cells were stained and observed, and the microcarriers were basically covered with cells. The results are as Figure 1 shown.

[0063] The cells were irradiated with LED ultraviolet light. Irradiation conditions: 60 μW / cm 2 , 8 mL was sampled at 0 h and allowed to settle. The supernatant was filtered through a 0.22-μm filter membrane and stored at 4°C; 20 mL of pure water was added to the cells to swell for 10 min, and then filtered through a 0.22-μm filter membrane and stored at 4°C. Subsequently, samples were taken from different bottles at 8 h, 12 h, 16 h, 18 h, 24 h, and 30 h respectively, and only the intracellular protein was collected to measure the protein concentration.

[0064] The cell state after 8 h of ultraviolet irradiation is as Figure 2 shown; the cell state after 12 h of ultraviolet irradiation is as Figure 3 shown; the cell state after 16 h of ultraviolet irradiation is as Figure 4 shown; the cell state after 24 h of ultraviolet irradiation is as Figure 5 shown; the cell state after 30 h of ultraviolet irradiation is as Figure 6 shown.

[0065] After the irradiation ended, cells were harvested at different time points. The culture was filtered through a 300-mesh filter bag to retain the microcarriers. The supernatant was centrifuged at 1200 rpm for 6 min and stored at 4°C. The cell pellet was washed three times with 150 mL of normal saline, lysed with 14 mL of pure water, and filtered through a 0.22-μm filter membrane. The harvested intracellular protein, namely the protein polymer in this application, was frozen at -80°C. The harvested intracellular protein was taken for gel electrophoresis analysis, and its SDS-PAGE results are as Figure 7 shown.

[0066] Experiment 2: Molecular sieve purification and protein polymer activity detection

[0067] Purification of protein polymer:

[0068] Instrument: AKTA explorer

[0069] Chromatography column: Nanomicro superdex 150 molecular sieve 8×500, column volume is about 30 mL

[0070] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, pure water

[0071] Ultraviolet absorption wavelengths: 280 nm, with 260 nm as a reference

[0072] Order of equilibration of the chromatography column:

[0073] First, rinse the chromatography column with 2 CV of pure water, and then equilibrate the chromatography column with 1×PBS for 2 CV, and zero the ultraviolet absorption value at 280 nm.

[0074] Sample preparation: Intracellular protein from the batch on January 18, 2024 (i.e., protein polymer, also known as Alituo or ALT), with a volume of about 20 mL. The 20 mL sample was concentrated to about 600 μL using an ultrafiltration concentrator tube with a molecular weight cut-off of 3 KD.

[0075] Experimental procedure: After equilibrating the chromatography column, load the sample using a 500-μL loading loop, with a loading flow rate of 0.4 mL / min and an elution flow rate of 0.2 mL / min with 1×PBS until the peak elutes. Collect the protein starting from an ultraviolet absorption value of 4 mAU. The elution curve is shown in Figure 8 . Take the proteins at the positions shown in Figure 8 2, 9, 12, 16, and 18 for activity detection, and name them 240305-Pure-2#, 240305-Pure-9#, 240305-Pure-12#, 240305-Pure-16#, and 240305-Pure-18# respectively.

[0076] Biological activity detection of protein polymer

[0077] Day 1

[0078] Cell seeding: Dilute PC12 low-differentiated cells with complete medium (5% FBS + DMEM), and seed 6000 cells per well (96-well plate). Incubate overnight at 37°C with 5% CO2.

[0079] The next day

[0080] Dilute the lyophilized samples of intracellular proteins stored at -80°C (about 800 μg / mL) and the purified samples of 240305-pure-2#, 240305-pure-9#, 240305-pure-12#, 240305-pure-16#, 240305-pure-18# with DMEM + 5% FBS medium so that the protein concentrations in each group of samples are basically the same.

[0081] Take 30% hydrogen peroxide and dilute it 15000-fold with DMEM + 5% FBS.

[0082] Hydrogen peroxide treatment: Discard 80 μL of culture supernatant per well, and add 50 μL of diluted hydrogen peroxide to the corresponding cultured cells per well. Incubate at room temperature for 25 min.

[0083] After hydrogen peroxide treatment, add 50 μL of DMEM + 5% FBS medium per well as the injury treatment control (NC).

[0084] Add 50 μL of diluted sample per well to the wells after hydrogen peroxide treatment.

[0085] For the untreated wells, discard the supernatant, and add 100 μL of complete medium per well as the cell growth control (PC).

[0086] Incubate at 37°C for 2 days.

[0087] The fifth day

[0088] Discard the culture supernatant, add 100 μL of two kinds of complete medium per well, and set up medium blank control wells. Add 10 μL of CCK8 per well and incubate at 37°C for 3.5 h. OD 450 Read the value. Calculate after deducting the medium blank.

[0089] The experimental results are shown in Table 1.

[0090] Table 1. Bioactivity experimental data of different samples

[0091]

[0092]

[0093] As can be seen from Table 1, in the PC12 cell oxidative damage model, samples 2#, 12#, and 16# all have strong oxidative damage repair abilities, and their repair abilities are better than those of the unpurified sample. The repair ability of sample 18# is lower than that of the unpurified sample.

[0094] The purified sample #12 (240305 - pure - #12) was subjected to SDS - PAGE electrophoresis analysis, and the electrophoresis results are as follows: Figure 9 As can be seen from the figure, the sample bands are mainly distributed between 11KD - 100KD. Among them, from the largest to the smallest molecular weight, the first band is between 75KD - 100KD; the second band is between 63KD - 75KD. Further analysis shows that the purified protein polymer contains the following proteins:

[0095] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens OX=9606GN=ALB PE=1SV=2;

[0096] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens OX=9606GN=TF PE - =1SV=3;

[0097] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens OX=9606GN=LUM PE=1SV=2.

[0098] The above 3 kinds are the main proteins, accounting for more than 40% of the total mass of the protein polymer. Other proteins include:

[0099] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens OX=9606GN=ACTA2 PE=1SV=1;

[0100] sp|P01009|A1AT_HUMAN Alpha - 1 - antitrypsin OS=Homo sapiens OX=9606GN=SERPINA1 PE=1SV=3;

[0101] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens OX=9606GN=TPM2 PE=1SV=1;

[0102] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens OX=9606GN=VIM PE=1SV=4;

[0103] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens OX=9606 GN=FN1 PE=1 SV=5;

[0104] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens OX=9606 GN=TPM1 PE=1 SV=2;

[0105] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens OX=9606 GN=FLNA PE=1 SV=4;

[0106] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens OX=9606 PE=1 SV=2;

[0107] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens OX=9606 GN=TNC PE=1 SV=3;

[0108] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens OX=9606 GN=A2M PE=1 SV=3;

[0109] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens OX=9606 GN=ACTB PE=1 SV=1;

[0110] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens OX=9606 GN=HBG1 PE=1 SV=2。

[0111] Experiment 3 HPLC-SEC Purification and Protein Polymer Activity Detection

[0112] HPLC-SEC Purification

[0113] Mobile phase: PBS

[0114] Detection conditions: injection volume 100 μL, column temperature 25 °C, flow rate 0.4 mL / min, wavelengths 280 nm and 260 nm; time: 48 min. It is understood that the aforementioned flow rate is the elution flow rate.

[0115] Sample: Tang Yi 3D intracellular protein sample on January 18, 2024 (unpurified sample); sample loading was repeated 2 times.

[0116] Collection time range is approximately: 1# 13.3 - 14.5 min; 2# 21.5 - 22 min; 3# 22 - 22.8 min; 4# 23 - 23.7 min; 5# 26.8 - 27.8 min; 6# 27.8 - 28.7 min; 7# 28.7 - 30 min; 8# 30 - 30.6 min; 9# 30.6 - 32 (no obvious peak at A280) min; 10# 33.8 - 34.8 min.

[0117] The elution curve is as Figure 10 shown. Proteins at positions 1 - 10 in the figure were taken for activity detection and were correspondingly named 240304SEC - 1# - 240304SEC - 10#.

[0118] Biological activity detection of protein polymers

[0119] The first day

[0120] Cell seeding: Dilute PC12 low - differentiated cells with complete medium (5% FBS + DMEM) and seed 6000 cells per well (96 - well plate). Incubate overnight at 37 °C with 5% CO2.

[0121] The second day

[0122] Dilute the lyophilized sample (about 800 μg / mL) and other purified samples with DMEM + 5% FBS medium to basically the same concentration.

[0123] Take 30% hydrogen peroxide and dilute it 15000 times with DMEM + 5% FBS.

[0124] Hydrogen peroxide treatment: Discard 80 μL of culture supernatant per well, and add 50 μL of diluted hydrogen peroxide to the corresponding cultured cells per well. Incubate at room temperature for 25 min.

[0125] After hydrogen peroxide treatment, add 50 μL of DMEM + 5% FBS medium per well as a damage treatment control (NC).

[0126] Add 50 μL of diluted sample per well to the wells after hydrogen peroxide treatment.

[0127] For the untreated wells, discard the supernatant and add 100 μL of 2 kinds of complete medium per well as cell growth controls (PC).

[0128] Incubate at 37°C for 2 days.

[0129] The fifth day

[0130] Discard the culture supernatant, add 100 μL / well of two complete media, and set up a media blank control well. Add 10 μL / well of CCK8 and incubate at 37°C for 3.5 h. OD 450 Read the value. Calculate after deducting the media blank.

[0131] The bioactivity results are shown in Table 2.

[0132] Table 2. Bioactivity experimental data of different samples

[0133]

[0134]

[0135] As can be seen from Table 2, in the PC12 cell oxidative damage model, samples 1#, 2#, 7#, 8#, and 10# all have strong oxidative damage repair ability. Their repair abilities are better than those of the unpurified sample 20240118 intracellular protein. The repair ability of sample 5# is lower than that of the unpurified sample 20240118 intracellular protein.

[0136] Experiment 4. Evaluation of the biological effects of protein polymers (alitoc / ALT)

[0137] Experiment 4.1 Changes in the concentrations of biomarker β-amyloid Aβ-40 and Aβ-42 after administration of protein polymers

[0138] Experimental design:

[0139] Screening: Use digital single molecule immunoarray analysis to detect the concentrations of β-amyloid Aβ-40 and Aβ-42 in the plasma of subjects, calculate the ratio of Aβ-42 / Aβ-40, and screen out 2 Alzheimer's subjects A and B with abnormal β-amyloid according to the existing reference intervals.

[0140] Administration: Take the lyophilized preparation of the protein polymer, prepare an injection solution, and administer the drug prepared from the protein polymer to the 2 subjects by intravenous injection. The dosing frequency is: once a day, 5 days a week, 2 days off, for a total of 2 weeks.

[0141] Biomarker monitoring: After the end of the drug administration period, detect the concentrations of β-amyloid Aβ-40 and Aβ-42 in the plasma of subjects A and B, and calculate the Aβ-42 / Aβ-40 ratio.

[0142] Data statistics:

[0143]

[0144] Conclusion:

[0145] Judging from the data before medication, the amyloid deposition of Alzheimer's disease in Subject B was more severe than that in Subject A. After the administration of the protein polymer to both subjects, the levels of amyloid Aβ-40 and Aβ-42 both decreased. Among them, the decrease in Subject B was greater than that in Subject A. This indicates that the protein polymer prepared in this application can effectively inhibit the malignant development of amyloid deposition and has a positive effect on the clearance of β-amyloid. In addition, the Aβ-42 / Aβ-40 values of both subjects increased after medication, indicating a trend of recovery from the abnormal state to the normal state.

[0146] Experiment 4.2 Changes in Biomarker p-Tau after Administration of Protein Polymer

[0147] 4.2.1 Experimental Purpose

[0148] To study the effect of the protein polymer (Alito / ALT, 20240408) on p-Tau on damaged motor neurons (MN), and to explore the therapeutic effect of Alito on neuron damage diseases caused by the pathological increase of p-Tau protein, including Alzheimer's disease and other neurodegenerative diseases.

[0149] 4.2.2 Experimental Preparation

[0150] Experimental Cells

[0151] Mouse BV2 cells, primary MN-E13 extracted and cultured on 2025.02.06, DIV7 MN

[0152] Test Samples

[0153] Alito lyophilized powder: batch number 20240408, prepared into a sample with a concentration of 70 μg / mL.

[0154] Experimental Reagents

[0155] LPS lipopolysaccharide (sigma, product number L2630, 1 mg / mL)

[0156] Experimental Materials

[0157] 1. Reagents:

[0158]

[0159]

[0160] 2. Consumables

[0161]

[0162]

[0163] 3. Instruments and Equipment

[0164] Instrument Name Model Brand Manufacturer Carbon Dioxide Incubator 3311 ThermoFisher Biological Safety Cabinet KS18 ThermoFisher Fluorescence Inverted Microscope D-35578 Leica Inverted Microscope Primovert ZEISS Tabletop Centrifuge ST40R Thermo

[0165] 4.2.3 Experimental Methods

[0166] 4.2.3.1 Extraction of Motor Neurons (MN)

[0167] S11: Sterilization of coverslips: Prepare a sterile 12-well plate, take 24 coverslips and soak them in absolute ethanol, light an alcohol lamp, and sterilize the coverslips by passing them over the flame.

[0168] S12: Coating of coverslips with polylysine PDL: Take 500 μl of 100 μg / mL PDL solution, cover the coverslips completely, and gently transfer them to an incubator at 37 °C for overnight coating.

[0169] S13: Coating of slides with laminin: Take 21 μl of laminin and dissolve it in 6 mL of HBSS buffer. Wash the PDL-coated slides 3 times with sterile ultrapure water, discard all the liquid, add 500 μl of laminin solution to each slide, incubate at 37 °C for coating, and wash 3 times with sterile ultrapure water before use.

[0170] S14: Isolation of spinal cord tissue: Take a mouse embryo, remove the thin sheath on the outer layer of its back skin, pierce and lift the spinal cord with forceps, transfer the spinal cord to a new dish containing HBSS, then open the central channel along the back of the spinal cord, remove the dorsal root ganglia (DRG) by removing the meninges from the cervical side, transfer the remaining spinal cord tissue to a new dish containing HBSS with a pipette, discard most of the liquid and retain a small amount of HBSS, and cut the spinal cord tissue into pieces.

[0171] S15: Digestion of cells with papain: Take 25 μl of papain and add it to 5 mL of Neurobasal medium. Transfer the spinal cord tissue fragments to the enzyme solution, then add 25 μl of Dnase enzyme (25 ng / mL), invert and shake well, place it flat in an incubator at 37 °C, incubate and digest for 30 min, and shake it 5 - 6 times in the middle.

[0172] S16: Cell collection: After digestion, centrifuge briefly, discard the supernatant, add 6 mL of Neurobasal medium, gently pipette up and down 10 times with a 5 mL pipette, let stand for 1 min, filter 3 mL of the upper layer through a 40 μm filter membrane and collect it into a new 15 mL centrifuge tube; add 3 mL of fresh Neurobasal medium, gently pipette up and down 10 times, let stand for 1 min, repeat the above filtration and collection process 2 times, and collect a total of 9 mL of cell suspension; centrifuge at 1000 rpm for 5 min, discard the supernatant and collect the cell pellet.

[0173] S17: Purification of motor neurons (MN) by density gradient centrifugation: Suspend the cell pellet in 1 mL of HBSS, gently spread it on the surface of 6 mL of 1.06 g / mL Optiprep solution, and centrifuge at 400xG for 5 min.

[0174] S18: MN culture: Take 1 mL of the uppermost cell solution, which is the purified MN, mix it with 9 mL of Neurobasal, centrifuge at 1200 rpm for 10 min, discard the supernatant, gently suspend the pellet in complete medium, count with a cell counting chamber, resuspend at 550,000 cells / well and transfer to the cover glass in a laminin-coated dish, and culture in a 37 °C incubator; after the MN adheres and grows for 1 h, discard the supernatant medium and non-adherent cells, and replace with 2 mL of fresh complete medium for continued culture.

[0175] Among them, the composition of the complete medium is: Neurobasal + 10% horse serum + 1% Glutamax Ⅰ + 1% B27.

[0176] S19: Continuously culture the MN with medium change, change half of the medium every 2 days and observe the cell growth.

[0177] 4.2.3.2 Preparation of conditioned medium (CM)

[0178] S21: Grouping: Control group (Ctr) and model group (LPS)

[0179] S22: Digest and plate BV2 cells at 800,000 cells per T25 flask for culture.

[0180] S23: After 24 h of culture: The Ctr control group cells are changed to 5 mL of normal medium and continuously cultured for 24 h; the LPS model group cells are changed to a medium containing 500 ng / mL LPS (5 mL of normal medium + 2.5 μL of LPS) for continuous stimulation culture for 24 h.

[0181] S24: Collect the culture medium supernatants of the cells in the Ctr control group and the LPS model group, centrifuge at 1000 rpm for 5 min, and the collected supernatant is CM-LPS and CM-Ctr. Discard the cell pellet.

[0182] 4.2.3.3 CM damage and administration of MN

[0183] Cultivate primary MN until the 7th day (obtain DIV7 MN), and perform CM medium replacement damage and culture according to the following protocol.

[0184] Grouping and damage conditions

[0185] Control group (Ctr group): Transfer DIV7 MN to CM-Ctr damage semi-medium (500 μl CM-Ctr medium + 500 μl complete medium) and culture for 24 h;

[0186] Model group (LPS group): Transfer DIV7 MN to CM-LPS damage semi-medium (500 μl CM-LPS medium + 500 μl complete medium) and culture for 24 h;

[0187] Alitor 300 ng / mL group (LPS + ALT300): Transfer DIV7 MN to CM-LPS damage semi-medium containing 300 ng / mL alitor (500 μl CM-LPS medium + 500 μl complete medium + 4.3 μl ALT) and culture for 24 h;

[0188] Alitor 600 ng / mL group (LPS + ALT600): Transfer DIV7 MN to CM-LPS damage semi-medium containing 600 ng / mL alitor (500 μl CM-LPS medium + 500 μl complete medium + 8.6 μl ALT) and culture for 24 h.

[0189] 4.2.3.4 Immunofluorescence staining

[0190] S41: Cell fixation: Take the culture plate, add the four groups of cells in 4.2.3.3 to the corresponding wells, preheat 4% PFA to room temperature, add 1 mL of 4% PFA to each well, and fix at room temperature for 20 min.

[0191] S42: Washing and permeabilization: Wash the above culture plate with PBS containing 0.2% Triton (PBST), transfer it to a shaker, shake and wash at 80 rpm for 5 min each time, and repeat washing 3 times.

[0192] S43: Blocking: Add 5% goat serum (PBS containing 0.2% Triton and 1‰ Proclean bacteriostatic agent) to the wells of the culture plate, transfer it to a shaker, and shake and wash at 80 rpm for 1 h.

[0193] S44: Incubate the primary antibody: Transfer the culture plate to a humidified chamber, cover it with sealing film, take 25 μl of the diluted antibody solution (mouse anti-Tuj1 (Bioss, 1:250), rabbit anti-p-Tau (HuaBio, 1:250), and 1‰ Proclean bacteriostatic agent) on the sealing film, and incubate overnight at 4°C.

[0194] S45: Incubate the secondary antibody: After mixing Rabbit 488 fluorescent secondary antibody (1:1000) and Mouse 594 fluorescent secondary antibody (1:300) with the secondary antibody diluent, add 1‰ Proclean bacteriostatic agent. Take 25 μl of the secondary antibody diluent on the sealing film, and incubate for 2 h at room temperature in the dark in a humidified chamber.

[0195] S46: DAPI staining: Take DAPI (1 μg / mL) staining solution on the sealing film and stain on a shaker for 8 min.

[0196] S47: Washing: Wash the culture plate with PBS containing 0.2% Triton (PBST), transfer it to a shaker, shake and wash at 80 rpm for 5 min each time, and repeat the washing 2 times.

[0197] S48: Transfer the stained cells to a glass slide, add 30 μl of anti-fluorescence quenching agent, and then place the coverslip on the glass slide.

[0198] S49: Sealing the coverslip: Seal the coverslip with nail polish.

[0199] 4.2.3.5 Photograph and count the results

[0200] Take pictures of the results of the immunofluorescence sections with a fluorescence inverted microscope (see Figure 11 ), randomly take pictures of 40 fields of view for each group, use the ImageJ program to calculate the average fluorescence intensity (Mean) of p-Tau on motor neurons in each field of view of each group, and then use Graphpad to perform a significant difference statistical analysis and organize the results as a bar chart (see Figure 12 ). The detailed data are shown in the following table:

[0201]

[0202]

[0203] 4.2.3.6 Experimental results

[0204] By statistically analyzing the mean fluorescence intensity of p-Tau in motor neurons in 40 randomly selected fields of view for each group of immunofluorescence sections and analyzing the significant differences, it was found that the conditioned medium of LPS-stimulated BV2 cells (CM-LPS) could cause abnormal elevation of p-Tau on neurons after damaging MN. Alito-300 ng / mL and Alito-600 ng / mL significantly inhibited the abnormal phosphorylation trend of Tau protein, reduced the elevation of p-Tau on motor neurons caused by neuroinflammation, inhibited the spread of inflammation in the nervous system, curbed the formation of neurofibrillary tangles, and had an obvious therapeutic effect on Alzheimer's disease and other neurodegenerative diseases caused by the pathological elevation of p-Tau protein on neurons.

[0205] See the specific results in Figures 11 - 12 , compared with the Ctr control group, the mean fluorescence intensity of p-Tau on MN in the LPS group increased significantly by 16.4% (P #### <0.0001), indicating that p-Tau on MN damaged by the conditioned medium (CM-LPS) formed by LPS-stimulated BV2 cells increased significantly; compared with the LPS model group, the mean fluorescence intensity of p-Tau on damaged MN in Alito-300 ng / mL (ALT-300 group) and Alito-600 ng / mL (ALT-600 group) decreased significantly by 28.1% and 31.6% respectively (P **** <0.0001), indicating that Alito could significantly reduce the hyperphosphorylated Tau protein (p-Tau) on damaged MN and had the effect of reversing the phosphorylation trend of Tau protein.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A protein polymer used as a drug for the preparation of a drug for treating Alzheimer's disease, characterized in that: The production process of the protein polymer comprises: S1) Using ultraviolet light to stimulate mesenchymal stem cells and culture them; S2) Lysing the mesenchymal stem cells, separating and purifying to obtain the protein polymer.

2. The use according to claim 1, characterized in that: The protein polymer satisfies at least one of the following characteristics: 1) During molecular sieve exclusion chromatography, under the conditions of size exclusion chromatography with a flow rate of 0.2 mL / min-0.4 mL / min and PBS as the eluent, the elution volume of the first component peak is 12 mL-13.2 mL, the elution volume of the second component peak is 15.2 mL-17 mL, the elution volume of the third component peak is 17 mL-20 mL, the elution volume of the fourth component peak is 29 mL-31 mL, and the elution volume of the fifth component peak is 31 mL-34 mL; 2) During SDS-PAGE detection, 4%-20% precast gel was used for sample separation and detection. The sample bands were mainly distributed between 11KD and 100KD. The molecular weight ranged from large to small. The first band was between 75KD and 100KD; the second band was between 63KD and 75KD. 3) In reverse phase HPLC detection, the sample volume was 25 μL, the column temperature was 30° C., the flow rate was 0.4 mL / min, the detection wavelength was 280 nm, the mobile phase A was 0.1% TFA aqueous solution, the mobile phase B was 0.1% TFA acetonitrile solution, and the gradient elution conditions were: Under the reversed-phase HPLC conditions, the elution time of the samples after separation is between 2min-20min. The elution time of the components of the first group of samples is between 12min-20min, of which the elution time of characteristic peak 1 is about 13min-17min. The elution time of the components of the second group of samples is 2min-5min, and the elution time of characteristic peak 2 is about 3min.

3. The use according to claim 1, characterized in that: The protein polymer comprises at least the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens OX=9606GN=ALB; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens OX=9606GN=TF; and sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens OX=9606GN=LUM; The weight of the above three proteins accounts for more than 40% of the total weight of the protein polymer.

4. The use according to claim 3, characterized in that: The protein polymer also includes at least one of the following proteins: sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens OX=9606GN=ACTA2 PE=1SV=1; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens OX=9606GN=SERPINA1 PE=1SV=3; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens OX=9606GN=TPM2 PE=1SV=1; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens OX=9606GN=VIM PE=1SV=4; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens OX=9606GN=FN1 PE=1SV=5; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens OX=9606GN=TPM1 PE=1SV=2; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens OX=9606GN=FLNA PE=1SV=4; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homo sapiensOX=9606PE=1SV=2; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens OX=9606GN=TNC PE=1SV=3; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens OX=9606GN=A2M PE=1SV=3; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1OS=Homo sapiens OX=9606GN=ACTBPE=1SV=1; and sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens OX=9606GN=HBG1 PE=1SV=2.

5. The use according to claim 2, characterized in that: The operation of the molecular sieve exclusion chromatography includes: after balancing the superdex 150 molecular sieve 8×500 molecular sieve chromatography column, loading the sample, eluting with PBS, the elution rate is 0.2 mL / min, collecting from the 280 nm ultraviolet absorption value of 4 mAU, and eluting and collecting five components with elution volumes of 12 mL-13.2 mL, 15.2 mL-17 mL, 17 mL-20 mL, 29 mL-31 mL, and 31 mL-34 mL.

6. The use according to claim 1, characterized in that: The ultraviolet irradiation stimulation time of mesenchymal stem cells is 1h-30h; the intensity of ultraviolet irradiation stimulation is 10μW / cm 2 -60μW / cm 2 , the wavelength of ultraviolet light is 290nm-340nm.

7. The use according to claim 6, characterized in that: The culture medium used by the mesenchymal stem cells during the ultraviolet irradiation stimulation is a serum-free MSCs culture medium.

8. The use according to claim 1, characterized in that: The mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.

9. The use according to claim 1, characterized in that: The protein polymer can be prepared into any dosage form of freeze-dried powder, injection, gel, spray, pill, tablet, powder, paste and microneedle preparation.

10. The use according to claim 9, characterized in that: The administration method of the protein polymer when used is selected from one of intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, oral administration and subcutaneous injection.

11. The use according to claim 1, characterized in that: The protein polymer is used to reduce the concentration of beta-amyloid protein Aβ-40 and Aβ-42 in plasma, and / or to increase the concentration ratio of beta-amyloid protein Aβ-42 to Aβ-40 in plasma.

12. The use according to claim 1, characterized in that: The protein polymer is used to inhibit the abnormal phosphorylation of Tau protein on motor neurons in a neuroinflammatory environment.

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