Terahertz electromagnetic wave stress cultured protein composition as well as preparation method and application thereof

Abstract

The invention relates to a protein composition for terahertz electromagnetic wave stress culture as well as a preparation method, a detection method and application of the protein composition. According to the invention, the frequency and stress time of terahertz electromagnetic waves are scientifically selected, and the protein composition obtained by culturing the stress stem cells has good cell oxidative damage repair and nerve damage repair effects, can be used for treating nerve cell function change or inflammation related diseases, and has a remarkable effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a protein composition cultured under terahertz electromagnetic wave stress, its preparation method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess self-replication and multi-lineage differentiation potential, and are widely found in tissues such as bone marrow, adipose tissue, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They are characterized by their wide availability, lack of need for matching, low infection rate, strong differentiation potential, high proliferation capacity, and convenient collection. MSCs can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN), exhibiting functions including regulating cell growth, apoptosis, and differentiation, antiviral activity, immune regulation, and tissue repair. There are reports of their use in treating acute lung injury, severe pneumonia, and acute respiratory distress syndrome. However, the need for cryopreservation during culture, expansion, storage, transportation, and use, and the requirement that cell viability be maintained for ≤12 hours, limits their application.

[0003] The terahertz (THz) band lies in the transition region from electronics to photonics, also known as the "terahertz gap" in the electromagnetic spectrum. The technological advantages of using terahertz electromagnetic waves to induce stress proteins in stem cells include: first, it is non-ionizing and physically safe; second, it is non-invasive and leaves no chemical residue; third, it has high specificity and controllability. Terahertz waves may resonate with specific vibrational modes of biomolecules such as proteins and DNA. Precisely controlling the frequency, power, and irradiation time of terahertz waves allows for "precise targeting" of specific signaling pathways or protein expression, making it a more selective tool than other stress methods (such as whole-body heating); fourth, it may activate unique protective pathways. Simulating thermal stress, it can activate some unique, yet-to-be-fully-discovered cell protection mechanisms by influencing cell membrane potential, hydration layer, or cytoskeleton, producing superior "pretreatment" or "enhancement" effects. Stem cells that have undergone terahertz "pretreatment" may contain more stress proteins, resulting in a higher survival rate and stronger function in the harsh microenvironment of transplanted lesions (such as ischemia, inflammation, and hypoxia).

[0004] Cells and microorganisms, when subjected to external stimuli and exogenous stressors (including temperature, electric current, radiation, and chemicals), will induce the production of stress proteins in response to stress. Current technology has not yet provided specific reports on the use of terahertz electromagnetic waves to induce the production of stress proteins in stem cells, or on the extraction of these protein compositions for applications such as oxidative damage repair or nerve damage repair. Summary of the Invention

[0005] The purpose of this invention is to provide a protein composition for terahertz electromagnetic wave stress culture. The proteins constituting the protein composition are selected from serum albumin, serum transferrin, lumican, actin, alpha-1 antitrypsin, tropomyosin beta chain, vimentin, fibronectin, tropomyosin alpha-1 chain, filamin-A, immunoglobulin gamma-1 heavy chain, tenascin, alpha-2 macroglobulin, human cytoplasmic actin 1, and hemoglobin γ-1 subunit. gamma-1, complement 3 (ComplementC3), or any combination thereof.

[0006] In a preferred embodiment of the present invention, the serum albumin content in the protein composition is greater than 30% by mass percentage, preferably 35-55%, and more preferably 40-50%.

[0007] In a preferred embodiment of the present invention, the serum transferrin content in the protein composition is greater than 5% by mass percentage, preferably 5-15%, and more preferably 8-13%.

[0008] In a preferred embodiment of the present invention, the protein composition contains 1%-6% α-1-antitrypsin, preferably 2-5%, by mass percentage.

[0009] In a preferred embodiment of the present invention, the immunoglobulin gamma-1 heavy chain contained in the protein composition is 1%-6% by mass, preferably 2-5%.

[0010] In a preferred embodiment of the present invention, the mass ratio of serum albumin: serum transferrin: α-1-antitrypsin: immunoglobulin γ-1 heavy chain in the protein composition is 10-20:1-10:1-5:1, preferably 12-18:2-5:1-3:1.

[0011] In a preferred embodiment of the present invention, the protein composition is a protein composition from stem cell lysates cultured under terahertz electromagnetic wave stress for a certain period of time, or a protein composition from stem cell lysates cultured under terahertz electromagnetic wave stress for a certain period of time, and the purified protein composition comprises at least one or a combination 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=Homosapiens;

[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 a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD.

[0029] In a preferred embodiment of the present invention, the molecular weight distribution of the protein in the protein composition is selected from any one of 75KD-100KD and 63KD-75KD or a combination thereof.

[0030] In a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD, wherein, from largest to smallest molecular weight, the molecular weight of the first protein detected is between 75KD and 100KD, and the molecular weight of the second protein is between 63 and 75KD.

[0031] In a preferred embodiment of the present invention, the protein composition contains a protein with the highest content having a molecular weight between 63 and 75 kDa.

[0032] In a preferred embodiment of the present invention, the preparation of the protein composition cultured under terahertz electromagnetic wave stress includes the following steps:

[0033] S-1, stem cells are cultured under terahertz electromagnetic wave stress, wherein the terahertz stress culture conditions are: the frequency of the terahertz electromagnetic wave is 0.1-10 THz, and the culture is carried out for 0.5-48 hours.

[0034] S-2 involves lysing the stress-cultured stem cells obtained in S-1 and separating and extracting the resulting stress-cultured protein composition.

[0035] In a preferred embodiment of the present invention, in step S-1, the initial concentration of the stem cells is (0.2-1) × 10⁻⁶ cells. 6 The concentration of cells / mL is preferably (0.25-0.6)×10⁻⁶. 6 per mL.

[0036] In a preferred embodiment of the present invention, the stem cells are selected from any one of mesenchymal stem cells, in vitro induced pluripotent stem cells, and MUSE cells.

[0037] In a preferred embodiment of the present invention, the mesenchymal stem cells are selected from any one of umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0038] In the preferred embodiment of the present invention, in step S-1, the frequency of the terahertz electromagnetic wave used for stem cell stress culture is 0.1-10THz, preferably 1-10THz, and more preferably 1-4THz.

[0039] In the preferred embodiment of the present invention, in step S-1, the stress culture time of stem cells under terahertz electromagnetic waves is 1-48h, preferably 1-36h, more preferably 8-18h, and even more preferably 8h, 12h, 16h, or 18h.

[0040] In a preferred embodiment of the present invention, in step S-1, the wavelength range of the terahertz electromagnetic wave is 30 to 3000 μm.

[0041] In a preferred embodiment of the present invention, the preparation process of the protein composition includes the following steps:

[0042] S-1: Cultivate stem cells and use terahertz electromagnetic waves to create a stress environment;

[0043] S-2: Lyse the stress-cultured stem cells and use the lysate directly as a drug;

[0044] S-3: The protein composition is obtained by separating and purifying the lysate of stress-cultured stem cells using an appropriate method, and the separated and purified protein composition is used as a drug;

[0045] The terahertz stress environment during stem cell culture is electromagnetic waves with a terahertz intensity of 0.1-10 THz and a wavelength of 30-3000 μm; the terahertz electromagnetic wave stress culture time for the stem cells is 0.5-48 h.

[0046] In a preferred embodiment of the present invention, in step S-2, the pyrolysis is selected from any one or a combination of ultrapure water swelling, repeated freeze-thaw cycles, ultrasonic pyrolysis, chemical pyrolysis, urea pyrolysis, and guanidine hydrochloride pyrolysis.

[0047] In a preferred embodiment of the present invention, in step S-2, the ultrapure water swelling and lysis is performed by adding an appropriate amount of ultrapure water to swell and lyse the cells, repeatedly blowing and lysing the stem cells for 5-10 minutes, and then filtering with a 0.22 μm filter membrane to obtain a filtrate containing a protein composition.

[0048] In a preferred embodiment of the present invention, the protein composition cultured under terahertz electromagnetic wave stress is purified.

[0049] In a preferred embodiment of the present invention, the purification method of the protein composition is selected from any one or a combination of molecular sieve chromatography and high-performance liquid chromatography (HPLC).

[0050] In a preferred embodiment of the present invention, the protein composition is purified using the following method:

[0051] When purifying using size exclusion chromatography with molecular sieves, under size exclusion chromatography conditions of a flow rate of 0.1–0.3 mL / min and elution buffer of PBS, the peak volume of the protein composition should be at least one peak before the elution volume reaches 0.1–2 column volumes; and / or,

[0052] When using HPLC purification, under reversed-phase HPLC conditions with a sample loading volume of 60–80 µL, a column temperature of 25–40 °C, a flow rate of 0.5–1 mL / min, a detection wavelength of 220–280 nm, mobile phase A being TFA aqueous solution, mobile phase B being TFA acetonitrile solution, and an elution time of 6–150 min, the peak elution time after sample separation is between 10 and 40 min.

[0053] In a preferred embodiment of the present invention, the molecular sieve exclusion chromatography operation includes: equilibrating a Superdex 150 molecular sieve 8×500 molecular sieve chromatography column, loading the sample, eluting with PBS, preferably at a elution rate of 0.2–0.4 mL / min, collecting protein fractions from a 280 nm UV absorbance of 4 mAU up to 1.2 column volumes.

[0054] In a preferred embodiment of the present invention, the protein composition obtained by molecular sieve purification has a main band located between 11KD and 100KD. Specifically, the molecular weight decreases as follows: the first band is located between 75KD and 100KD; the second band is located between 63 and 75KD; the third band is located between 48KD and 63KD; and the fourth band is located around 48KD.

[0055] In a preferred embodiment of the present invention, the HPLC purification operation uses a reversed-phase liquid chromatography column, with mobile phase A being an aqueous solution of 0.1% TFA and mobile phase B being a 71.4% acetonitrile solution of 0.075% TFA.

[0056] Another objective of this invention is to provide a method for preparing a protein composition under terahertz electromagnetic stress culture, comprising: stem cell expansion; stress culture of stem cells in a terahertz-conditioned stimulation medium; collection of the stress-cultured stem cells for stem cell lysis; and separation and purification of the protein composition from the lysate.

[0057] In a preferred embodiment of the present invention, the preparation of the protein composition cultured under terahertz electromagnetic wave stress includes the following steps:

[0058] S-1: Stem cells are cultured under terahertz electromagnetic wave stress, wherein the terahertz stress culture conditions are: the frequency of the terahertz electromagnetic wave is 0.1-10 THz, and the culture is carried out for 0.5-48 hours.

[0059] S-2: Lyse the stress-cultured stem cells obtained in S-1 and separate and extract the resulting stress-cultured protein composition.

[0060] In a preferred embodiment of the present invention, in step S-1, the initial concentration of the stem cells is (0.2-1) × 10⁻⁶ cells. 6 The concentration of cells / mL is preferably (0.25-0.6)×10⁻⁶. 6 per mL.

[0061] In a preferred embodiment of the present invention, the stem cells are selected from any one of mesenchymal stem cells, in vitro induced pluripotent stem cells, and MUSE cells.

[0062] In a preferred embodiment of the present invention, the mesenchymal stem cells are selected from any one of umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0063] In the preferred embodiment of the present invention, in step S-1, the frequency of the terahertz electromagnetic wave used for stem cell stress culture is 0.1-10THz, preferably 1-10THz, and more preferably 1-4THz.

[0064] In the preferred embodiment of the present invention, in step S-1, the stress culture time of stem cells under terahertz electromagnetic waves is 1-48h, preferably 1-36h, more preferably 8-18h, and even more preferably 8h, 12h, 16h, or 18h.

[0065] In a preferred embodiment of the present invention, in step S-1, the wavelength range of the terahertz electromagnetic wave is 30 to 3000 μm.

[0066] In a preferred embodiment of the present invention, the preparation process of the protein composition includes the following steps:

[0067] S1) Cultivate stem cells and use terahertz electromagnetic waves to create a stress environment;

[0068] S2) Lyse the stress-cultured stem cells and use the lysate directly as a drug;

[0069] S3) The protein composition is obtained by separating and purifying the lysate of stress-cultured stem cells using an appropriate method, and the separated and purified protein composition is used as a drug;

[0070] The terahertz stress environment during stem cell culture is electromagnetic waves with a terahertz intensity of 0.1-10 THz and a wavelength of 30-3000 μm.

[0071] The terahertz electromagnetic stress culture time for the stem cells was 0.5–48 h.

[0072] In a preferred embodiment of the present invention, in step S-2, the pyrolysis is selected from any one or a combination of ultrapure water swelling, repeated freeze-thaw cycles, ultrasonic pyrolysis, chemical pyrolysis, urea pyrolysis, and guanidine hydrochloride pyrolysis.

[0073] In a preferred embodiment of the present invention, in step S-2, the ultrapure water swelling and lysis is performed by adding an appropriate amount of ultrapure water to swell and lyse the cells, repeatedly blowing and lysing the stem cells for 5-10 minutes, and then filtering with a 0.22 μm filter membrane to obtain a filtrate containing a protein composition.

[0074] In a preferred embodiment of the present invention, the protein composition cultured under terahertz electromagnetic wave stress is purified.

[0075] In a preferred embodiment of the present invention, the purification method of the protein composition is selected from any one or a combination of molecular sieve chromatography and high-performance liquid chromatography (HPLC).

[0076] In a preferred embodiment of the present invention, the protein composition is purified using the following method:

[0077] When purifying using size exclusion chromatography with molecular sieves, under size exclusion chromatography conditions of a flow rate of 0.1–0.3 mL / min and elution buffer of PBS, the peak volume of the protein composition should be at least one peak before the elution volume reaches 0.1–2 column volumes; and / or,

[0078] When using HPLC purification, under reversed-phase HPLC conditions with a sample loading volume of 60–80 µL, a column temperature of 25–40 °C, a flow rate of 0.5–1 mL / min, a detection wavelength of 220–280 nm, mobile phase A being TFA aqueous solution, mobile phase B being TFA acetonitrile solution, and an elution time of 6–150 min, the peak elution time after sample separation is between 10 and 40 min.

[0079] In a preferred embodiment of the present invention, the molecular sieve exclusion chromatography operation includes: equilibrating a Superdex 150 molecular sieve 8×500 molecular sieve chromatography column, loading the sample, eluting with PBS at a preferred elution rate of 0.2–0.4 mL / min, collecting protein fractions from a 280 nm UV absorbance of 4 mAU until a column volume of 1.2 is reached.

[0080] In a preferred embodiment of the present invention, the protein composition obtained by molecular sieve purification has a main band located between 11KD and 100KD. Specifically, the molecular weight decreases as follows: the first band is located between 75KD and 100KD; the second band is located between 63 and 75KD; the third band is located between 48KD and 63KD; and the fourth band is located around 48KD.

[0081] In a preferred embodiment of the present invention, the HPLC purification operation uses a reversed-phase liquid chromatography column, with mobile phase A being an aqueous solution of 0.1% TFA and mobile phase B being a 71.4% acetonitrile solution of 0.075% TFA.

[0082] In the preferred embodiment of the present invention, during the HPLC purification operation, the elution time of characteristic peak 1 is 13-17 min, the elution time of the second group of sample components is 2-5 min, and the elution time of characteristic peak 2 is 20-22 min.

[0083] In a preferred embodiment of the present invention, the HPLC purification operation uses an XBridge ProteinBEH C4 column, 300 Å, 3.5 μm, 4.6 mm * 150 mm.

[0084] In a preferred embodiment of the present invention, the operating conditions for the HPLC purification are as follows:

[0085] .

[0086] In a preferred embodiment of the present invention, the protein composition cultured under terahertz electromagnetic wave stress comprises proteins selected from serum albumin, serum transferrin, lumican, actin, alpha-1 antitrypsin, tropomyosin beta chain, vimentin, fibronectin, tropomyosin alpha-1 chain, filamin-A, immunoglobulin gamma-1 heavy chain, tenascin, alpha-2 macroglobulin, human cytoplasmic actin 1, and hemoglobin γ-1 subunit. gamma-1, complement 3 (ComplementC3), or any combination thereof.

[0087] In a preferred embodiment of the present invention, the serum albumin content in the protein composition is greater than 30.0% by mass percentage, preferably 35-55%, and more preferably 40-50%.

[0088] In a preferred embodiment of the present invention, the serum transferrin content in the protein composition is greater than 5.0% by mass percentage, preferably 5-15%, and more preferably 8-13%.

[0089] In a preferred embodiment of the present invention, the protein composition contains 1%-6% α-1-antitrypsin, preferably 2-5%, by mass percentage.

[0090] In a preferred embodiment of the present invention, the immunoglobulin gamma-1 heavy chain contained in the protein composition is 1%-6% by mass, preferably 2-5%.

[0091] In a preferred embodiment of the present invention, the mass ratio of serum albumin: serum transferrin: α-1-antitrypsin: immunoglobulin γ-1 heavy chain in the protein composition is 10-20:1-10:1-5:1, preferably 12-18:2-5:1-3:1.

[0092] In a preferred embodiment of the present invention, the protein composition is a protein composition of stem cell lysate after terahertz electromagnetic wave stress culture for a certain period of time or a protein composition of stem cell lysate after terahertz electromagnetic wave stress culture for a certain period of time.

[0093] The purified protein composition contains at least one or a combination of the following proteins:

[0094] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0095] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0096] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0097] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0098] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0099] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0100] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0101] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0102] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0103] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0104] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homosapiens;

[0105] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0106] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0107] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0108] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0109] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0110] In a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD.

[0111] In a preferred embodiment of the present invention, the molecular weight distribution of the protein in the protein composition is selected from any one of 75KD-100KD and 63KD-75KD or a combination thereof.

[0112] In a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD, wherein, from largest to smallest molecular weight, the molecular weight of the first protein detected is between 75KD and 100KD, and the molecular weight of the second protein is between 63 and 75KD.

[0113] In a preferred embodiment of the present invention, the protein composition contains a protein with the highest content having a molecular weight between 63 and 75 kDa.

[0114] Another object of the present invention is to provide a method for detecting protein compositions cultured under terahertz electromagnetic stress, selected from any one or a combination of the following methods:

[0115] 1) Size exclusion chromatography: Under size exclusion chromatography conditions with a flow rate of 0.1–0.3 mL / min and PBS as the eluent, the peak volume of the protein composition should be at least one peak before the peak volume is 0.1–2 times the column volume; or

[0116] 2) HPLC detection method: Sample loading volume 60-80 µL, column temperature 25-40℃, flow rate 0.5-1 mL / min, detection wavelength 220-280 nm, mobile phase A is TFA aqueous solution, mobile phase B is TFA acetonitrile solution, elution time is 6-150 min under reversed-phase HPLC conditions, and the peak time after sample separation is between 10-40 min.

[0117] In a preferred embodiment of the present invention, the molecular sieve exclusion chromatography operation includes: equilibrating a Superdex 150 molecular sieve 8×500 molecular sieve chromatography column, loading the sample, eluting with PBS at a preferred elution rate of 0.2–0.4 mL / min, collecting protein fractions from a 280 nm UV absorbance of 4 mAU until a column volume of 1.2 is reached.

[0118] In a preferred embodiment of the present invention, the protein composition obtained by molecular sieve purification has a main band located between 11KD and 100KD. Specifically, the molecular weight decreases as follows: the first band is located between 75KD and 100KD; the second band is located between 63 and 75KD; the third band is located between 48KD and 63KD; and the fourth band is located around 48KD.

[0119] In a preferred embodiment of the present invention, the HPLC purification operation uses a reversed-phase liquid chromatography column, with mobile phase A being an aqueous solution of 0.1% TFA and mobile phase B being a 71.4% acetonitrile solution of 0.075% TFA.

[0120] In a preferred embodiment of the present invention, the protein composition cultured under terahertz electromagnetic wave stress comprises proteins selected from serum albumin, serum transferrin, lumican, actin, alpha-1 antitrypsin, tropomyosin beta chain, vimentin, fibronectin, tropomyosin alpha-1 chain, filamin-A, immunoglobulin gamma-1 heavy chain, tenascin, alpha-2 macroglobulin, human cytoplasmic actin 1, and hemoglobin γ-1 subunit. gamma-1, complement 3 (ComplementC3), or any combination thereof.

[0121] In a preferred embodiment of the present invention, the serum albumin content in the protein composition is greater than 30.0% by mass percentage, preferably 35-55%, and more preferably 40-50%.

[0122] In a preferred embodiment of the present invention, the serum transferrin content in the protein composition is greater than 5.0% by mass percentage, preferably 5-15%, and more preferably 8-13%.

[0123] In a preferred embodiment of the present invention, the protein composition contains 1%-6% α-1-antitrypsin, preferably 2-5%, by mass percentage.

[0124] In a preferred embodiment of the present invention, the immunoglobulin gamma-1 heavy chain contained in the protein composition is 1%-6% by mass, preferably 2-5%.

[0125] In a preferred embodiment of the present invention, the mass ratio of serum albumin: serum transferrin: α-1-antitrypsin: immunoglobulin γ-1 heavy chain in the protein composition is 10-20:1-10:1-5:1, preferably 12-18:2-5:1-3:1.

[0126] In a preferred embodiment of the present invention, the protein composition is a protein composition of stem cell lysate after terahertz electromagnetic wave stress culture for a certain period of time or a protein composition of stem cell lysate after terahertz electromagnetic wave stress culture for a certain period of time.

[0127] The purified protein composition contains at least one or a combination of the following proteins:

[0128] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0129] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0130] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0131] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0132] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0133] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0134] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0135] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0136] sp|P09493|TPM1 _HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0137] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0138] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homosapiens;

[0139] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0140] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0141] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0142] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0143] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0144] In a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD.

[0145] In a preferred embodiment of the present invention, the molecular weight distribution of the protein in the protein composition is selected from any one of 75KD-100KD and 63KD-75KD or a combination thereof.

[0146] In a preferred embodiment of the present invention, the molecular weight of the protein in the protein composition is mainly between 11KD and 100KD, wherein, from largest to smallest molecular weight, the molecular weight of the first protein detected is between 75KD and 100KD, and the molecular weight of the second protein is between 63 and 75KD.

[0147] In a preferred embodiment of the present invention, the protein composition contains a protein with the highest content having a molecular weight between 63 and 75 kDa.

[0148] Another object of the present invention is to provide the use of the protein composition cultured under terahertz electromagnetic stress of the present invention in the preparation of drugs for oxidative damage repair and / or nerve damage repair.

[0149] In a preferred embodiment of the present invention, the application includes the preparation of drugs for treating neurodegenerative diseases, nervous system diseases, stroke, arthritis, enteritis, post-traumatic recovery, autism, depression, and pulmonary fibrosis.

[0150] In the preferred embodiment of the present invention, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), cerebrovascular disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebrovascular disease, and different types of spinocerebellar ataxia (SCA).

[0151] This invention uses liquid chromatography-mass spectrometry (LC-MS) to determine protein components and content. The specific detection conditions are as follows:

[0152] .

[0153] Mass spectrometry data were acquired using Xcalibur software, and the resulting data were analyzed using Spectronaut software. The Uniport Human protein database (containing 20,342 protein sequences) was used to search for proteins and obtain identification information.

[0154] Compared with the prior art, the beneficial effects of the present invention are:

[0155] 1. This invention scientifically selects the frequency and stress time of terahertz electromagnetic waves, and the protein composition obtained by stress stem cell culture has good cell oxidative damage repair and nerve damage repair effects. It can be used to treat nerve cell function changes or inflammation-related diseases, and has significant effects.

[0156] 2. The protein composition of the present invention is easy to store and transport after freeze-drying, has good stability, and is suitable for industrial production. Attached Figure Description

[0157] Figure 1 Photographs of cell states before and after terahertz electromagnetic wave stress culture: a) 0h stress culture, b) 12h stress culture;

[0158] Figure 2 SDS-PAGE results of protein compositions obtained from terahertz electromagnetic stress culture;

[0159] Figure 3 Photographs of cell states before and after terahertz electromagnetic wave stress culture: a) 0h stress culture, b) 8h stress culture;

[0160] Figure 4 SDS-PAGE results of the purified protein composition of this invention;

[0161] Figure 5Experimental Example 1: Study on the effect of the protein composition of the present invention on the repair of cellular oxidative damage;

[0162] Figure 6 Experimental Example 2: Study on the effect of the protein composition of the present invention on the repair of cellular oxidative damage;

[0163] Figure 7 Experimental Example 3: Study on the effect of the protein composition of the present invention on the repair of cellular nerve damage.

[0164] Example 1: Preparation of the protein composition of the present invention and study on the effects of terahertz stress on cell morphology and proteins.

[0165] Human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) were cultured in 2L of HK-G050 (PRF) 3D medium from Tangyi Huike Biotechnology, with a total cell count of approximately 5 × 10⁻⁶ cells. 8 Each sample was dispensed into four T225 vials, each containing 500 mL of cell culture medium (containing 1.25 × 10⁶ cells). 8 Cells in each vial were subjected to 1 THz terahertz electromagnetic stress. One vial was randomly selected from those cultured for 0 h and 12 h under terahertz electromagnetic stress for staining and observation (using a 40x magnifying glass). The results are as follows: Figure 1 a, Figure 1 As shown in b, the contents of cells decrease after stress.

[0166] Cells cultured under terahertz electromagnetic stress for 0 h and 12 h were respectively swelled and lysed with an appropriate amount of ultrapure water. The filtrate, containing the protein composition, was filtered through a 0.22 μm filter membrane and analyzed using conventional SDS-PAGE gel electrophoresis. The results are shown in the figure below. Figure 2 .

[0167] Example 2: Preparation of the protein composition of the present invention and study on the effect of terahertz stress on protein concentration.

[0168] One small cell line of P8 generation HUC-MSCs was revived and added to 16 T25 culture flasks, with 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium added to each flask. The 16 flasks of cells were cultured under different terahertz electromagnetic wave conditions and divided into 16 groups, as detailed in Table 1.

[0169] Table 1

[0170]

[0171] Taking group 6 (cultured for 8 hours under 1THz terahertz electromagnetic wave conditions) as an example, cell morphology images after 0h and 8h of terahertz stress culture are shown below. Figure 3 a and Figure 3b. It can be seen that terahertz electromagnetic wave culture has an impact on cell morphology, causing certain effects and stress on the cells, which in turn can lead to the production of stress proteins in the cells under this stress environment.

[0172] After culture, the supernatant of each flask of cells was carefully removed, and the cells were washed twice with 1 mL of physiological saline. Then, 1 mL of pure water was added to the cells to swell them and the stem cells were repeatedly lysed by pipetting for 10 min. The cells were then filtered through a 0.22 μm filter membrane and the filtrate was collected. The protein concentration and filtrate volume in the filtrate were detected by the BCA method, as shown in Table 2.

[0173] Table 2

[0174]

[0175] Example 3 Purification of the protein composition of the present invention

[0176] Instrument: AKTA explorer

[0177] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume (CV) approximately 30 mL.

[0178] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water

[0179] Equilibrium chromatography column sequence:

[0180] First, rinse the chromatography column with 2CV purified water, then equilibrate the 2CV column with 1×PBS to zero the 280 nm UV absorbance.

[0181] Sample preparation: 20 mL of each protein composition prepared according to Example 2 was reduced to 600 μL using an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD.

[0182] Experimental Procedure: After equilibrating the chromatography column, load the sample using a 100 μL loop at a flow rate of 0.4 mL / min. Elute with 1×PBS at a flow rate of 0.2 mL / min until the peak is reached. Collect the protein fraction starting from a UV absorbance of 4 mAU and continuing until 1.2 column volumes are collected. Freeze-dry the collected protein to obtain the purified protein compositions for each group.

[0183] The purified protein composition from group 6 of Example 2 (cultured for 8 hours under 1THz terahertz electromagnetic wave conditions) was subjected to SDS-PAGE electrophoresis. The results are shown in the figure. Figure 4 .

[0184] Depend on Figure 4It can be seen that the sample bands are mainly distributed in the range of 11 KD to 100 KD. Among them, the molecular weight decreases from large to small. The first band is located between 75 KD and 100 KD; the second band is located between 63 KD and 75 KD; the third band is located between 48 KD and 63 KD; and the fourth band is located at around 48 KD.

[0185] The purified protein composition of group 6 was analyzed by mass spectrometry using the liquid chromatography-mass spectrometry method of the present invention, and the following proteins were obtained:

[0186] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0187] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0188] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0189] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0190] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0191] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0192] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0193] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0194] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0195] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0196] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homosapiens;

[0197] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0198] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0199] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0200] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0201] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0202] The protein composition contained 46.27% Serum albumin, 12.22% Serotransferrin, 4.28% Alpha-1-antitrypsin, and 2.70% Immunoglobulin gamma-1 heavy chain.

[0203] Example 4: Purification of the protein composition of the present invention

[0204] 1. Solution preparation

[0205] Mobile phase A (0.1% TFA aqueous solution): Take 1000 mL of ultrapure water, add 1 mL of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase A.

[0206] Mobile phase B (71.4% acetonitrile solution of 0.075% TFA): Take 286 ml of ultrapure water, add 714 mL of acetonitrile and 0.75 ml of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase B.

[0207] Protein composition sample: Weigh the protein composition obtained in Example 2, dilute it with PBS pH 7.2 buffer, mix well, and prepare a concentration of 1 mg / mL.

[0208] 2. Chromatographic conditions

[0209] The chromatographic column was an XBridge Protein BEH C4, 300 Å, 3.5 μm, 4.6 mm * 150 mm, with a column temperature of 40℃. Mobile phase A consisted of 0.1% TFA solution; mobile phase B consisted of 0.075% TFA in 71.4% acetonitrile solution. The detector was 220 nm. Chromatographic conditions are shown in Table 3. The peak detected at 20.2 min represents the purified protein composition.

[0210] Table 3

[0211]

[0212] Experimental Example 1: Study on the effect of the protein composition of the present invention on the repair of cellular oxidative damage

[0213] Day 1: Cell Plating: Dilute PC12 (rat adrenal pheochromocytoma cells) low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate at 6000 cells / well (96-well plate). Incubate overnight at 37℃ with 5% CO2.

[0214] Day 2: Dilute the unpurified protein composition of Example 2 and the purified protein composition of Example 3 (800 g / L) with DMEM + 5% FBS medium. g / mL).

[0215] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0216] Hydrogen peroxide treatment: Discard 80 μL / well of cell culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and incubate at room temperature for 25 min.

[0217] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as a damage treatment control.

[0218] Add 50 μL / well of the diluted sample to the well after hydrogen peroxide treatment.

[0219] Discard the supernatant from the untreated wells and add 100 μL of complete culture medium per well as a cell growth control (PC).

[0220] Incubate for 2 days under standard cell culture conditions at 37°C with 5.0% CO2.

[0221] Day 5: Discard the culture supernatant, add 100 μL / well of DMEM complete medium, and set up blank control wells. Add 10 μL / well of CCK8, incubate at 37℃ for 3.5 h, and measure OD using a microplate reader. 450 Read the values, subtract the culture medium blank, and calculate cell viability.

[0222] Experimental results are as follows Figure 5 As shown, in the PC12 cell oxidative damage model, all samples had strong oxidative damage repair capabilities, with purified samples showing better repair capabilities than unpurified samples.

[0223] Experimental Example 2: Study on the effect of the protein composition of the present invention on the repair of cellular oxidative damage

[0224] Day 1: Cell Plating: Dilute PC12 (rat adrenal pheochromocytoma cells) low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate at 6000 cells / well (96-well plate). Incubate overnight at 37℃ with 5% CO2.

[0225] Day 2: Dilute the unpurified protein composition of Group 7 of Example 2 (cultured for 18 h under 1 THz terahertz electromagnetic wave conditions) and the purified protein composition of Example 4 (approximately 100 μg / mL) with DMEM + 5% FBS medium.

[0226] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0227] Hydrogen peroxide treatment: Discard 80 μL / well of cell culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and incubate at room temperature for 25 min.

[0228] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as a damage treatment control.

[0229] Add 50 μL / well of the diluted sample to the well after hydrogen peroxide treatment.

[0230] Discard the supernatant from the untreated wells and add 100 μL of complete culture medium per well as a cell growth control (PC).

[0231] Incubate for 2 days under standard cell culture conditions at 37°C with 5.0% CO2.

[0232] Day 5: Discard the culture supernatant, add 100 μL / well of DMEM complete medium, and set up blank control wells. Add 10 μL / well of CCK8, incubate at 37℃ for 3.5 h, and measure OD using a microplate reader. 450 Read the values, subtract the culture medium blank, and calculate cell viability. See the results below. Figure 6 .

[0233] Experimental Example 3: Study on the effect of the protein composition of the present invention on the repair of cellular oxidative damage

[0234] Adjust SH-SY5Y cells (neuroblastoma subclonal cells from human bone marrow) to 10 5 Density per mL, 100 L / well inoculated into 96-well plates.

[0235] The cell model was established 24 hours after cell seeding. The modeling process is as follows:

[0236] Dilute hydrogen peroxide (theoretical concentration 1M) to 200 mL with complete culture medium (RPM 1640). M needs to be prepared fresh each time it is used.

[0237] The experiment was divided into 4 groups: normal cell group, model control group, drug group 1 (the unpurified protein composition of group 7 of Example 2 (cultured for 18h under 1THz terahertz electromagnetic wave conditions) was diluted with complete culture medium to a concentration of 100ng / mL) and drug group 2 (the purified protein composition of Example 4 was diluted with complete culture medium to a concentration of 100ng / mL), with 4 replicates per group.

[0238] Aspirate the culture supernatant from all wells. Add 200 ml of the supernatant to the model control group and the drug-treated group. hydrogen peroxide, 100 L / well, damage for about 30 minutes, cell modeling is complete.

[0239] Drug administration: Immediately after modeling, aspirate the supernatant as thoroughly as possible. For the drug administration group, administer the drug at the concentration described above, 100 mg / well. L, the model control group and normal cell group were replaced with normal culture medium 100. L / well, incubated at 37°C, 5% CO2 for 72 h, and the neurofilament protein (NF) content in the supernatant was measured. NFL is a neuron-specific cytoskeletal protein that is crucial for axonal structural integrity and function. Measuring NFL content can be used to assess the neuronal damage repair capacity.

[0240] According to the instructions for use of Jianglai Biotechnology reagent kit, a standard curve was prepared, and the OD values ​​of the samples were analyzed. 450 The absorbance value was used to calculate the sample concentration. The NFL content in the sample was calculated based on the fitted standard curve, and the restoration rate was then calculated.

[0241] NFL repair rate % = (Content in model control group - Content in drug-treated group) / Content in model control group * 100%.

[0242] Depend on Figure 7 It is known that abnormally elevated NFL protein levels indicate neurodegeneration, and the protein composition of the present invention can repair nerve damage.

[0243] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A protein composition for terahertz electromagnetic stress culture, wherein the proteins constituting the protein composition are selected from serum albumin, serum transferrin, lumican, actin, alpha-1 antitrypsin, tropomyosin beta chain, vimentin, fibronectin, tropomyosin alpha-1 chain, filamin-A, immunoglobulin gamma-1 heavy chain, tenascin, alpha-2 macroglobulin, human cytoplasmic actin 1, hemoglobin subunit gamma-1, complement 3, etc. Any one or a combination thereof of C3).

2. The protein composition according to claim 1, wherein the serum albumin content in the protein composition is greater than 30% by mass percentage, preferably 35-55%, more preferably 40-50%.

3. The protein composition according to any one of claims 1-2, wherein the content of serum transferrin in the protein composition is greater than 5% by mass percentage, preferably 5-15%, more preferably 8-13%.

4. The protein composition according to any one of claims 1-3, wherein the protein composition contains 1%-6% α-1-antitrypsin, preferably 2-5%, by mass percentage.

5. The protein composition according to any one of claims 1-4, wherein the protein composition contains 1%-6% by mass of immunoglobulin gamma-1 heavy chain, preferably 2-5%.

6. The method for preparing the protein composition of terahertz electromagnetic wave stress culture as described in any one of claims 1-5, comprising stem cell expansion, stress culture of stem cells in terahertz conditioned stimulation medium, collection of stress cultured stem cells for stem cell lysis, and separation and purification of the protein composition from the lysate.

7. The method of claim 6, wherein the preparation of the protein composition cultured under terahertz electromagnetic wave stress comprises the following steps: S-1: Stem cells cultured under terahertz electromagnetic stress, wherein... The terahertz stress culture conditions are as follows: The frequency of terahertz electromagnetic waves is 0.1-10 THz, and the stress culture time is 0.5h-48h. S-2: Lyse the stress-cultured stem cells obtained in S-1 and separate and extract the resulting stress-cultured proteome. Compound.

8. The method according to any one of claims 6-7, wherein the purification method of the protein composition is selected from molecular sieve chromatography, high-performance liquid chromatography (HPLC), or a combination thereof.

9. The method for detecting the protein composition cultured under terahertz electromagnetic stress as described in any one of claims 1-5, wherein the method is selected from any one or a combination of the following methods: 1) Size exclusion chromatography: Size exclusion chromatography is performed using molecular sieves at a flow rate of 0.1–0.3 mL / min and PBS as the eluent. Under analytical conditions, before the peak volume is 0.1-2 times the column volume, the peak containing the protein composition is one or more peaks; or 2) HPLC detection method: Sample loading volume 60–80 µL, column temperature 25–40℃, flow rate 0.5–1 mL / min. The detection wavelength was 220–280 nm, the mobile phase A was TFA aqueous solution, the mobile phase B was TFA acetonitrile solution, and the elution time was 6–150 min under reversed-phase HPLC conditions. The peak elution time after sample separation was between 10 and 40 min.

10. The use of the protein composition cultured under terahertz electromagnetic stress as described in any one of claims 1-5 in the preparation of oxidative damage repair and / or nerve damage repair drugs.

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