Application of protein polymer in preparation of medicine for treating autism

Abstract

The invention belongs to the field of medicines, and discloses application of a protein polymer in preparation of a medicine for treating autism. The inventor finds that a protein polymer generated after mesenchymal stem cells are stimulated by a stress environment, such as ultraviolet radiation, has a certain treatment effect on autism, and provides possibility for treatment of autism.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of a protein polymer in the preparation of a drug for treating autism. Background Art

[0002] Autism, also known as autism, is a developmental disorder caused by a disorder of the nervous system. It is characterized by the inability to express normal language and social activities in the early stage, and often involves stereotyped, repetitive and restrictive movements and behaviors. The current incidence rate in the world is about 1%, with a greater proportion of male patients than female patients. However, female patients have more severe symptoms than male patients, and at least 70% of autistic patients usually have other diseases (Lai MC, Lombardo MV, Baron-Cohen S. Search strategy and selection criteria [J]. Lancet, 2014, 383: 896-910.).

[0003] Treatment of autism is usually multidisciplinary, and studies have shown that systematic intensive behavioral therapy (for example, strengthening interactions and meaningful communication) brings more benefits. Psychiatrists and educators, through behavioral analysis, systematically adopt corresponding behavioral treatments according to different behavioral 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 the core symptoms. Drugs targeting different systems involved in autism are being studied (oxytocin system, cholinergic system, and glutamate system). Antipsychotic drugs can effectively reduce repetitive behaviors in children with autism, but there is still a lack of evidence on the effectiveness of treating autism in adolescents and adults.

[0004] Stem cells are generally cultured under conventional conditions. The inventors have found that after culturing stem cells under appropriate stress conditions, multiple proteins can be separated from the lysates of the stress-cultured stem cells. The separated proteins have the effect of repairing nervous system functions and treating nervous system diseases. Summary of the invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide an application of a protein polymer in the preparation of a drug for treating autism.

[0006] The technical solution adopted by the present invention is:

[0007] A use of a protein polymer in preparing a drug for treating autism, wherein the preparation of the protein polymer comprises the following steps:

[0008] S1) Cultivating stem cells and using appropriate methods to create a stress environment to stimulate the stem cells;

[0009] S2) Lysing the stem cells cultured under the stress conditions, and separating and purifying the protein polymer from the lysate.

[0010] In some application examples, the protein polymer includes at least one of the following proteins:

[0011] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0012] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0013] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0014] sp|P62736|ACTA_HUMANActin, aortic smooth muscle OS=Homo sapiens;

[0015] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0016] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0017] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0018] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0019] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0020] sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens;

[0021] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0022] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0023] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0024] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0025] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0026] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0027] In some application examples, the sum of the masses of the sp|P02768|ALBU_HUMAN Serum albumin OS=Homosapiens and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer accounts for no less than 40% of the total mass of the protein polymer.

[0028] In some application examples, the stress condition includes at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH, ​​low pH, 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 successively or simultaneously when they do not conflict. For example, ultraviolet irradiation can be performed simultaneously with high oxygen or low oxygen, or ultraviolet irradiation stress can be performed first, followed by high CO2 stress treatment.

[0029] In some application examples, the ultraviolet irradiation satisfies at least one of the following conditions:

[0030] The irradiation time is 1 to 30 hours;

[0031] Irradiation intensity is 10~100μW / cm 2 ;

[0032] The wavelength of ultraviolet light is 290 to 340 nm.

[0033] In some application examples, the high temperature is 37.5°C to 42.0°C; the low temperature is 2.0-25.0°C; the low oxygen is an oxygen content of 3.0%-16.0% by volume; the high oxygen is an oxygen content of 22.0%-100.0% by volume; the high pH is 7.6-10.0; the low pH is 1.0-6.8; the intensity of the ultrasound does not exceed the ultrasound intensity that the cells can tolerate; the irradiation intensity or dose of the X-rays, microwaves, α-rays, β-rays, γ-rays, and ion beams does not exceed the intensity or dose that the cells can tolerate; the electromagnetic field is an intensity that does not exceed the intensity that the cells can tolerate; the high CO2 is a CO2 content of 5.1%-15.0%; the low CO2 is a CO2 content of 1.0%-4.9%.

[0034] The serum-free stem cell culture medium contains fewer unknown components and less exogenous protein, so the obtained protein polymer has higher safety and is also conducive to simplifying the purification operation of the protein polymer. In some application examples, the culture medium used for the culture is a serum-free stem cell culture medium.

[0035] In some application examples, 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.

[0036] In some application examples, the sources of the mesenchymal stem cells 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, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0037] In some application examples, the lysis method of the stress cultured stem cells is a method that can lyse the stem cells, including chemical methods and / or physical methods. The method of separating and extracting proteins from the lysate includes, but is not limited to, at least one of chromatography, spectroscopy, volumetric method, dialysis, salting out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.

[0038] In some application examples, the protein polymer satisfies at least one of the following properties:

[0039] 1) During molecular sieve exclusion chromatography, under the conditions of size exclusion chromatography with a flow rate of 0.1-0.3 mL / min and PBS as the eluent, the elution volume of the first component peak was 12.0-13.2 mL, the elution volume of the second component peak was 15.2-17.0 mL, the elution volume of the third component peak was 17.0-20.0 mL, the elution volume of the fourth component peak was 29.0-31.0 mL, and the elution volume of the fifth component peak was 31.0-34.0 mL;

[0040] 2) During SDS-PAGE detection, 4-20% precast gel was used for sample separation and detection. The sample bands were mainly distributed in the range of 11KD to 100KD, and the molecular weight was from large to small. The first band was located between 75KD and 100KD; the second band was located between 63KD and 75KD;

[0041] 3) In reverse phase HPLC detection, the sample volume is 80-120 μL, the column temperature is 20-30° C., the flow rate is 0.2-0.8 mL / min, the detection wavelength is 260 nm and / or 280 nm, the mobile phase is PBS solution, and the peak time after sample separation is between 10 and 40 min, among which the peak time of characteristic peak 1 is 13-17 min, and the peak time of characteristic peak 2 is 20-22 min.

[0042] In some application examples, the drug is an injection, a nasal spray preparation or an oral preparation.

[0043] In some application examples, the drug is administered by a method selected from intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

[0044] In some application examples, the drug is in the form of lyophilized powder, injection, gel, pill, tablet, powder or paste.

[0045] The beneficial effects of the present invention are:

[0046] The inventors have found that protein polymers derived from mesenchymal stem cells after being exposed to a stress environment, such as ultraviolet irradiation, have a certain therapeutic effect on autism, providing a possibility for the treatment of autism. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a photograph of the cell state after irradiation in Example 1.

[0048] Figure 2 It is the SDS-PAGE electrophoresis picture of the protein harvested in Example 1.

[0049] Figure 3 This is the elution curve of Example 2.

[0050] Figure 4 It is the SDS-PAGE electrophoresis diagram of sample No. 12 (pure-12#) after purification in Example 3.

[0051] Figure 5 This is the elution curve of Example 3.

[0052] Figure 6 This is the time point for VPA model establishment and behavioral testing.

[0053] Figure 7 It is the result of a free social experiment.

[0054] Figure 8 This is the result of the three-box experiment. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below in conjunction with embodiments.

[0056] For the convenience of explanation, mesenchymal stem cells are used as examples in the embodiments, but in practical applications, they are not limited to mesenchymal stem cells. Other stem cells can also produce similar or better stress proteins 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] In the embodiments, umbilical cord mesenchymal stem cells are used as an example to illustrate the problem. In actual applications, sources of mesenchymal stem cells include but are not limited to umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0058] After the stem cells are subjected to stress conditions, in theory, they can be induced to produce corresponding stress proteins. For the convenience of explanation, in the embodiments, ultraviolet irradiation is used as a stress condition to prepare proteins in stem cell lysates. In practical applications, other stress conditions can achieve similar or better effects. In order to prepare a variety of proteins in stressed stem cell lysates, other stress conditions can also use stress methods such as at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, rays, rays, rays, ion beams, high CO2, and low CO2. Various stress treatments can be carried out one after another, or simultaneously when they do not conflict.

[0059] The method that can be used for separating protein from cell lysate can be used for separating and purifying protein polymers belonging to the present invention from the stem cells of stress culture in theory. For convenience of explanation, chromatography and volumetric method (molecular sieve) are used in some embodiments of the present invention. In practical applications, any method that can separate and extract the target protein from the lysate can also be used, including but not limited to at least one of the methods such as chromatography, spectroscopy, volumetric method, dialysis, salting out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, centrifugation, etc.

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

[0061] Resuscitate a small crystal of P8 HUC-MSC (human umbilical cord mesenchymal stem cells) into a T25 culture flask and add 2.5 ml of Huakai mesenchymal stem cell serum-free culture medium.

[0062] The cells were irradiated with 300nm LED UV for 6h. The cells were carefully cleaned of supernatant, washed twice with 1ml physiological saline, added with 660μl pure water and repeatedly blown for 10min, filtered through a 0.22μm filter membrane, and stored at 4℃.

[0063] UV irradiation conditions are as follows:

[0064] Intermediate medium T25 bottle <![CDATA[UVA(μW / cm 2 )]]> 11.71 <![CDATA[UVB(μW / cm 2 )]]> 26.81 Peak wavelength (nm) 320.4

[0065] The cell status after irradiation is Figure 1 The protein polymer stock solution (also called Aleeto or ALT) was harvested, with a protein concentration of 0.718 mg / ml and a volume of 500 μl. The SDS-PAGE results of the harvested protein are shown in Figure 2 shown.

[0066] Example 2: Molecular sieve purification and activity detection of protein polymers

[0067] 2-1 Molecular sieve purification of protein polymers:

[0068] Instrument: AKTAexplorer

[0069] Chromatographic column: Nano-micron superdex150 molecular sieve 8×500, column volume is about 30mL

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

[0071] UV absorption wavelength: 280nm, 260nm as reference

[0072] Equilibration column sequence:

[0073] The column was first rinsed with 2CV of pure water, and then equilibrated with 1×PBS for 2CV, and the UV absorption value at 280nm was returned to zero.

[0074] Sample preparation: Repeat the method in Example 1 to harvest a total of 20 mL of protein. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD to reduce the 20 mL sample to about 600 μl.

[0075] Experimental process: After balancing the column, select a 500μl sample loop for sample loading, the sample loading flow rate is 0.4mL / min, the 1×PBS elution flow rate is 0.2mL / min, and the elution is carried out to the peak. Collect the protein from the UV absorbance value of 4mAU. For details of the elution curve, see Figure 3 .Pick Figure 3 The proteins at positions 2, 9, 12, 16, and 18 are freeze-dried and stored.

[0076] 2-2 Biological Activity Detection of Protein Polymers

[0077] Day 1: Cell plating: Complete medium (5% FBS + DMEM) was used to dilute PC12 poorly differentiated cells, and 6000 cells / well were plated (96-well plate); incubate at 37°C 5% CO2 overnight;

[0078] The next day: dilute the lyophilized sample and the sample before purification (about 800 μg / ml) with DMEM+5% FBS medium;

[0079] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS;

[0080] Hydrogen peroxide treatment: discard 80 μL / well culture supernatant, add 50 μL / well diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min;

[0081] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as the injury treatment control;

[0082] 50 μL / well of diluted samples were added to the wells treated with hydrogen peroxide;

[0083] The supernatant of untreated wells was discarded, and 100 μL / well of complete medium was added as cell growth control (PC);

[0084] Incubate at 37°C for 2 days;

[0085] Day 5: Discard the culture supernatant, add complete culture medium at 100 μL / well, set up a culture medium blank control well, add CCK8 at 10 μL / well, and incubate at 37°C for 3.5 h. OD 450 Read the sample and calculate the OD value after subtracting the culture medium blank.

[0086] The experimental results are shown in Table 1.

[0087] Table 1 Biological activity experimental data of different samples

[0088]

[0089] As shown in Table 1, in the PC12 cell oxidative damage model, the proteins separated from the above bands all have certain therapeutic effects. Samples 2#, 12#, and 16# have strong oxidative damage repair capabilities, and their repair capabilities are better than those of unpurified samples. The repair ability of sample 18# is lower than that of unpurified samples.

[0090] The purified 12# sample was subjected to SDS-PAGE electrophoresis analysis. The electrophoresis results were as follows: Figure 4 As shown in the figure, the sample bands are mainly distributed in the range of 11KD to 100KD, and the molecular weights are from large to small, with the first band located between 75KD and 100KD, and the second band located between 63KD and 75KD.

[0091] Further analysis showed that the purified protein polymer also included at least one of the following proteins:

[0092] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0093] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0094] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0095] sp|P62736|ACTA_HUMANActin,aortic smooth muscle OS=Homo sapiens;

[0096] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0097] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0098] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0099] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0100] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0101] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0102] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0103] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0104] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0105] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0106] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0107] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

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

[0109] Based on the difference in hydrophobicity, reverse phase chromatography can be used to separate the protein polymers obtained by ultraviolet irradiation stimulation of the present invention. Under initial conditions, the concentration of organic components in the mobile phase is low, the hydrophobic interaction between the protein polymer and the stationary phase is strong, and the protein polymer is almost completely adsorbed by the stationary phase; when the organic components in the mobile phase reach a specific concentration, the protein polymer is completely eluted from the stationary phase and no longer interacts with the stationary phase. Therefore, a very small change in the organic components of the mobile phase can significantly affect the reverse phase retention behavior of the protein polymer.

[0110] 3-1 HPLC-SEC purification

[0111] Test product information: The protein polymer (ALEETO stock solution) prepared by the method of Example 1 was 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] The collection time range is approximately: 1#13.3~14.5min; 2#21.5~22min; 3#22~22.8min; 4#23~23.7min; 5#26.8~27.8min; 6#27.8~28.7min; 7#28.7~30min; 8#30~30.6min; 9#30.6~32 (no obvious peak of A280)min; 10#33.8~34.8min.

[0115] The elution curve is Figure 5 As shown, the proteins at positions 1-10 shown in the figure are taken and freeze-dried for storage.

[0116] 3-2 Biological Activity Detection of Protein Polymers

[0117] Day 1: Cell plating: Complete medium (5% FBS + DMEM) was used to dilute PC12 poorly differentiated cells, and 6,000 cells were plated per well (96-well plate). Incubate at 37°C with 5% CO2 overnight.

[0118] The next day: the lyophilized sample and the sample before purification were diluted with DMEM+5% FBS medium (about 800ug / ml).

[0119] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

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

[0121] Untreated control: 50 μL / well of DMEM+5% FBS medium was added as the injury treatment control.

[0122] 50 μL / well of the diluted sample was added to the wells treated with hydrogen peroxide.

[0123] The supernatant of the untreated wells was discarded, and 100 μL / well of complete culture medium was added as a cell growth control (PC).

[0124] Incubate at 37°C for 2 days.

[0125] Day 5: Discard the culture supernatant, add complete culture medium at 100 μL / well, set up a culture medium blank control well, add CCK8 at 10 μL / well, and incubate at 37°C for 3.5 h. OD 450 Read the sample and calculate the OD value after subtracting the culture medium blank.

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

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

[0128]

[0129]

[0130] As shown in Table 2, in the PC12 cell oxidative damage model, samples 1#, 2#, 7#, 8#, and 10# all have strong oxidative damage repair capabilities, and their repair capabilities are better than those of the samples before purification. The repair ability of sample 5# is lower than that of the sample before purification.

[0131] The technical solution of the present invention is further explained below in conjunction with experiments.

[0132] Establishment of autism rat model

[0133] 1 Establishment of VPA autism rat model

[0134] 1.1 Experimental animals

[0135] The Wistar rats (male and female) used in this study were purchased from Weitonglihua Laboratory Animal Co., Ltd. The rats were kept under the following conditions: light hours from 7:00 to 19:00, ambient temperature of 23±2°C, humidity of 50±10%, and adequate diet was provided to the animals during the culture process.

[0136] After both male and female rats reached 12 weeks of age, they were placed in breeding cages overnight and checked the next morning for vaginal plug shedding. Female rats with vaginal plug shedding were marked as gestational day 1 (embryonic day 1, E1) and subsequently housed individually.

[0137] All animal experiments complied with relevant ethical requirements.

[0138] 1.2 Preparation of sodium valproate solution and administration of model

[0139] 1.2.1 Preparation of VPA solution

[0140] Accurately weigh 2.0 g of valproic acid sodium salt (Sigma: P4543) powder, dissolve it 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 it at 4°C away from light.

[0141] 1.2.2 Establishment of VPA autism rat model

[0142] Accurately weigh 2.0g of valproic acid sodium salt (Sigma: P4543) powder, dissolve it in 0.9% normal saline (NS), and dilute to 10ml to prepare a VPA solution with a concentration of 200mg / ml, and store it at 4℃ in the dark. Weigh the female mice at 12.5 days of pregnancy, and inject VPA solution (VPA group) or an equal amount of normal saline (NS group) intraperitoneally at 450mg / kg. After the injection, return them to the cage until the pregnant mice give birth.

[0143] The offspring were housed in the same cage with the mother mouse from birth (P0) to 20 days (P20), and were weaned and housed in separate cages on the 21st day (P21). Only male offspring were kept as research subjects in this experiment, and the number of offspring in each cage was maintained at 2 to 6. Only male offspring were selected as research subjects in this study, and they were housed separately from the mother mouse on the 21st day (PND21) after birth. Offspring from the same litter were housed together (3-4 / cage). The time points for VPA model establishment and behavioral testing are shown in Figure 6 .

[0144] 2. Method of administration

[0145] The route of administration is nasal drops.

[0146] 3. Recording of early physical development indexes of offspring (Developmental indexes)

[0147] Record the physical development indicators of the offspring in the early stage of growth after birth, including:

[0148] 1) Weight: Record the weight of the offspring from the 1st to the 15th day after birth

[0149] 2) Pinna detachment: Starting from the second day after the offspring are born, the separation of the pinnae on both sides of each mouse is observed and recorded.

[0150] 3) Incisor eruption: Starting from the 8th day after birth, the eruption of the upper and lower incisors of each mouse was observed and recorded.

[0151] 4) Eye opening: Starting from the 9th day after birth, the eye opening of each pup was observed and recorded.

[0152] 4 Behavioral tests

[0153] 4.1 Juvenile social behaviors

[0154] 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 medialamygdala depending on the relevance of the social stimulus: male juvenile versus female adult conspecifics, Psychoneuroendocrinology, 2013 Jun; 38(6): 916-26), this study tested the free social behavior of juvenile offspring. Experimental process: In a quiet, dark environment, prepare a clean test box (39cm×25cm×20cm), put the test mouse in, and let it freely explore and adapt to the device for 10 minutes. Then put the model mouse of the same gender and age in the mouse box, and use an infrared camera to record the activities of the test mouse and the model mouse within 10 minutes. When reviewing the video, pay attention to the collection of the following evaluation indicators:

[0155] 1) Latency of social interaction: Record the time when the test mouse first actively follows and sniffs the model mouse, and calculate the latency of the test mouse to initiate normal social activities.

[0156] 2) Duration of normal social interaction (social exploration): record the time the test mouse actively follows and sniffs the model mouse, as well as the duration of normal social interaction between the test mouse and the model mouse. The activity state of the test mouse in this behavior is stable, normal, and not anxious.

[0157] 3) Duration of abnormal attack and fighting (fighting): record the time when the test rat abnormally pounces and attacks the model rat, as well as the duration of abnormal fighting with the model rat. The activity state of the test rat in this behavior is abnormal and anxious. There are two classic behavioral actions in the fighting process: pouncing: the test rat violently attacks, pounces, and pounces on the neck and back area of ​​the model rat; pinning: the test rat pins the model rat under its body, facing the abdomen of the model rat.

[0158] 4) Duration of attacking behavior: record the duration of the two actions of the test mouse pouncing and pinning the model mouse.

[0159] 4.2 Three chamber test of offspring puberty

[0160] 35 to 42 days after the offspring are born, a three-box social experiment is used to test the social behavior and social memory of the test mice. The experimental device consists of three plexiglass boxes. An infrared camera is used to record the activities of the test mice in the experimental device. The specific experimental steps are as follows:

[0161] 1) Adaptation phase: First, the test mouse was placed in the middle box and allowed to freely explore the three boxes of the experimental device for 5 minutes.

[0162] 2) First test phase / social preference test phase: The test mouse was taken out and a metal cage was placed in one of the two boxes. The cage contained a wild-type model mouse 1 of the same sex and age as the test mouse, which served as social stimulus 1 (S1); a clean, identical metal cage was placed in the other box as a non-social stimulus (NS). The test mouse was then placed in the middle box and allowed to freely explore the three-box apparatus for 10 minutes.

[0163] 3) Second test phase / social novelty recognition (short-term social memory) test phase: The test mouse was taken out, and the box containing social stimulus 1 remained unchanged; a wild-type model mouse 2 of the same sex and age was placed in the empty metal cage in the other box as social stimulus 2 (S2). The test mouse was then placed in the middle box and allowed to freely explore the three-box apparatus for 10 minutes.

[0164] After the test of each subject, wipe the three-box device and the metal cages placed in the two boxes with 75% alcohol to eliminate the interference of residual odor on subsequent experiments. When reviewing the video, pay attention to the collection of the following evaluation indicators:

[0165] 1) During the adaptation phase, the time the test mice stayed in the three boxes.

[0166] 2) During the social preference test phase, the duration of the mice sniffing the S1 metal cage and the NS metal cage, as well as the duration of their stay in the three boxes.

[0167] 3) In the social novelty recognition test phase, the duration of the test mice sniffing the S1 metal cage and the S2 metal cage, as well as the time they stayed in the three boxes.

[0168] 5. Statistics and analysis of data

[0169] 1) First, the Shapiro-Wilk method was used to test the normal distribution of the experimental data. For data that conformed to the 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 data that were not normally distributed, Mann-Whitney U Test, Kruskal-Wallis Test, and Spearman's rho were used for analysis.

[0170] 2) Multiple testing methods include: LSD, Tukey, and Bonferroni.

[0171] 3) For multi-factor models, the General Linear Model (GLM) was used to construct a “main effects” model to analyze the main effects of the factors.

[0172] 4) For all statistical analyses, a two-sided p < 0.05 was considered statistically significant.

[0173] 5) All data were analyzed using SPSS version 26, and all graphs were drawn in Prism version 9.

[0174] 6) Unless otherwise specified, all statistical results are expressed as “mean ± standard error (Mean ± SEM)”.

[0175] Experimental results:

[0176] The free social experiment found that compared with the control group, the VPA model rats had a longer latency to initiate social interaction and a shorter social exploration time, showing a significant weakening of social tendency. Compared with the untreated group, the VPA model rats treated with Alito did not have a shorter social latency, but their social exploration time increased significantly ( Figure 7 ).

[0177] The three-box experiment found that the social preference index and social novelty index of VPA model rats decreased significantly compared with the control group. The above two indexes of VPA model rats treated with Alito were significantly improved compared with the untreated group ( Figure 8 ).

[0178] The above is a further detailed description of the present invention, which should not be regarded as a limitation to the specific implementation of the present invention.

[0179] For ordinary technicians in the technical field to which the present invention belongs, simple deduction or substitution without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Use of a protein polymer in the preparation of a drug for treating autism, characterized in that: The preparation of the protein polymer comprises the following steps: S1) Cultivating stem cells and using appropriate methods to create a stress environment to stimulate the stem cells; S2) Lysing the stem cells cultured under the stress conditions, and separating 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_HUMANActin, aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMANAlpha-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-1chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

3. The use according to claim 2, characterized in that: The sum of the masses of the sp|P02768|ALBU_HUMAN SerumalbuminOS=Homo sapiens and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer accounts for no less than 40% of the total mass of the protein polymer.

4. The use according to claim 1, characterized in that: The stress conditions include at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, α rays, β rays, γ rays, ion beams, high CO2, and low CO2.

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

6. The 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 cells are selected from 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; preferably, the source of the mesenchymal stem cells can be umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

7. The use according to any one of claims 1 to 6, characterized in that: The method for lysing the stress-cultured stem cells is a method that can lyse the stem cells, and the method for separating and extracting proteins from the lysate includes at least one of chromatography, spectroscopy, volumetric method, dialysis, salting out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.

8. The use according to any one of claims 1 to 7, 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 0.1-0.3 mL / min and PBS as the eluent, the elution volume of the first component peak was 12.0-13.2 mL, the elution volume of the second component peak was 15.2-17.0 mL, the elution volume of the third component peak was 17.0-20.0 mL, the elution volume of the fourth component peak was 29.0-31.0 mL, and the elution volume of the fifth component peak was 31.0-34.0 mL; 2) During SDS-PAGE detection, 4-20% precast gel was used for sample separation and detection. The sample bands were mainly distributed in the range of 11KD to 100KD, and the molecular weight was from large to small. The first band was located between 75KD and 100KD; the second band was located between 63KD and 75KD; 3) In reverse phase HPLC detection, the sample volume is 80-120 μL, the column temperature is 20-30° C., the flow rate is 0.2-0.8 mL / min, the detection wavelength is 260 nm and / or 280 nm, the mobile phase is PBS solution, and the peak time after sample separation is between 10 and 40 min, among which the peak time of characteristic peak 1 is 13-17 min, and the peak time of characteristic peak 2 is 20-22 min.

9. The use according to any one of claims 1 to 8, characterized in that: The drug is an injection, a nasal spray preparation or an oral preparation; or the drug is administered by a method selected from intrathecal administration, intravenous administration, a combination of intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

10. The use according to claim 9, characterized in that: The dosage form of the drug is lyophilized powder, injection, gel, pill, tablet, powder or paste.

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