Application of protein polymer in preparation of drug for treating autism

Abstract

Provided is an application of a protein polymer in the preparation of a drug for treating autism. Research has found that protein polymers derived from mesenchymal stem cells stimulated under stress conditions, such as ultraviolet irradiation, have a certain therapeutic effect on autism, providing a possibility for the treatment of autism.

Description

Use of a protein polymer in the preparation of a medicament for treating autism

[0001] The present disclosure claims priority to the Chinese patent application No. 202411379350.3, filed on September 30, 2024, with the Chinese Patent Office, and entitled "Use of a protein polymer in the preparation of a medicament for treating autism", the entire contents of the above application being hereby incorporated by reference in the present disclosure. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and particularly relates to use of a protein polymer in the preparation of a medicament for treating autism. BACKGROUND

[0003] Autism, also known as autism spectrum disorder (ASD), is a neurodevelopmental disorder characterized by impaired social interaction and communication, and stereotyped, repetitive, and restricted behaviors. The worldwide prevalence of autism is about 1%, and the proportion of male patients is higher than that of female patients. However, the symptoms of female patients are more severe than those of male patients. At least 70% of autism patients also have other diseases (Lai M C, Lombardo M V, Baron-Cohen S. Search strategy and selection criteria. Lancet, 2014, 383: 896-910.).

[0004] Autism treatment is usually multidisciplinary. Studies have shown that systematic reinforcement behavior therapy (e.g., strengthening interaction and meaningful communication) brings more benefits. Psychologists and educators, through behavior analysis, systematically take corresponding behavior therapy according to different behavior problems, which helps to manage children at home and school. There is currently a lack of drugs that can change the course of autism and change core symptoms. Drugs targeting different systems involved in autism (oxytocin system, cholinergic system, and glutamate system) are being studied. Antipsychotic drugs can effectively reduce repetitive behaviors in children with autism. The effectiveness of antipsychotic drugs in the treatment of adolescents and adults with autism is still lacking evidence.

[0005] Stem cells are usually cultured under conventional conditions. The inventors found that after culturing stem cells under appropriate stress conditions, protein polymers can be isolated from the lysates of the lysed stress-cultured stem cells. The isolated protein polymers have the efficacy of repairing nervous system function and treating nervous system diseases. SUMMARY

[0006] The purpose of the present application is to overcome at least one deficiency of the prior art and provide use of a protein polymer in the preparation of a medicament for treating autism.

[0007] The technical scheme adopted by the present application is:

[0008] Application of a protein polymer in preparation of a drug for treating autism, the preparation of the protein polymer comprising the following steps:

[0009] S1) culture stem cells and stimulate stem cells using a suitable method to create a stress environment;

[0010] S2) lysing the stem cells cultured under stress conditions, and separating and purifying the protein polymer from the lysate.

[0011] In some examples of application, the protein polymer comprises at least one of the following proteins:

[0012] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0013] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0014] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0015] sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens;

[0016] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0017] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0018] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0019] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0020] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0021] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0022] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens;

[0023] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0024] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0025] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0026] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0027] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0028] In some examples of application, the sum of the mass of the sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer is not less than 40% of the total mass of the protein polymer.

[0029] In some examples of application, the stress condition comprises at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, α-ray, β-ray, γ-ray, ion beam, high CO2, low CO2. Various stress treatments can be performed in sequence, or simultaneously when not conflicting. For example, ultraviolet irradiation can be performed simultaneously with high oxygen or low oxygen, or ultraviolet irradiation stress is performed first, followed by high CO2 stress treatment.

[0030] In some examples of application, the ultraviolet irradiation satisfies at least one of the following conditions:

[0031] The irradiation time is 1h-30h;

[0032] The irradiation intensity is 10μW / cm 2 -100μW / cm 2 ;

[0033] The wavelength of the ultraviolet light is 290nm-340nm.

[0034] In some examples of application, the high temperature is 37.5℃-42.0℃; the low temperature is 2.0℃-25.0℃; the low oxygen is 3.0%-16.0% of oxygen volume content; the high oxygen is 22.0%-100.0% of oxygen volume content; the high pH value is 7.6-10.0; the low pH value is 1.0-6.8; the intensity of the ultrasound is not more than the intensity of ultrasound that the used cells can tolerate; the irradiation intensity or dose of the X-ray, microwave, α-ray, β-ray, γ-ray, ion beam is not more than the intensity or dose that the used cells can tolerate; the electromagnetic field is not more than the intensity that the used cells can tolerate; the high CO2 is 5.1%-15.0% of CO2 content; the low CO2 is 1.0%-4.9% of CO2 content.

[0035] The unknown components in the serum-free stem cell culture medium are less, and the content of exogenous proteins is less, so that the obtained protein polymer has higher safety, and it is also beneficial to simplify the purification operation of the protein polymer. In some examples of application, the culture medium used in the culture is a serum-free stem cell culture medium.

[0036] In some examples of application, the stem cells include but are not limited to at least one of embryonic stem cells, mesenchymal 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.

[0037] In some examples of application, the mesenchymal stem cell sources include but are not limited to umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0038] In some examples of application, the lysis method for the stress-cultured stem cells is a method that can lyse stem cells, including chemical methods and / or physical methods. The method for separating and extracting protein polymers from the lysate includes but is not limited to at least one of chromatography, spectroscopy, volumetry, dialysis, salting-out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.

[0039] In some examples of application, the protein polymer satisfies at least one of the following properties:

[0040] 1) In the molecular sieve exclusion chromatography, under the exclusion chromatography condition of a flow rate of 0.1 mL / min to 0.3 mL / min and an eluent of PBS, the elution volume of the first component is 12.0 mL to 13.2 mL, the elution volume of the second component is 15.2 mL to 17.0 mL, the elution volume of the third component is 17.0 mL to 20.0 mL, the elution volume of the fourth component is 29.0 mL to 31.0 mL, and the elution volume of the fifth component is 31.0 mL to 34.0 mL;

[0041] 2) In the SDS-PAGE detection, using 4% to 20% precast gel for sample separation and detection, the sample bands are mainly distributed in 11 KD to 100 KD, and in order of decreasing molecular weight, the first band is located between 75 KD and 100 KD, and the second band is located between 63 KD and 75 KD;

[0042] 3) In the reverse phase HPLC detection, under the condition of a sample volume of 80 μL to 120 μL, a column temperature of 20°C to 30°C, a flow rate of 0.2 mL / min to 0.8 mL / min, a detection wavelength of 260 nm and / or 280 nm, and a mobile phase of PBS solution, the peak time of the sample after separation is between 10 min and 40 min, the peak time of characteristic peak 1 is 13 min to 17 min, and the peak time of characteristic peak 2 is 20 min to 22 min.

[0043] In some examples of application, the pharmaceutical dosage form is an injection, a nasal spray preparation, or an oral preparation.

[0044] In some examples of application, the pharmaceutical dosage form is a lyophilized powder, an injection, a gel, a pill, a tablet, a powder, or a paste.

[0045] In some examples of application, the administration mode of the pharmaceutical is selected from one of intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

[0046] The present application has the following beneficial effects:

[0047] The inventors have found that the protein polymer derived from mesenchymal stem cells after stimulation by a stress environment, such as ultraviolet irradiation, has a certain therapeutic effect on autism, which provides a possibility for the treatment of autism. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a photograph of the state of the irradiated cells in Example 1.

[0049] Figure 2 is a SDS-PAGE electrophoretogram of the harvested protein in Example 1.

[0050] Figure 3 is an elution curve of Example 2.

[0051] Figure 4 is an elution curve of Example 3.

[0052] Figure 5 is the time points of VPA model establishment and behavioral detection.

[0053] Figure 6 is the result of free social experiment.

[0054] Figure 7 is the result of three-box experiment. DETAILED DESCRIPTION

[0055] The technical solutions of the present application are further illustrated below in combination with examples.

[0056] For the convenience of illustration, mesenchymal stem cells are used as an example in the examples, but in actual application, it is not limited to mesenchymal stem cells, and other stem cells can also produce similar or better stress protein polymers after stress induction. 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.

[0057] Umbilical cord mesenchymal stem cells are used as an example in the examples, and in actual application, mesenchymal stem cell sources include, but are not limited to, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0058] Theoretically, stem cells can be induced to produce corresponding stress product protein polymers after being treated under stress conditions. For the convenience of illustration, ultraviolet irradiation is used as a stress condition to prepare protein polymers in stem cell lysates in the examples. In actual application, other stress conditions can achieve similar or better results. In order to prepare a variety of protein polymers in the lysate of stressed stem cells, other stress conditions can also use at least one of the following stress methods: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, ray, ray, ray, ion beam, high CO2, and low CO2. Various stress treatments can be performed in sequence, or simultaneously when they do not conflict.

[0059] The methods that can be used to isolate protein polymers from cell lysates can theoretically be used to isolate and purify the protein polymers of the present application from stressed cultured stem cells. For ease of illustration, chromatography and size exclusion (molecular sieving) are used in some of the examples of the present application. In actual practice, any method that can isolate and extract the desired protein polymers from the lysate can be used, including but not limited to at least one of chromatography, spectroscopy, size exclusion, dialysis, salting out, organic solvent precipitation, acid extraction, base extraction, ultrafiltration, chromatography, electrophoresis, centrifugation, and the like.

[0060] Example 1: Culture of human umbilical cord mesenchymal stem cells

[0061] Recover one vial of P8 generation HUC-MSC (human umbilical cord mesenchymal stem cells) into a T25 culture flask, and add 2.5 mL of HUC-MSC serum-free medium.

[0062] Irradiate the cells with 300 nm LED ultraviolet light for 6 hours. Carefully remove the supernatant, wash twice with 1 mL of normal saline, add 660 μL of pure water and repeatedly pipette to lyse for 10 minutes, pass through a 0.22 μm filter, and store at 4°C.

[0063] The ultraviolet irradiation conditions are as follows:

[0064] The cell state after irradiation is shown in Figure 1. The protein polymer stock solution was harvested, and the protein concentration was 0.718 mg / mL with a volume of 500 μL. The harvested protein SDS-PAGE results are shown in Figure 2.

[0065] Example 2: Purification of protein polymers by size exclusion and activity detection

[0066] 2-1 Purification of protein polymers by size exclusion:

[0067] Instrument: AKTA explorer

[0068] Chromatography column: Nano micro superdex 150 molecular sieve 8x500, column volume about 30 mL;

[0069] Reagents: 0.1 M NaOH, 20% ethanol, 1x PBS, purified water;

[0070] Ultraviolet absorption wavelength: 280 nm, 260 nm as reference;

[0071] Equilibrate the chromatography column sequence:

[0072] First, flush the chromatography column with 2 CV (i.e., 2 times the column volume) of pure water, then equilibrate the chromatography column with 2 CV of 1x PBS, and set the 280 nm ultraviolet absorption value to zero.

[0073] Sample preparation: repeat the method in Example 1, harvest protein volume of 20 mL. Use ultrafiltration concentration tube with molecular weight cut-off of 3KD to concentrate 20 mL sample to about 600 μL.

[0074] Experimental procedure: after equilibrating the chromatography column, choose 500 μL sample loop to load, loading flow rate of 0.4 mL / min, elute with 1xPBS, flow rate of 0.2 mL / min, elute to peak. Collect protein, start collection from UV absorbance value of 4 mAU, see Figure 3 for elution profile. Elution volume for first component (2# position) to peak is 12 mL ~ 13.2 mL; elution volume for second component (9# position) to peak is 15.2 mL ~ 17 mL; elution volume for third component (12# position) to peak is 17 mL ~ 20 mL; elution volume for fourth component (16# position) to peak is 29 mL ~ 31 mL; elution volume for fifth component (18# position) to peak is 31 mL ~ 34 mL. Conversion shows that elution time for each of the five components is 60 min ~ 66 min, 76 min ~ 85 min, 85 min ~ 100 min, 145 min ~ 155 min, 155 min ~ 170 min, respectively. Lyophilize the elution components at positions 2, 9, 12, 16, and 18 in Figure 3.

[0075] 2-2 Bioactivity detection of protein polymer

[0076] Day 1: cell plating: dilute PC12 low differentiation cells with complete medium (5% FBS + DMEM), 6000 cells / well plated (96 well plate); 37°C, 5% CO2 overnight;

[0077] Day 2: dilute lyophilized sample and sample before purification (about 800 μg / mL) with DMEM + 5% FBS medium;

[0078] Take 30% hydrogen peroxide, dilute 15000 times with DMEM + 5% FBS;

[0079] Hydrogen peroxide treatment: discard 80 μL / well of culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min;

[0080] Untreated control: add 50 μL / well of DMEM + 5% FBS medium as damage treatment control;

[0081] Add 50 μL / well of diluted sample to the wells after hydrogen peroxide treatment;

[0082] Discard the supernatant from the untreated wells, and add 100 μL / well of complete medium as cell growth control (PC);

[0083] Incubate at 37°C for 2 days;

[0084] Day 5: Discard the culture supernatant, add 100 μL / well of complete culture medium, and set up a medium blank control well. Add 10 μL / well of CCK8, and incubate at 37°C for 3.5 h. OD 450 Readings. Calculate the OD value after deducting the medium blank.

[0085] The experimental results are shown in Table 1.

[0086] Table 1: Biological activity experimental data of different samples

[0087] As can be seen from Table 1, in the PC12 cell oxidative damage model, compared with NC, PC, and the medium blank, the unpurified sample has the ability to repair oxidative damage. Further, the purified sample has stronger oxidative damage repair ability, for example, samples 2#, 12#, and 16# all have strong oxidative damage repair ability, and their repair abilities are better than that of the unpurified sample. The repair ability of sample 9# is slightly higher than that of the unpurified sample, and the repair ability of sample 18# is lower than that of the unpurified sample.

[0088] Take the purified 12# sample for SDS-PAGE electrophoresis analysis. From the SDS-PAGE electrophoresis, it can be seen that the sample bands are mainly distributed in 11KD-100KD, and the molecular weight decreases from large to small, the first band is located between 75KD-100KD, and the second band is located between 63KD-75KD.

[0089] Further analysis shows that the protein polymer obtained by purification also includes the following proteins:

[0090] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0091] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0092] The above two are main proteins, accounting for more than 40% of the total mass of the protein polymer. Other proteins include:

[0093] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0094] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0095] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0096] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0097] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0098] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0099] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0100] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0101] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens;

[0102] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0103] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0104] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0105] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0106] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0107] Further analysis showed that the content of serum albumin protein was at least 38% of the total content of protein polymers, and the content of serotransferrin protein was at least 2% of the total content of protein polymers.

[0108] Example 3: HPLC-SEC purification and detection of protein polymer activity

[0109] Based on the difference in hydrophobicity, the protein polymers obtained by stimulating with ultraviolet irradiation can be separated using reverse phase chromatography. Under initial conditions, the organic component in the mobile phase has a low concentration, and the protein polymers have strong hydrophobic interaction with the stationary phase, and are almost completely adsorbed on the stationary phase; when the organic component in the mobile phase reaches a certain concentration, the protein polymers are completely eluted from the stationary phase and no longer interact with the stationary phase. Therefore, a slight change in the organic component of the mobile phase can significantly affect the reverse phase retention behavior of the protein polymers.

[0110] 3-1 HPLC-SEC purification

[0111] Test sample information: the protein polymers prepared by the method of Example 1 were diluted to 1 mg / mL with PBS.

[0112] Mobile phase: PBS;

[0113] Detection conditions: injection volume 100 μL, column temperature 25°C, flow rate 0.4 mL / min, wavelength 280 nm, 260 nm; time: 48 min;

[0114] Collection time range: about 1# 13.3 min-14.5 min; 2# 21.5 min-22 min; 3# 22 min-22.8 min; 4# 23 min-23.7 min; 5# 26.8 min-27.8 min; 6# 27.8 min-28.7 min; 7# 28.7 min-30 min; 8# 30 min-30.6 min; 9# 30.6 min-32 (A280 has no obvious peak) min; 10# 33.8 min-34.8 min.

[0115] The elution curve is shown in Figure 4, and the proteins at positions 1-10 in Figure 4 were freeze-dried and stored.

[0116] 3-2 Detection of biological activity of protein polymers

[0117] Day 1: cell plating: dilute PC12 low differentiation cells with complete culture medium (5% FBS + DMEM) at 6000 cells / well (96-well plate). 37°C, 5% CO2 overnight.

[0118] Day 2: Dilute the freeze-dried sample and the sample before purification (about 800 μg / mL) with DMEM + 5% FBS medium.

[0119] Take 30% hydrogen peroxide, dilute 15000 times with DMEM + 5% FBS.

[0120] Hydrogen peroxide treatment: discard 80 μL / well of culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min.

[0121] Untreated control: add 50 μL / well of DMEM + 5% FBS medium as damage treatment control.

[0122] Add 50 μL / well of diluted sample to the hydrogen peroxide treated well.

[0123] Discard the supernatant of the untreated well, and add 100 μL / well of complete culture medium as a cell growth control (PC).

[0124] Incubate at 37°C for 2 days.

[0125] Day 5: discard the culture supernatant, add 100 μL / well of complete culture medium, and set up a medium blank control well. Add 10 μL / well of CCK8, and incubate at 37°C for 3.5 h. OD 450 Readings. Subtract the medium blank to calculate the OD value.

[0126] The experimental results are shown in Table 2.

[0127] Table 2 Biological activity experiment data of different samples

[0128] As can be seen from Table 2, in the PC12 cell oxidative damage model, the unpurified sample is compared with NC, PC, and the medium blank, and it can be seen that the unpurified sample has oxidative damage repair ability. Further, the purified sample has stronger oxidative damage repair ability, for example, samples 1#, 2#, 7#, 8#, and 10# all have strong oxidative damage repair ability, and their repair ability is better than that of the unpurified sample. The repair ability of sample 5# is lower than that of the sample before purification.

[0129] The technical solutions of the present application are further illustrated below in combination with experiments.

[0130] Establishment of a rat model of autism

[0131] 1. Establishment of a VPA autism rat model

[0132] 1.1 Experimental animals

[0133] The Wistar strain rats (male and female) used for modeling in this study were purchased from Vantianlihua Experimental Animal Company. The rats were raised under the following conditions: light period 7:00-19:00, ambient temperature 23±2℃, humidity 50±10%, and sufficient food was provided to the animals during the culture process.

[0134] After the male and female rats reached the age of 12 weeks, the male and female rats were placed in a mating cage overnight, and the next morning the presence or absence of vaginal plug shedding was checked. The female rats found to have vaginal plug shedding were marked as the first day of pregnancy (first day of embryonic period, E1), and then were individually raised.

[0135] All animal experiments were in accordance with the relevant ethical requirements.

[0136] 1.2 Preparation of sodium valproate solution and modeling administration

[0137] 1.2.1 Preparation of VPA solution

[0138] Accurately weigh 2.0 g of sodium valproate salt (Valproic acid sodium salt, Sigma: P4543) powder, dissolve in 0.9% normal saline (NS), and make up to 10 mL to prepare a VPA solution with a concentration of 200 mg / mL, and store at 4℃ in the dark.

[0139] 1.2.2 Establishment of VPA autism rat model

[0140] Accurately weigh 2.0 g of sodium valproate salt (Valproic acid sodium salt, Sigma: P4543) powder, dissolve in 0.9% normal saline (NS), and make up to 10 mL to prepare a VPA solution with a concentration of 200 mg / mL, and store at 4℃ in the dark. At 12.5 days of pregnancy, the female rats were weighed and injected intraperitoneally with VPA solution (VPA group) or an equal amount of normal saline (NS group) at 450 mg / kg. After the injection was completed, the rats were returned to the feeding cage until the pregnant rats gave birth.

[0141] The offspring were raised in the same cage as the mother rats from birth (P0) to 20 days (P20), and were weaned and raised in separate cages on day 21 (P21). Only male offspring were retained as research subjects in this experiment, and the number of offspring raised in each cage was maintained at 2-6. Only male offspring were selected as research subjects in this study, and were separated from the mother rats at 21 days after birth (PND21) and raised together (3-4 per cage). The time points for VPA model establishment and behavioral testing are shown in Figure 5.

[0142] 2 Administration method

[0143] The administration method is nasal drops.

[0144] 3. Recording of early physical development indexes of offspring

[0145] The various physical development indexes of the early growth of the offspring after birth are recorded, including:

[0146] 1) Body weight: The body weight of the offspring from the first day to the 15th day after birth is recorded.

[0147] 2) Pinna detachment: From the second day after the birth of the offspring, the detachment of the pinna of each mouse is observed and recorded.

[0148] 3) Incisor eruption: From the eighth day after the birth of the offspring, the eruption of the upper and lower incisors of each mouse is observed and recorded.

[0149] 4) Eye opening time: From the ninth day after the birth of the offspring, the eye opening of each mouse is observed and recorded.

[0150] 4. Behavioral tests

[0151] 4.1 Juvenile social behavior experiment of offspring

[0152] According to the literature (The pleasures of play: pharmacological insights into social reward mechanisms, Trends Pharmacol Sci, 2010 Oct; 31(10): 463-9; Oxytocin mediates rodent social memory within the lateral septum and the medial amygdala depending on the relevance of the social stimulus: male juvenile versus female adult conspecifics, Psychoneuroendocrinology, 2013 Jun; 38(6): 916-26), the offspring of the juvenile period (juvenile) are detected for free social behavior. Experimental process: In a quiet and dark environment without interference, prepare a clean test box (39 cm x 25 cm x 20 cm), put the test mice into it, and make them freely explore and adapt to the device for 10 minutes. Then put the model mice of the same sex and age into the mouse box, use an infrared camera to record the activities of the test mice and the model mice within 10 minutes. When reviewing the video, pay attention to the collection of the following evaluation indicators:

[0153] 1) Latency of social interaction: record the time point when the test mice first actively follow and sniff the model mice, and calculate the latency of the test mice initiating normal social activities.

[0154] 2) Duration of normal social interaction (social exploration): record the time when the test mice actively follow and sniff the model mice, and the duration of normal social interaction between the test mice and the model mice. The activity state of the test mice in this behavior is stable, normal, and not anxious.

[0155] 3) Duration of abnormal fighting (fighting): record the time when the test mice abnormally bite and attack the model mice, and the duration of abnormal fighting with the model mice. The activity state of the test mice in this behavior is abnormal and anxious. The fighting process includes two classic behavior actions: pouncing: the test mice fiercely attack, pounce on, and bite the neck and back area of the model mice; pinning: the test mice pin the model mice under their bodies, opposite to the abdominal area of the model mice.

[0156] 4) Duration of attacking and pinning behavior: The duration of both pouncing and pinning actions of the subject mice were recorded.

[0157] 4.2 Three chamber test for the offspring at puberty

[0158] The social behavior and social memory of the subject mice were tested by three-chamber social test at 35-42 days after the birth of the offspring. The experimental device consisted of three organic glass boxes. An infrared camera was used to record the activities of the subject mice in the experimental device. The specific experimental steps were as follows:

[0159] 1) Adaptation stage: The subject mice were first placed in the middle box to freely explore the three boxes of the experimental device for 5 minutes.

[0160] 2) First test stage / social preference test stage: The subject mice were taken out, and a metal cage was placed in one of the two side boxes, containing a wild-type model mouse of the same sex and age as the subject mouse, serving as social stimulus 1 (S1); a clean metal cage was placed in the other side box, serving as non-social stimulus (NS). Then the subject mice were placed in the middle box to freely explore the three-box device for 10 minutes.

[0161] 3) Second test stage / social novelty recognition (short-term social memory) test stage: The subject mice were taken out, and the box containing social stimulus 1 was not changed; a metal cage containing a wild-type model mouse of the same sex and age as the subject mouse was placed in the other side box, serving as social stimulus 2 (S2). Then the subject mice were placed in the middle box to freely explore the three-box device for 10 minutes.

[0162] After the test of each subject mouse, the three-box device and the metal cages placed in the two boxes were wiped with 75% alcohol to eliminate the interference of residual odor on subsequent experiments. When reviewing the video, the following evaluation indicators were collected:

[0163] 1) In the adaptation stage, the duration of the subject mice in the three boxes.

[0164] 2) In the social preference test stage, the duration of the subject mice sniffing the S1 metal cage and the NS metal cage, and the duration of the subject mice in the three boxes.

[0165] 3) In the social novelty recognition test stage, the duration of the subject mice sniffing the S1 metal cage and the S2 metal cage, and the duration of the subject mice in the three boxes.

[0166] 5. Statistics and analysis of data

[0167] 1) First, Shapiro-Wilk method was used to test the normal distribution of the data obtained in the experiment. For data conforming to normal distribution, Independent-Samples T Test, Paired-Samples T Test, One-Way ANOVA, Two-Way ANOVA, Pearson Chi-Square Test, and Pearson Correlation were used for analysis; for non-normal distribution data, Mann-Whitney U Test, Kruskal-Wallis Test, and Spearman’s rho were used for analysis.

[0168] 2) The method of multiple testing includes: LSD, Tukey, Bonferroni.

[0169] 3) For multi-factor models, General Liner Model was used to construct the “main effects” model to analyze the main effects of factors.

[0170] 4) For all statistical analysis, it is statistically significant when two-sided p < 0.05.

[0171] 5) All data were analyzed by SPSS version 26, and all graphs were completed in Prism version 9.

[0172] 6) Except for special instructions, the statistical results of all data are represented by “Mean ± SEM”.

[0173] Experimental results:

[0174] The free social interaction experiment found that, compared with the control group, the VPA model rats had prolonged social initiation latency, shortened social exploration time, and showed a significant decrease in social inclination. Compared with the untreated group, the social initiation latency of the VPA model rats treated with the protein polymer did not shorten, but the social exploration time significantly increased (Figure 6).

[0175] The three-box experiment found that, compared with the control group, the social preference index and social novelty index of the VPA model rats significantly decreased. Compared with the untreated group, the above two indexes of the VPA model rats treated with the protein polymer were significantly improved (Figure 7).

[0176] The above is a further detailed description of the present application, which cannot be regarded as a limitation on the specific implementation of the present application.

[0177] It is obvious to the person skilled in the art that simple modifications and replacements can be made to the present application without departing from the concept of the present application, and all these modifications and replacements shall fall within the scope of the present application.

Claims

1. Use of a protein polymer in the manufacture of a medicament for the treatment of autism, characterised in that, The preparation of the protein polymer comprises the following steps: S1) culturing stem cells and stimulating the stem cells with a suitable method to create a stress environment; S2) lysing the stem cells cultured under stress conditions and isolating and purifying the protein polymer from the lysate.

2. The use according to claim 1, wherein the protein polymer comprises at least one of the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS = Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS = Homo sapiens; sp|P51884|LUM_HUMAN Lumican OS = Homo sapiens; sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS = Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS = Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS = Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS = Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS = Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS = Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS = Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS = Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS = Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS = Homo sapiens; sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS = Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS = Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS = Homo sapiens.

3. Use according to claim 2, characterized in that, The sum of the mass of the sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer accounts for not less than 40% of the total mass of the protein polymer.

4. Use according to claim 1, characterized in that, The stress condition comprises at least one of ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, α-ray, β-ray, γ-ray, ion beam, high CO2, low CO2.

5. Use according to claim 4, characterized in that, The ultraviolet irradiation satisfies at least one of the following conditions: The irradiation time is 1h-30h; The irradiation intensity was 10 μW / cm 2 ~ 100 μW / cm 2 ; The wavelength of the ultraviolet is 290nm-340nm; The high temperature is 37.5℃-42.0℃; the low temperature is 2.0℃-25.0℃; the low oxygen is the oxygen volume content of 3.0%-16.0%; the high oxygen is the oxygen volume content of 22.0%-100.0%; the high pH value is 7.6-10.0; the low pH value is 1.0-6.8; the intensity of the ultrasound does not exceed the ultrasound intensity that the used cell can tolerate; the irradiation intensity or dose of the X-ray, infrared irradiation, microwave, α-ray, β-ray, γ-ray, ion beam does not exceed the intensity or dose that the used cell can tolerate; the electromagnetic field is not more than the intensity that the used cell can tolerate; the high CO2 is the CO2 content of 5.1%-15.0%; the low CO2 is the CO2 content of 1.0%-4.9%.

6. Use according to any one of claims 1 to 5, characterized in that, The culture medium used in the culture is a serum-free stem cell culture medium; and / or the stem cell is at least one selected from the group consisting of embryonic stem cell, mesenchymal stem cell, in vitro induced pluripotent stem cell, hematopoietic stem cell, neural stem cell, bone marrow stem cell, liver stem cell, muscle satellite cell, skin epidermal stem cell, intestinal epithelial stem cell, retinal stem cell, pancreatic stem cell, MUSE cell; preferably, the mesenchymal stem cell source includes umbilical cord-derived human mesenchymal stem cell, bone marrow-derived mesenchymal stem cell, human placenta-derived mesenchymal stem cell, adipose tissue-derived mesenchymal stem cell, skin-derived mesenchymal stem cell.

7. Use according to any one of claims 1 to 6, characterized in that, The method for separating and extracting the protein polymer from the lysate comprises at least one of chromatography, spectroscopy, volumetry, dialysis, salting-out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, centrifugation.

8. Use according to any one of claims 1 to 7, characterized in that, The protein polymer satisfies at least one of the following properties: 1) under the conditions of the size exclusion chromatography, the elution volume of the first component is 12.0 mL-13.2 mL, the elution volume of the second component is 15.2 mL-17.0 mL, the elution volume of the third component is 17.0 mL-20.0 mL, the elution volume of the fourth component is 29.0 mL-31.0 mL, and the elution volume of the fifth component is 31.0 mL-34.0 mL, under the conditions of the flow rate of 0.1 mL / min-0.3 mL / min and the eluent of PBS; 2) under the conditions of the SDS-PAGE detection, the sample separation and detection are performed using 4%-20% precast gel, and the sample bands are mainly distributed in 11 KD-100 KD, wherein the first band is located between 75 KD-100 KD, and the second band is located between 63 KD-75 KD; 3) under the conditions of the reverse phase HPLC detection, the sample volume is 80 μL-120 μL, the column temperature is 20°C-30°C, the flow rate is 0.2 mL / min-0.8 mL / min, the detection wavelength is 260 nm and / or 280 nm, the mobile phase is PBS solution, and the peak time of the sample separation is between 10 min-40 min, wherein the peak time of the characteristic peak 1 is 13 min-17 min, and the peak time of the characteristic peak 2 is 20 min-22 min.

9. Use according to any one of claims 1 to 8, characterized in that, The pharmaceutical dosage form is injection, nasal spray preparation or oral preparation; preferably, the pharmaceutical dosage form is lyophilized powder, injection, gel, pill, tablet, powder or paste.

10. Use according to any one of claims 1 to 8, characterized in that, The administration mode of the drug is selected from one of intrathecal administration, intravenous administration, intrathecal and intravenous combined administration, intracranial administration, nasal spray administration, mucosal administration and oral administration.

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