A method for preparing an autologous psoriatic skin tissue immunomodulator and use thereof

Abstract

The application relates to the technical field of biological medicine, in particular to a preparation method and application of an autologous psoriasis skin tissue immune remodeling stimulator, which comprises the following steps: S1, placing an autologous psoriasis lesion single-cell suspension in a solution of imino acid and non-reducing disaccharide, performing constant-temperature treatment, and obtaining a mixed suspension; S2, adding a serine protease inhibitor into the mixed suspension, freezing the mixed suspension containing the serine protease inhibitor in liquid nitrogen, then transferring the mixed suspension into a buffer solution to melt, and centrifuging to obtain a precipitate; S3, resuspending the precipitate in physiological saline to obtain a resuspension, and the physiological saline contains an imidazole ring-containing dipeptide and a guanidino group-containing amino acid; and S4, performing heating treatment on the resuspension, then performing cooling, and thus the autologous psoriasis skin tissue immune remodeling stimulator is obtained. The application can solve the problem of conformational collapse caused by the lack of site shielding at high temperature, and realize conformational locking.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and in particular to a method for preparing an autologous psoriasis skin tissue immune remodeling agonist and its application. Background Technology

[0002] In the preparation of autologous psoriatic skin tissue immune remodeling agonists, the system must undergo transphase shearing physical disruption under extremely low temperatures of liquid nitrogen, followed by terminal inactivation treatment under high pressure and humid heat at 108°C. These dual extreme thermodynamic conditions lead to lipid membrane fragmentation and a surge in specific surface area, forcing antigen proteins such as S100A8 / A9 to thermally defold and expose their hydrophobic cores. Preventing irreversible hydrophobic aggregation and carbonyl cross-linking of the thermally defolded target antigens, and locking them in an effective three-dimensional conformation that exposes tolerance epitopes, is a fundamental requirement for inducing immune-tolerant Treg cells and preventing a biochemical shift in pharmacodynamics towards pro-inflammatory activation.

[0003] Chinese invention patent application CN110432259A, entitled "A cryoprotectant and a cell cryopreservation solution containing the same and its application in cell cryopreservation," discloses a cell cryopreservation scheme. The system comprises L-proline at a concentration of 1 to 2 mol / L, trehalose at a concentration of 0.2 to 0.3 mol / L, and DuPont phosphate buffer. The process involves mixing cells with the cryopreservation solution and storing them in liquid nitrogen at -196°C. Within the system, L-proline regulates the redox potential of cells and stabilizes biomolecules, while trehalose forms a protective film on the cell membrane surface, thereby reducing ice crystal formation and maintaining the morphology and structure of cryopreserved cells and the activity of membrane surface proteins.

[0004] When this existing technology is applied to the conditions required for an autologous psoriatic skin tissue immune remodeling agonist, cells completely lose their structural integrity and generate a large number of lipid fragments under extremely low temperature transphase transition shearing. When the system enters a 108°C high-pressure humid heat environment, the free lipid fragments undergo chain peroxidation, releasing a large number of highly reactive electrophilic carbonyl groups. The high temperature simultaneously drives the unfolding of target antigens such as S100A8 / A9, exposing internal hydrophobic residues and free amino groups. The electrophilic carbonyl groups then infiltrate into the hydrophobic microdomains of the antigen, forming irreversible covalent crosslinks with the amino groups through Schiff base condensation reactions. This abnormal crosslinking process directly leads to the collapse of the three-dimensional spatial conformation of the target antigen and large-scale hydrophobic aggregation, forcing an irreversible biochemical shift in the pharmacodynamic characteristics of the formulation from inducing immune tolerance to pro-inflammatory activation. Summary of the Invention

[0005] To address the conformational collapse caused by the lack of fixed-point shielding at high temperatures and to achieve tolerance conformational locking, this application provides a method for preparing an autologous psoriasis skin tissue immune remodeling agonist and its application.

[0006] In a first aspect, this application provides a method for preparing an autologous psoriasis skin tissue immune remodeling agonist, which adopts the following technical solution: A method for preparing an autologous psoriasis skin tissue immune remodeling agonist, comprising: Step S1: The autologous psoriasis lesion single cell suspension is placed in a solution containing imino acids at a concentration of 10-15 mmol / L and non-reducing disaccharides at a concentration of 100-150 mmol / L, and is kept at a constant temperature of 41-43℃ to obtain a mixed suspension. Step S2: Add serine protease inhibitor to the mixed suspension, and freeze the mixed suspension containing the serine protease inhibitor in liquid nitrogen. Then, transfer it to a buffer solution at a temperature of 37°C, which serves as an external thawing heat transfer bath, to thaw. Centrifuge to obtain the precipitate. Step S3: The precipitate is resuspended in physiological saline to obtain a resuspension, wherein the physiological saline contains an imidazole cyclic dipeptide at a concentration of 20-25 mmol / L and a guanidine amino acid at a concentration of 60-75 mmol / L. Step S4: The resuspension is heated under an absolute pressure of 0.12-0.16 MPa and a temperature of 105-110°C, and then cooled in an environment with a temperature below 0°C to obtain the autologous psoriasis skin tissue immune remodeling agonist.

[0007] Optionally, in step S1, the imino acid is L-proline and the non-reducing disaccharide is trehalose; in step S3, the imidazole-containing dipeptide is L-carnosine and the guanidine-containing amino acid is L-arginine hydrochloride.

[0008] Optionally, in step S2, the serine protease inhibitor is benzyl sulfonyl fluoride, and the concentration of benzyl sulfonyl fluoride in the mixed suspension containing benzyl sulfonyl fluoride is 0.8–1.2 mmol / L.

[0009] Optionally, before step S1, the isolated autologous psoriasis lesion tissue is immersed in a digestive solution and dissociated at a temperature of 36.5–37.5°C for 15–20 minutes. After centrifugation and washing, a single-cell suspension of the autologous psoriasis lesion tissue is obtained. The digestive fluid contains 0.2%–0.3% trypsin, 0.05%–0.15% type I collagenase, and 0.5–1.5 mmol / L metal ion chelating agent.

[0010] Optionally, in step S1, the duration of the isothermal treatment is 70 to 80 minutes; In step S2, the duration of freezing the mixed suspension containing the serine protease inhibitor in liquid nitrogen is 3 to 8 minutes, and the duration of thawing in the buffer solution at a temperature of 37°C, which serves as the external thawing heat transfer bath environment, is 3 to 8 minutes. The freezing and thawing are alternately cycled 2 to 3 times.

[0011] Optionally, in step S2, the step of obtaining the precipitate by centrifugation includes: centrifuging at a temperature of 0-5°C with a centrifugal force of 750-850g for 10-20 minutes, and discarding the supernatant.

[0012] Optionally, before the heat treatment in step S4, the resuspended liquid is further packaged into a pressure-resistant container and sealed and vented; in step S4, the absolute pressure of the heat treatment is 0.13 to 0.15 MPa, the temperature of the heat treatment is 107.5 to 108.5 °C, and the duration of the heat treatment is 20 to 25 minutes.

[0013] Optionally, in step S4, the step of cooling by moving the container into an environment with a temperature below 0°C includes: moving the pressure-resistant container after the heat treatment into an ice-salt bath with a temperature of -25 to -15°C.

[0014] Optionally, in step S3, the precipitate contains lipid fragments released by the freezing and thawing process, as well as S100A8 / A9 antigen protein, and the physiological saline is a sodium chloride aqueous solution with a mass fraction of 0.85% to 0.95%.

[0015] Secondly, the application of an autologous psoriasis skin tissue immune remodeling agonist prepared by the aforementioned preparation method in the preparation of a drug for treating psoriasis is provided in this application, using the following technical solution: the application of an autologous psoriasis skin tissue immune remodeling agonist prepared by the aforementioned preparation method in the preparation of a drug for treating psoriasis.

[0016] In summary, this application includes the following beneficial technical effects: 1. Through isothermal treatment at 41–43°C with 10–15 mmol / L imino acids and 100–150 mmol / L non-reducing disaccharides in step S1, combined with resuspension in physiological saline containing 20–25 mmol / L imidazole-ring dipeptide and 60–75 mmol / L guanidinyl amino acids in step S3, and heat treatment at an absolute pressure of 0.12–0.16 MPa and a temperature of 105–110°C in step S4, the primary amino group and imidazole ring of the imidazole-ring dipeptide are subjected to... During the heat phase, a competitive reaction occurs between the carbonyl group derived from lipid peroxidation and the guanidino amino acid reduces the probability of non-specific contact between heat-exposed proteins through hydration. Together, they inhibit the hydrophobic aggregation and carbonyl cross-linking of the S100A8 / A9 antigen protein, thereby controlling the relative abundance of S100A8 / A9-specific Schiff base cross-linked modified peptides in the range of 12.5% ​​to 15.2%, and locking the apparent abundance of α-helical residuals in the range of 15.8% to 17.5%, thus achieving effective locking of the tolerance conformation.

[0017] 2. In step S2, 0.8–1.2 mmol / L of benzyl sulfonyl fluoride is added to the mixed suspension, and the mixture is alternately frozen in liquid nitrogen for 3–8 minutes and thawed in a buffer solution at 37°C for 3–8 minutes for 2–3 cycles. During the thawing phase, benzyl sulfonyl fluoride inhibits the hydrolysis of S100A8 / A9 antigen proteins by serine proteases released during cell wall disruption. The phase transition shear stress of freezing and thawing causes S100A8 / A9 to entangle on the nuclear membrane and skeletal debris, forming large insoluble complex clumps. Combined with low-speed centrifugation at 750–850 g at 0–5°C to achieve co-precipitation retention, the detectable content of S100A8 / A9 antigen proteins can be increased to 38.4–42.5 μg / mg total protein, avoiding degradation loss of antigen proteins before subsequent heat treatment.

[0018] 3. In step S3, the precipitate is resuspended in physiological saline containing 20–25 mmol / L L-carnosine and 60–75 mmol / L L-arginine hydrochloride. The imidazole ring of L-carnosine and the primary amino group capture the electrophilic carbonyl group released by lipid peroxidation in a high-pressure and humid heat environment. The guanidino group of L-arginine hydrochloride enhances the stability of the hydration layer and shields exposed hydrophobic residues in aqueous solution. The two work synergistically to maintain the total protein extraction yield at 4.15–4.68 mg / g tissue, while inhibiting malondialdehyde equivalent at 0.82–0.95 nmol / mg protein and protein carbonyl content at 1.25–1.45 nmol / mg protein, providing a low-oxidative-damage antigenic basis for the induction of immune-tolerant Treg cells. Attached Figure Description

[0019] Figure 1 Transmission electron microscope images of lipid fragments and S100A8 / A9 antigen protein complexes provided for embodiments of this application; Figure 2 A bar chart showing the relative abundance of specific Schiff base crosslinked modified peptides provided in the embodiments of this application; Figure 3 A bar chart comparing the thickness of the mouse epidermis as provided in the embodiments of this application. Detailed Implementation

[0020] The following combination Figures 1-3 This application will be described in further detail.

[0021] Embodiments of this application provide a method for preparing an autologous psoriasis skin tissue immune remodeling agonist, comprising the following stages: Raw materials and reagents Autologous psoriasis lesion tissue was derived from clinically diagnosed psoriasis patients. Tissue collection, transportation, and in vitro processing complied with medical ethics requirements, and informed consent was obtained from the subjects. Each autologous psoriasis lesion tissue sample was only validated in vitro with peripheral blood mononuclear cells from the same subject to avoid interference with immunological results due to differences in the major histocompatibility complex of allogeneic tissues.

[0022] L-proline, trehalose, L-carnosine, and L-arginine hydrochloride were prepared using pharmaceutical-grade raw materials with a purity of not less than 99.0%. Trypsin and type I collagenase were prepared using cell culture-grade raw materials, respectively, and low-endotoxin cell culture-grade raw materials were used. Ethylenediaminetetraacetic acid (EDTA) was used as the metal ion chelating agent. Benzyl sulfonyl fluoride with a purity of not less than 99.0% was prepared as a 100 mmol / L stock solution using anhydrous ethanol and added to the subsequently obtained mixed suspension before freezing in liquid nitrogen to achieve a concentration of 0.8–1.2 mmol / L. Phosphate buffer preheated to 37°C (pH 7.2–7.4, hereinafter referred to as "the 37°C buffer") was used only as the external thawing heat transfer bath environment. Physiological saline was prepared as a 0.85%–0.95% sodium chloride aqueous solution. All aqueous solutions were prepared using pyrogen-free water for injection.

[0023] L-proline, an imino acid, is used in this process to regulate water molecule migration and ice crystal growth during freeze-thaw cycles. Trehalose, a non-reducing disaccharide, is used in this process to increase liquid-phase osmotic pressure and stabilize the hydration layer on the lipid membrane surface. Trehalose lacks a free reducing terminal, making it less prone to non-enzymatic glycosylation reactions with amino compounds during heat treatment compared to reducing sugars.

[0024] L-Carnosine is an imidazole-ring-containing dipeptide. Its primary amino group and imidazole ring can competitively react with some lipid peroxidation-derived carbonyl groups, and can buffer local proton changes to some extent. L-Arginine hydrochloride is a guanidino-containing amino acid that exhibits strong hydration in aqueous solution, reducing the probability of non-specific contact between heat-exposed proteins. This interaction involves electrostatic interactions, hydrogen bonding, aromatic surface interactions, and solvent preference, and cannot be attributed to a single cation-π interaction.

[0025] Benzyl sulfonyl fluoride is a serine protease inhibitor. Pre-distributing it in a suspension allows it to immediately and simultaneously inhibit serine proteases released into the system during cell lysis during the melting phase. The hydrolysis rate of Benzyl sulfonyl fluoride in aqueous solution increases with increasing temperature and pH. Its effective action time at near-neutral conditions and 37°C is typically on the order of tens of minutes. A melting duration of 3–8 minutes, shorter than this timescale, is beneficial for preserving inhibitory activity; an excessively long melting duration increases the risk of hydrolysis of Benzyl sulfonyl fluoride and degradation of the S100A8 / A9 antigen protein.

[0026] S100A8 and S100A9 belong to the S100 calcium-binding protein family, with approximately 65% ​​and 67% homology in amino acid sequence between humans and mice, respectively (UniProtP05109 / P06702; P27005 / P31725). Species differences exist between the two in the TLR4 / MD-2 complex binding domain and downstream NF-κB signaling pathway activation. Therefore, in this application, the in vitro immune effect evaluation uses human peripheral blood mononuclear cells from the same subject to directly verify the immunophenotypic remodeling effect of the human-derived formulation; the in vivo pharmacodynamics section uses a mouse homology model only as an auxiliary verification of process activity retention, and the in vivo efficacy of the human-derived formulation is not claimed within the scope of this application.

[0027] Preparation method In one specific embodiment, the method for preparing an autologous psoriatic skin tissue immune remodeling agonist provided in this application specifically includes core steps S1 to S4. Before performing step S1, it is usually necessary to prepare a single-cell suspension of skin lesions as a pretreatment, the specific process of which is as follows: The excised autologous psoriasis lesion tissue was placed in a sterile cryogenic transport solution and processed within 2 hours of collection. The operator removed surface blood and significant subcutaneous fat, and cut the tissue into pieces with sides no larger than 1 mm.

[0028] Add the digestive solution at a ratio of 10 mL per gram of tissue. The digestive solution contains 0.2%–0.3% trypsin, 0.05%–0.15% type I collagenase, and 0.5–1.5 mmol / L metal ion chelating agent.

[0029] The tissue block was immersed in the digestive solution and dissociated by shaking at 40–60 rpm for 15–20 minutes at 36.5–37.5°C. Below 36.5°C, the apparent reaction rates of trypsin and type I collagenase decreased, making it difficult to form a homogeneous autologous psoriasis lesion single-cell suspension within a short time. Above 37.5°C or when the dissociation time exceeded 20 minutes, the risk of non-target protein hydrolysis of cell surface proteins and S100A8 / A9 antigen proteins increased.

[0030] Dissociation was terminated using phosphate-buffered saline containing soybean trypsin inhibitor, and the sample was filtered through a 70 μm cell sieve. The filtrate was centrifuged at 4°C and 400 g for 5 minutes. The supernatant was discarded, and the precipitate was washed twice with phosphate-buffered saline to obtain a single-cell suspension from autologous psoriasis lesions. Cell concentration was determined using the trypan blue exclusion method, and the cell concentration was adjusted to 1.0 × 10⁻⁶ cells / cells. 6 per mL.

[0031] After obtaining the above-mentioned autologous psoriasis lesion single-cell suspension, the following core preparation steps were performed sequentially: In step S1, the autologous psoriasis lesion single-cell suspension is placed in a solution containing 10–15 mmol / L imino acid and 100–150 mmol / L non-reducing disaccharide. The imino acid used is L-proline, and the non-reducing disaccharide is trehalose. The autologous psoriasis lesion single-cell suspension is then subjected to isothermal treatment at 41–43°C for 70–80 minutes to obtain a mixed suspension.

[0032] Temperatures of 41–43°C can enhance membrane transport and molecular diffusion rates and induce measurable heat stress responses. L-proline can enter cells via amino acid transport systems, but trehalose typically cannot diffuse freely across intact mammalian cell membranes. Therefore, this step does not presuppose direct transmembrane translocation of trehalose or the necessary opening of mechanosensitive channels, but rather relies on moderate cell dehydration, extracellular hydration layer stabilization, and L-proline transport to jointly regulate freeze-thaw behavior. After the pre-heat stress treatment at 41–43°C in step S1, the S100A8 / A9 macromolecules in the system will pre-expose some of their internal hydrophobic residues.

[0033] In step S2, benzyl sulfonyl fluoride is added to the mixed suspension, and the mixed suspension containing 0.8–1.2 mmol / L benzyl sulfonyl fluoride is placed in a sealed container and frozen in liquid nitrogen for 3–8 minutes. Then, it is transferred to a 37°C buffer solution, which serves as an external thawing heat transfer bath, to thaw for 3–8 minutes. The freezing and thawing cycle is repeated 2–3 times.

[0034] Driven by the phase transition shear stress of subsequent liquid nitrogen freezing and thawing, a large amount of free S100A8 / A9 nonspecifically and firmly cross-links and entangles on massive intact nuclear membranes and cytoskeleton remnants, forming large, insoluble complex clumps. When the thawing time is less than 3 minutes, ice nuclei may remain in the center of the sample, resulting in inconsistent starting states in subsequent cycles. When the thawing time is longer than 8 minutes, the contact time between the released serine protease and the S100A8 / A9 antigen protein increases, while the degree of hydrolysis of benzyl sulfonyl fluoride also increases. When the number of cycles is less than 2, the release of lipid fragments and S100A8 / A9 antigen complexes is insufficient; when the number of cycles exceeds 3, the specific surface area of ​​lipid fragments continues to increase, increasing the risk of lipid peroxidation and protein mechanical damage.

[0035] After freezing and thawing, the mixed suspension was centrifuged at 750–850 g for 10–20 minutes at 0–5°C. The supernatant was discarded, and the precipitate was retained. Based on the mechanism of the large-mass complex fixation generated by the initial targeted induction, co-precipitation can be achieved at 0–5°C using only a low-speed centrifugation force of 750–850 g, preventing the loss of free small molecules with the supernatant. The precipitate contains lipid fragments released during freezing and thawing, as well as entangled and fixed S100A8 / A9 antigen-protein complexes.

[0036] In step S3, the precipitate is resuspended in physiological saline to obtain a resuspension. The specific resuspension ratio is: per 1.0 × 10 6 The precipitate corresponding to the initial cell volume was resuspended in 1.5–2.0 mL of the saline solution to provide a reproducible absolute molar basis for the protectant concentration. The saline solution contained 20–25 mmol / L of an imidazole-containing dipeptide and 60–75 mmol / L of a guanidine-containing amino acid. The imidazole-containing dipeptide was L-carnosine, and the guanidine-containing amino acid was L-arginine hydrochloride. The saline solution was a 0.85%–0.95% (w / w) sodium chloride aqueous solution. The mixture was stirred using a low-shear rotary mixer for 5 minutes to form a homogeneous resuspension.

[0037] In step S4, the resuspension is dispensed into pressure-resistant containers that can withstand 110°C and an absolute pressure of not less than 0.20 MPa. The liquid volume of each pressure-resistant container does not exceed 80% of its nominal volume. The operator removes any obvious air bubbles and seals the pressure-resistant container.

[0038] The pressure-resistant container is placed in a pressurized humid heat treatment device and subjected to heat treatment at an absolute pressure of 0.12–0.16 MPa and a temperature of 105–110 °C. Preferably, the absolute pressure is 0.13–0.15 MPa, the temperature is 107.5–108.5 °C, and the duration is 20–25 minutes. This heat treatment disrupts the original spatial folding structure of the protein. Its objective is not to maintain the native conformation of the antigen protein, but rather to control disordered aggregation, carbonyl addition, and insoluble cross-linking, ultimately locking the S100A8 / A9-related antigen protein within an effective three-dimensional residual conformation that exposes the tolerable non-native epitopes.

[0039] After the heat treatment, the pressure vessel was moved into an ice-salt bath at -25 to -15°C for cooling until the core temperature of the sample was below 0°C. Cooling can shorten the duration of the sample in the high reaction rate temperature range and limit the residual heat-driven oxidation and cross-linking reactions.

[0040] To better understand the technical essence of this application and the beneficial effects of each protective agent combination, the technical solution of this application is described in detail below through several specific embodiments and comparative examples. It should be understood that the following embodiments are only used to further illustrate this application and are not intended to limit the scope of protection of this application.

[0041] Example 1 This embodiment provides a method for preparing an immune remodeling agonist from autologous psoriasis skin tissue, and the specific preparation parameters are as follows: A digestive solution was prepared using 0.2% trypsin, 0.05% type I collagenase, and 0.5 mmol / L metal ion chelating agent, and then dissociated at 36.5 °C for 15 minutes.

[0042] Step S1 used 10 mmol / L L-proline, 100 mmol / L trehalose, 41°C, and 70 minutes. In Step S2, the freezing and thawing cycles were repeated twice; further centrifugation was performed at 5°C with a centrifugal force of 750 g for 10 minutes. Step S3 used physiological saline containing 20 mmol / L L-carnosine and 60 mmol / L L-arginine hydrochloride. This physiological saline was a 0.85% sodium chloride aqueous solution, and the resuspension ratio was 1.0 × 10⁻⁶ mg / L. 6 1.5 mL of the physiological saline was added to each initial cell volume. Step S4 was performed at an absolute pressure of 0.12 MPa, 105 °C, and for 20 minutes, followed by cooling in an ice-salt bath at -15 °C.

[0043] Example 2 This embodiment provides a method for preparing an immune remodeling agonist from autologous psoriasis skin tissue, and the specific preparation parameters are as follows: A digestive solution was prepared using 0.25% trypsin, 0.10% type I collagenase, and 1.0 mmol / L metal ion chelating agent, and then dissociated at 37.0 °C for 18 minutes.

[0044] Step S1 used 12.5 mmol / L L-proline, 125 mmol / L trehalose, 42°C, and 75 minutes. In Step S2, the freezing and thawing cycles were repeated three times; further centrifugation was performed at 4°C with a centrifugation force of 800 g for 15 minutes. Step S3 used physiological saline containing 22.5 mmol / L L-carnosine and 67.5 mmol / L L-arginine hydrochloride, which was a 0.90% sodium chloride aqueous solution, with a resuspension ratio of 1.0 × 10⁻⁶ mg / L. 6 1.8 mL of the physiological saline was added to each initial cell volume. Step S4 was performed at 0.14 MPa absolute pressure, 108 °C for 22.5 minutes, and cooled in an ice-salt bath at -20 °C.

[0045] Example 3 This embodiment provides a method for preparing an immune remodeling agonist from autologous psoriasis skin tissue, and the specific preparation parameters are as follows: A digestive solution was prepared using 0.3% trypsin, 0.15% type I collagenase, and 1.5 mmol / L metal ion chelating agent, and then dissociated at 37.5 °C for 20 minutes.

[0046] Step S1 used 15 mmol / L L-proline, 150 mmol / L trehalose, 43°C, and 80 minutes. In Step S2, the freezing and thawing cycles were repeated three times; further centrifugation was performed at 0°C with a centrifugal force of 850 g for 20 minutes. Step S3 used physiological saline containing 25 mmol / L L-carnosine and 75 mmol / L L-arginine hydrochloride. This physiological saline was a 0.95% sodium chloride aqueous solution, and the resuspension ratio was 1.0 × 10⁻⁶ mg / L. 6 2.0 mL of this physiological saline was added to the starting cell volume. Step S4 was performed at an absolute pressure of 0.15 MPa, 110 °C, and 25 minutes, followed by cooling in an ice-salt bath at -25 °C.

[0047] Comparative Example 1 To verify the synergistic effect of specific cryoprotectants and the differences between conventional cryopreservation protocols, a comparative example was established to simulate a conventional cell cryopreservation protocol involving the passive mixing of L-proline and trehalose. The specific parameters are as follows: Step S1 uses 1.0 mol / L L-proline and 0.20 mol / L trehalose, without isothermal treatment at 41–43 °C. No benzyl sulfonyl fluoride is added to this suspension. The precipitate is resuspended in a 0.90% sodium chloride aqueous solution at a concentration of 1.0 × 10⁻⁶.6 The initial cell volume was increased by adding 1.8 mL of the sodium chloride aqueous solution, which was free of L-carnosine and L-arginine hydrochloride. The remaining steps followed the parameters of Example 2.

[0048] Comparative Example 2 Comparative Example 2 uses the preparation method of Example 2, the only difference being that the physiological saline in step S3 does not contain L-carnosine, and the concentration of L-arginine hydrochloride is maintained at 67.5 mmol / L.

[0049] Comparative Example 3 Comparative Example 3 uses the preparation method of Example 2, the only difference being that the physiological saline in step S3 does not contain L-arginine hydrochloride, and the L-carnosine concentration is maintained at 22.5 mmol / L.

[0050] Comparative Example 4 Comparative Example 4 is a blank comparative example. Comparative Example 4 uses the preparation method of Example 2, the only difference being that the physiological saline in step S3 does not contain L-carnosine and L-arginine hydrochloride.

[0051] Comparative Example 5 Comparative Example 5 is the lower boundary comparative example. Step S1 used 9 mmol / L L-proline and 90 mmol / L trehalose. Step S3 used 18 mmol / L L-carnosine and 54 mmol / L L-arginine hydrochloride. The remaining steps used the parameters of Example 2.

[0052] Comparative Example 6 Comparative Example 6 is the upper boundary comparative example. Step S1 used 16.5 mmol / L L-proline and 165 mmol / L trehalose. Step S3 used 27.5 mmol / L L-carnosine and 82.5 mmol / L L-arginine hydrochloride. The remaining steps used the parameters of Example 2.

[0053] Test methods To further verify the physicochemical properties and immunophenotypic remodeling effects of the autologous psoriasis skin tissue immune remodeling agonist prepared in the above embodiments of this application, the samples of Examples 1 to 3 and Comparative Examples 1 to 6 were tested and evaluated using the following methods: Each embodiment and comparative example used at least six independent autologous psoriasis lesion tissue samples. Three technical replicates were set for each sample. Test results are expressed as mean ± standard deviation. One-way ANOVA and Tukey multiple comparisons were used for inter-group comparisons; when the distribution was not normal, the Kruskal-Wallis test and Dunn multiple comparisons were used. The significance level was set at P < 0.05.

[0054] The Z-mean particle size and polydispersity index of the resuspended solution were determined using a dynamic light scattering instrument at 25°C. Before testing, the solution was only mixed by slow inversion; ultrasonic treatment was not used.

[0055] Total protein concentration was determined using the micro-BCA method, and the total protein extraction yield (mg / g tissue) was calculated based on the initial wet weight. Malondialdehyde equivalent was determined using the thiobarbituric acid reactant method. Protein carbonyl content was determined using the 2,4-dinitrophenylhydrazine derivatization method.

[0056] The relative abundance of S100A8 / A9-specific Schiff base cross-linked modified peptides was quantitatively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The extracted target macromolecules were digested with trypsin and desalted before injection. This application explicitly defines the characteristic peptide sequence of human S100A9 rich in free amino groups as NIETIINTFHQYSVK. Using this as the evaluation benchmark, the mass-to-charge ratio of its free precursor ion was extracted and monitored (the monitoring channel was set to m / z 903.97, [M+2H]). 2+ The study also identified a characteristic mass shift modification peak derived from Schiff base addition (mass shift increment +54.0 Da, exhibiting a double-charge characteristic response at m / z 930.97). The percentage of the peak area relative to the total abundance of specific sequence free and modified peptides was calculated to evaluate the cross-linking blocking effect of specific targeting molecules. This mass spectrometry abundance data effectively confirmed the site-directed protection of the core small molecule amino group.

[0057] Circular dichroism (CD) chromatography was used to detect the residual state of S100A8 / A9 after high-pressure hydrothermal heating and disintegration of the native α-helical conformation. The samples were scanned in the far-ultraviolet wavelength range of 190–250 nm, and the absolute molar ellipticity [θ] at 208 nm and 222 nm was extracted. This was compared with the unheated native S100A8 / A9 labeled protein (the basal state [θ] measured in this experiment). 222 Recorded -12,450±120 deg·cm 2 ·dmol -1 ), calculate the measured apparent abundance of α-helical residues (such as the thermal residues [θ] in Example 2). 222 The value is -2,180±150 deg·cm 2 ·dmol -1 These spectral parameters directly verify that the product ultimately exhibits a partially unwound, non-natural state, rather than undergoing complete thermal destruction.

[0058] The content of S100A8 / A9 antigen proteins can be detected by enzyme-linked immunosorbent assay (ELISA) of the S100A8 / A9 complex. High molecular weight aggregates of S100A8 / A9 antigen proteins are detected by non-reducing polyacrylamide gel electrophoresis and Western blotting.

[0059] The morphology of lipid fragments and S100A8 / A9 antigen protein complexes was observed using transmission electron microscopy, and the number of aggregates with a particle size greater than 1 μm was calculated.

[0060] Bacterial endotoxins were detected using a dynamic colorimetric assay with Limulus amebocyte lysate (LAL). Peripheral blood mononuclear cells from the same subject were used to evaluate the in vitro immune response. After 72 hours of culture, CD4 counts were measured by flow cytometry. + CD25 + FoxP3 + Treg cells and CD4 + IL-17A + The proportion of Th17 cells was determined, and the levels of inflammatory cytokines in the culture supernatant were measured.

[0061] Data Recording and Result Determination The results of the parallel test on the test bench are shown in the table below:

[0062] Based on the above independent parallel test results, the specific protective agent combination in this application directly determines the degree of locking of the residual conformation of the antigen after heating and the direction of immunophenotypic remodeling. For example... Figure 1 As shown, transmission electron microscopy reveals an irregular, membrane-like morphology of lipid fragments. Numerous S100A8 / A9 antigen-protein complexes are tightly adhered in granular form to the surface and edges of these lipid fragments. This microscopic pattern objectively confirms the physical mechanism by which cells undergo cell wall fragmentation and form large, insoluble aggregates under the shear stress driven by phase transition. The total protein extraction yield directly reflects the effectiveness of the technology in preventing material denaturation and loss: after undergoing destructive conditions such as 108°C high-pressure humid heat, which easily leads to denaturation and aggregation, the protein extraction yields of Examples 1 to 3 remained stable in the range of 4.15–4.68 mg / g tissue, significantly higher than that of the blank matrix control example 4 (only 1.62 ± 0.25 mg / g tissue), which experienced large-scale thermal cross-linking and sedimentation due to the lack of a protective agent. This performance data confirms that the process has a definite anti-substrate precipitation and depletion effect.

[0063] Supplemental liquid chromatography-tandem mass spectrometry (LC-MS / MS) and circular dichroism (CD) characterization results confirmed the site-directed crosslinking blocking and conformational locking mechanism of the specific macromolecules in this application. Combined with... Figure 2As shown, under extreme high-pressure and humid heat conditions at 108℃, thanks to the synergistic protection of the imidazole cyclic dipeptide and the guanidino acid, the column heights of Examples 1 and 2 were suppressed to a low baseline, with their relative abundances locked at 15.2% and 12.5%, respectively. The measured data confirmed that the relative abundance of the S100A8 / A9 specific Schiff base crosslinked modified peptides in Examples 1 to 3 was stably controlled in the range of 12.5% ​​to 15.2%. In contrast, the column heights of Comparative Example 4, which lacked the dual synergistic components, and Comparative Example 1, which was cryopreserved using conventional methods, showed a precipitous increase, surging to 65.4% ± 6.2% and 62.4% ± 5.5%, respectively, due to reaction capacity failure. The corresponding circular dichroism chromatogram data showed that after thermal decomposition, Examples 1 to 3 did not completely lose their secondary structure, but successfully locked in 15.8%–17.5% of the apparent abundance of α-helical residues; while Comparative Examples 1 and 4, due to disordered molecular cross-linking and thermal decomposition, dropped to 4.1% ± 0.8% and 5.2% ± 1.1%, respectively. The above measured physicochemical spectral parameters directly confirmed the microscopic effectiveness of the system in preventing hydrophobic aggregation, and confirmed that the target antigen was effectively locked in a tolerant non-natural epitope state, providing material basis evidence for downstream activation of the Treg pathway.

[0064] More importantly, based on the pre-stated low-endotoxin cell culture-grade raw materials and pyrogen-free water for injection, the results of the Limulus Amebocyte Lysate (LAL) reagent dynamic colorimetric assay showed that the residual bacterial endotoxin in the systems of each embodiment was stably suppressed to an extremely low level of 0.05±0.01 to 0.08±0.02 EU / mg total protein. Given that trace amounts of bacterial endotoxin (LPS) are themselves extremely potent pro-inflammatory activators of TLR4 receptors, this extremely low baseline noise logically disproves the hypothesis that phenotypic differences in homologous monocyte activation experiments stem from exogenous background noise, thus establishing a closed loop for pharmacodynamic attribution leading to a substantial biochemical shift in immune tolerance remodeling.

[0065] In terms of efficacy in remodeling immune tolerance, compared to conventional cryopreservation (Comparative Example 1), which resulted in a significant increase in Th17 targeting and a large release of inflammatory factors due to uncontrolled antigen unfolding, Examples 1 to 3 reversed the pro-inflammatory activation kinetic pathway overall. CD4 in the culture system + CD25 + FoxP3 + The proportion of Treg immune-tolerant cell populations that differentiated and expanded was increased to 13.8%–15.2%, and the expression of the anti-inflammatory target factor interleukin-10 was significantly high (both not less than 410 pg / mL). Simultaneously, pro-inflammatory CD4+... + IL-17A + The proportion of Th17 cells, interleukin-17A, and tumor necrosis factor-α expression were all suppressed to very low baseline levels of inactivation.

[0066] In vivo pharmacodynamic evaluation (miquimod-induced homologous remodeling model of psoriasis in mice, for auxiliary validation) A psoriatic dermatitis model was established using 6-8 week old BALB / c mice. After shaving the back of the mice, 5% imiquimod (IMQ) cream was applied for 6 consecutive days. To verify the preliminary biological activity of the formulation prepared by the process described in this application in vivo, after successful modeling, skin lesions from the back of syngeneic modeling mice were harvested, and a mouse homologous immune remodeling agonist was prepared in parallel using the same process as in Example 2 of this application. Starting from day 2 of modeling, the treatment group received subcutaneous injections of 100 μL of the mouse homologous immune remodeling agonist (total protein dose of 50 μg / kg) daily, the positive control group received 0.1% dexamethasone ointment, and the model group received an equal volume of physiological saline. This intervention was continued for 5 consecutive days. On day 7, the PASI (Psoriasis Area and Severity Index) clinical score was calculated, and the back skin lesions were subjected to H&E staining to measure the epidermal thickness.

[0067] Test results showed that the normal control group mice had a PASI score of 0 and an epidermal thickness of 14.2±2.1 μm; the model group exhibited severe erythema, scaling, and pathological thickening, with the PASI score surging to 9.3±0.8 and the epidermal thickness reaching 88.5±10.4 μm; the positive control group had a PASI score of 3.2±0.5 and an epidermal thickness decreasing to 33.4±4.8 μm. Figure 3 As shown in the thickness gradient characteristics of each group, after intervention with the mouse homologous remodeling agonist prepared in this application, the pathological features of mouse skin lesions were significantly alleviated. The columnar height of the treatment group was significantly reduced compared with the model group, and the actual epidermal thickness was reduced to 38.2±5.4μm (P<0.01 compared with the model group), which directly verified the therapeutic potential of the immune remodeling agonist to reverse pathological epidermal hyperplasia and restore normal tissue physiological structure in vivo.

[0068] In summary, the analysis of the experimental and test results shows that the method provided in this application, through the synergistic combination of a specific protective agent and specific high-pressure humid heat and low-temperature conditions, successfully solves the technical problem of irreversible hydrophobic aggregation of target antigens under high-temperature and extremely low-temperature phase transition conditions, achieves effective locking of the tolerance conformation, demonstrates excellent ability to induce immune-tolerant Treg cells, and has significant potential for clinical application translation.

[0069] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for preparing an autologous psoriasis skin tissue immune remodeling agonist, characterized in that, include: Step S1: The autologous psoriasis lesion single cell suspension is placed in a solution containing imino acids at a concentration of 10-15 mmol / L and non-reducing disaccharides at a concentration of 100-150 mmol / L, and is kept at a constant temperature of 41-43℃ to obtain a mixed suspension. Step S2: Add serine protease inhibitor to the mixed suspension, and freeze the mixed suspension containing the serine protease inhibitor in liquid nitrogen. Then, transfer it to a buffer solution at a temperature of 37°C, which serves as an external thawing heat transfer bath, to thaw. Centrifuge to obtain the precipitate. Step S3: The precipitate is resuspended in physiological saline to obtain a resuspension, wherein the physiological saline contains an imidazole cyclic dipeptide at a concentration of 20-25 mmol / L and a guanidine amino acid at a concentration of 60-75 mmol / L. Step S4: The resuspension is heated under an absolute pressure of 0.12-0.16 MPa and a temperature of 105-110°C, and then cooled in an environment with a temperature below 0°C to obtain the autologous psoriasis skin tissue immune remodeling agonist.

2. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: In step S1, the imino acid is L-proline and the non-reducing disaccharide is trehalose; in step S3, the imidazole-containing dipeptide is L-carnosine and the guanidine-containing amino acid is L-arginine hydrochloride.

3. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: In step S2, the serine protease inhibitor is benzyl sulfonyl fluoride, and the concentration of benzyl sulfonyl fluoride in the mixed suspension containing benzyl sulfonyl fluoride is 0.8–1.2 mmol / L.

4. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: Before step S1, the isolated autologous psoriasis lesion tissue is immersed in a digestive solution and dissociated at a temperature of 36.5-37.5°C for 15-20 minutes. After centrifugation and washing, a single-cell suspension of the autologous psoriasis lesion tissue is obtained. The digestive fluid contains 0.2%–0.3% trypsin, 0.05%–0.15% type I collagenase, and 0.5–1.5 mmol / L metal ion chelating agent.

5. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: In step S1, the duration of the isothermal treatment is 70 to 80 minutes; In step S2, the duration of freezing the mixed suspension containing the serine protease inhibitor in liquid nitrogen is 3 to 8 minutes, and the duration of thawing in the buffer solution at a temperature of 37°C, which serves as the external thawing heat transfer bath environment, is 3 to 8 minutes. The freezing and thawing are alternately cycled 2 to 3 times.

6. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: In step S2, the step of obtaining the precipitate by centrifugation includes: centrifuging at a temperature of 0-5°C with a centrifugal force of 750-850g for 10-20 minutes, and discarding the supernatant.

7. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: Before the heat treatment in step S4, the resuspended liquid is further packaged into pressure-resistant containers and sealed and vented; in step S4, the absolute pressure of the heat treatment is 0.13 to 0.15 MPa, the temperature of the heat treatment is 107.5 to 108.5 °C, and the duration of the heat treatment is 20 to 25 minutes.

8. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 7, characterized in that: In step S4, the step of cooling by moving the container into an environment with a temperature below 0°C includes: moving the pressure-resistant container after the heat treatment into an ice-salt bath with a temperature of -25 to -15°C.

9. The method for preparing the autologous psoriasis skin tissue immune remodeling agonist according to claim 1, characterized in that: In step S3, the precipitate contains lipid fragments released by the freezing and thawing processes, as well as S100A8 / A9 antigen proteins, and the physiological saline is a sodium chloride aqueous solution with a mass fraction of 0.85% to 0.95%.

10. The use of an autologous psoriatic skin tissue immune remodeling agonist prepared by any one of claims 1 to 9 in the preparation of a medicament for treating psoriasis.

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