Stem cell exosome immune regulation function improving system based on synthetic biological regulation

Abstract

The invention discloses a stem cell exosome immune regulation function improving system based on synthetic biological regulation, and particularly relates to the technical field of biological medicine and cell engineering. Comprising a port synthetic gene loop module, a dynamic parameter culture parameter regulation and control module, a controlled promoter subsystem module, a stem cell exosome collection and purification module, a stem cell exosome quality and function detection module and a stem cell exosome quality and function detection feedback module. The synthetic gene loop module is used for introducing a miRNA module capable of inducing regulation expression, designing a synthetic promoter module for expressing miR-146a, and combining the synthetic promoter module with a transcription factor to regulate and transfect human umbilical cord mesenchymal stem cells; on the basis of a cell synthesis flux optimization theory, a brand new dynamic culture parameter regulation and control system is established, a plurality of key culture parameters are precisely regulated and controlled, the secretion amount of the exosome is greatly multiplied, the problem that the secretion amount is limited is solved, and the clinical dosage requirement can be better met.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cell engineering technology, specifically to a stem cell exosome immune regulation enhancement system based on synthetic biology regulation. Background Technology

[0002] As an important medium for intercellular communication, stem cell exosomes have shown broad application prospects in regulating immune inflammation, tissue repair, and anti-aging. Among them, exosomes derived from mesenchymal stem cells (MSCs) have attracted much attention due to their low immunogenicity and ability to carry a variety of immunologically active molecules such as miRNAs, proteins, and lipids.

[0003] Traditional methods for treating stem cell exosomes include: enhancing their anti-inflammatory and immunomodulatory functions through LPS pretreatment; using static culture methods to seed stem cells (such as MSCs) in culture dishes or flasks; employing three-dimensional spheroid culture, where stem cells aggregate into 3D spheres using hanging drop methods, microcarriers, or bioreactors to enhance intercellular signal transduction, but this method is prone to hypoxia; and using microcarriers (such as Cytodex) to expand the adhesion area of ​​stem cells, thereby enhancing intercellular signal transduction and increasing cell density.

[0004] However, existing stem cell exosomes face several key technical challenges in practical applications: batch-to-batch compositional differences in exosomes under conventional culture conditions make it difficult to consistently maintain anti-inflammatory characteristics such as high IL-10 / low TNF-α and low IL-6 expression, leading to unstable immunomodulatory efficacy; traditional static culture and exosome secretion using microcarriers result in low secretion rates, and three-dimensional spheroid culture lacks dynamic regulation of culture parameters; current methods only induce the expression of immune-related molecules through exogenous stimuli (such as LPS pretreatment), lacking control over functional enhancement; and there is no mature strategy to precisely regulate exosome immune function at the cellular level using synthetic biology. Therefore, there is an urgent need for a controllable enhancement system for exosome immune regulation at the stem cell level using synthetic biology techniques to improve its practical application value in immune diseases, chronic inflammation, and anti-aging. Summary of the Invention

[0005] To overcome the aforementioned deficiencies of the prior art, embodiments of the present invention provide a stem cell exosome immune regulation enhancement system based on synthetic biology regulation to address the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a stem cell exosome immune regulation enhancement system based on synthetic biology regulation, comprising: Synthetic gene circuit module: Introduce an inducible non-coding RNA module in stem cells, design a synthetic promoter module expressing miR-146a to jointly regulate and transfect human umbilical cord mesenchymal stem cells with transcription factors, construct a synthetic gene circuit module containing transfected cell lines with proven stability, and transfer it to the dynamic culture parameter regulation module and the control promoter system module. The dynamic culture parameter regulation module seeds transfected cell lines containing stable and validated cells from the synthetic gene loop module into a dynamic stem cell culture device. It uses Doxycycline to activate the promoter expression system, induces the synthesis of target miRNA, and regulates environmental parameters during stem cell culture based on the cell synthesis throughput optimization theory, thus establishing a dynamic culture parameter regulation system. Controlled promoter system module: Through the controlled promoter system, the expression of immune regulatory genes in stem cells is controlled in terms of time and dosage, and the results of time and dosage control are transmitted to the synthetic gene loop module and the dynamic culture parameter regulation module; Exosome collection and purification module: Collects stem cell exosomes obtained from the synthetic gene circuit module, dynamic parameter culture parameter regulation module, and control promoter system module, and uses ultracentrifugation combined with membrane filtration process, combined with charge-selective purification of stem cell exosomes; Quality and Function Detection Module: Used to detect the quality and functional data of purified exosomes. Quality data includes exosome particle size distribution, marker protein and miRNA expression profiles. Functional data includes assessment of IL-10 upregulation rate and TNF-α downregulation rate through macrophage co-culture experiments, and assessment of exosome regulatory indicators of inflammatory response through anti-inflammatory models. Stem cell exosome quality and function detection feedback module: compares the quality and function data of exosomes with the corresponding thresholds, and feeds back the abnormal comparison results to the management terminal for human-computer interaction.

[0007] The technical effects and advantages of this invention are as follows: 1. This invention is the first to propose introducing a specific miRNA module that can be induced to regulate expression in stem cells and combining it with a regulatory transcription factor to construct a unique synthetic gene circuit. This circuit can precisely target and enhance the content of anti-inflammatory molecules in exosomes, effectively solving the problem of unstable anti-inflammatory function of exosomes in the prior art, making the function of exosomes more consistent and the expression profile controllable. 2. Based on the theory of cell synthesis flux optimization, this invention establishes a novel dynamic culture parameter control system, which breaks through the limitations of traditional culture methods. By precisely controlling multiple key culture parameters, it achieves a significant increase in exosome secretion, solves the problem of limited secretion, and can better meet clinical dosage requirements. 3. This invention employs an advanced control promoter system, which enables precise timing and dosage control of the expression of immune regulation-related genes. This overcomes the current lack of control in functional empowerment, indirectly enhances the loading capacity of exosomes, effectively improves the large batch-to-batch variation of exosomes, and increases batch-to-batch stability, which is beneficial for its clinical application. Attached Figure Description

[0008] Figure 1 This is a schematic diagram of the overall process of the present invention.

[0009] Figure 2 This is a schematic diagram of the exosome collection and purification module of the present invention. Detailed Implementation

[0010] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] Please see Figure 1 As shown, this invention provides a stem cell exosome immune regulation enhancement system based on synthetic biology regulation, including a synthetic gene circuit module, a dynamic parameter culture parameter regulation module, a control promoter system module, a stem cell exosome collection and purification module, a stem cell exosome quality and function detection module, and a stem cell exosome quality and function detection feedback module.

[0012] The synthetic gene circuit module is interconnected with the dynamic parameter culture parameter regulation module and the control promoter system module. The stem cell exosome collection and purification module is connected to the synthetic gene circuit module and the stem cell exosome quality and function detection module, respectively. The stem cell exosome quality and function detection feedback module is connected to the stem cell exosome quality and function detection module and the synthetic gene circuit module, respectively.

[0013] Synthetic gene circuit module: A non-coding RNA module capable of inducible regulation of expression is introduced into stem cells. A synthetic promoter module expressing miR-146a is designed and co-regulated with transcription factors and transfected into human umbilical cord mesenchymal stem cells. A synthetic gene circuit module containing a transfected cell line with validated stability is constructed and transferred to a dynamic culture parameter regulation module and a control promoter system module, including the following steps: This embodiment requires specific explanation of the following: non-coding RNA modules include miRNA, lncRNA, etc.; miRNA modules include miR-146a, miR-21, let-7, etc.; transfection refers to the technique of introducing exogenous genes (such as synthetic modules carrying miR-146a) into cells, and common methods include electroporation, liposome transfection, etc.; regulatory transcription factors such as NF-κB repressor; human umbilical cord mesenchymal stem cells (hUC-MSCs) naturally have high exosome production, and their efficiency is further improved after synthetic biology modification. The content of immunomodulatory molecules (such as miR-146a) carried by their exosomes is higher than that of MSCs from other sources.

[0014] S1.1: First, an inducible non-coding RNA module is introduced into stem cells to design a synthetic promoter module (such as the TRE system) for expressing miR-146a. Then, through transcription factor (TF) binding site prediction technology and experimental studies (such as chromatin immunoprecipitation, ChIP), target transcription factors that bind to the designed synthetic promoter and regulate miR-146a expression are screened, and a gene encoding the target transcription factor is constructed. Finally, the designed synthetic promoter module for expressing miR-146a, the transcription factor encoding gene, and the required regulatory elements (such as internal ribosome entry sites IRES, terminators, etc.) are linked to the selected vector in the required order to construct a complete synthetic gene loop vector. Simultaneously, the efficiency of miR-146a expression η(E1) is calculated, where η(E1) = C. tar / C tot C tar The expression level of target miR-146a was obtained by quantitative analysis of miR-146a using specific detection methods (such as qPCR) in the experiment. C tot The total miRNA expression level in stem cells was obtained through the corresponding detection method and used as the denominator to calculate the relative expression ratio of miR-146a in the total miRNA. If the miR-146a expression efficiency η(E1) does not meet the corresponding expected value, the sequences of the designed miR-146a expression promoter module, transcription factor encoding gene and required regulatory element are re-sequencing. This embodiment specifically describes the TRE (tetracycline response element) system design and synthesis promoter module. This module contains a tetracycline response element and a minimal promoter sequence, which can initiate the expression of downstream genes under the induction of tetracycline or its analogues (such as doxycycline). The vector, such as a plasmid vector or a viral vector, may contain elements such as a replication origin and selection marker genes (such as antibiotic resistance genes) to enable stable transfection and selection in cells.

[0015] This embodiment needs to specifically explain that bioinformatics analysis (such as gene expression profiling and transcription factor binding site prediction) is an existing technology, and transcription factor (TF) binding site prediction (such as using JASPAR, MEME Suite, and ChIP-seq data) is a conventional method for analyzing gene regulatory networks. Integrating bioinformatics analysis with experimental research (such as chromatin immunoprecipitation technology ChIP) can solve the shortcomings of relying solely on bioinformatics prediction (such as TF binding sites) or gene expression profiling analysis, which lacks functional verification.

[0016] S1.2: First, the constructed synthetic gene loop vector was transfected into hUC-MSCs using transfection technology. Simultaneously, the stem cell transfection efficiency η(E2) was calculated, where η(E2) = N. t / N0,N t The number of stem cells expressing the synthetic promoter module of miR-146a to be successfully transfected can be determined by methods such as fluorescent labeling (e.g., detecting the number of fluorescently positive cells by flow cytometry after transfection with an expression vector tagged with a fluorescent protein) or specific gene expression detection (e.g., detecting the expression of specific genes after transfection by PCR). N0 is the initial total number of stem cells used for the transfection experiment, accurately counted by a cell counter before the experiment begins. If the transfection efficiency η(E) of some cells does not meet the corresponding expected value, the transfection process is repeated until the expected value is reached. Transfection conditions can be adjusted, or different transfection methods can be tried to compare the transfection efficiency of different methods and select the most suitable transfection method. Then, according to the selection marker gene in the synthetic gene loop vector, the transfected hUC-MSCs are screened and amplified using the corresponding selection agent (e.g., antibiotics). The expression levels C of miR-146a and target transcription factor proteins are recorded at n different passage numbers. miR and C TF The gene expression stability coefficient η(S) was calculated, and finally, a synthetic gene circuit module containing transfected cell lines with verified stability was constructed based on the gene expression stability coefficient. This embodiment specifically describes the verification of cell line stability: Stable transfected cell lines were cultured through continuous passages. qRT-PCR was used to detect miR-146a expression, and Western blot was used to detect transcription factor protein expression. The expression levels of miR-146a and transcription factors were measured at different passage numbers, and the gene expression stability coefficient η(S) was calculated to assess the stability of gene expression in the cell lines. The expression level of miR-146a was recorded using qRT-PCR at n (n≥3) different passage numbers, denoted as C. miR-i , i=1,2,...,n, calculate the average expression level μ(C) of miR-146a.miR ) and standard deviation σ(C miR The protein expression levels of the target transcription factor were detected using Western blot technology and denoted as C. TF-i Let i = 1, 2, ..., n, and calculate the average expression level μ(C) of the target transcription factor protein. TF ) and standard deviation σ(C TF ), to obtain the gene expression stability coefficient η(S), η(S) = 1 / {a1×[σ(C miR ) / μ(C miR )]+a2×[σ(C TF ) / μ(C TF )]}, a1 and a2 are the corresponding weights, which can be set according to the importance of miR-146a and transcription factors in the study. For example, if the two are equally important, then a1=a2=0.5; if the gene expression stability coefficient η(S) is less than or equal to the corresponding threshold, it means that the cell line stability verification has passed, otherwise it means that the verification has failed, and the process of synthesizing gene circuit modules is repeated. This embodiment specifically illustrates that by constructing such a synthetic gene circuit, the expression of molecules related to the anti-inflammatory function of exosomes in stem cells can be precisely regulated, thereby enhancing the anti-inflammatory properties of exosomes.

[0017] The dynamic culture parameter control module involves seeding transfected cell lines with proven stability from the synthetic gene circuit module into a dynamic stem cell culture device. Doxycycline is used to activate the promoter expression system, inducing the synthesis of target miRNAs. Based on cell synthesis throughput optimization theory, environmental parameters during stem cell culture are regulated to establish a dynamic culture parameter control system, including the following steps: This embodiment specifically explains that the existing technology is based on the theory of cell synthesis flux optimization. By analyzing factors such as the synthesis pathways, reaction rates, and enzyme activities of intracellular metabolites, a mathematical model is established to optimize the cell synthesis flux in order to increase the yield of the target product. The dynamic culture parameter control module, based on the theory of cell synthesis flux optimization, can regulate the O2 concentration, nutrient supply rhythm, and micro-shear stress during the stem cell culture process to achieve a doubling of exosome secretion.

[0018] S2.1: Initial Culture Conditions Setup: First, set the initial culture parameter set TPS required for the stem cell dynamic culture equipment (such as a bioreactor) and the corresponding value for each parameter. TPS = [tp1, tp2, ..., tp...] n1 ], tp n1 The n1st parameter value is given; then, the transfected cell line containing the stable cell line is seeded into a dynamic stem cell culture device (such as a bioreactor). This embodiment requires specific explanation of the initial culture parameters, such as the temperature typically maintained at 37℃ (suitable for the growth of most mammalian stem cells), pH set between 7.2 and 7.4, oxygen concentration for mesenchymal stem cell culture at 1-5%, and stirring speed set at a low level (e.g., 30-50 rpm). It also includes nutrient supply rhythm and micro-shear stress. Taking glucose as an example, the nutrient supply rhythm is as follows: 4 g / L is added daily for days 1-3 (high nutrition promotes proliferation); 2 g / L is added every other day for days 4-6, then 1 g / L (reducing nutrition); glucose is stopped on day 7, and an antioxidant is used instead. Shear stress is controlled by adjusting the stirring speed, for example, by setting it to "5 dynes / cm², 10 dynes / cm²", to avoid mechanical damage to the cells.

[0019] In this embodiment, it should be specifically noted that the stem cell dynamic culture equipment has been cleaned, disinfected, and calibrated to ensure that the equipment can accurately control environmental parameters such as temperature, pH value, dissolved oxygen concentration, and stirring speed; at the same time, culture medium containing doxycycline (Dox) and other conventional cell culture reagents, such as fetal bovine serum, basal culture medium, and antibiotics, have been prepared.

[0020] S2.2: Activating the promoter expression system using Doxycycline: The Doxycycline induction concentration and induction time were obtained experimentally. Doxycycline solution corresponding to the required induction concentration was added to the dynamic stem cell culture device to achieve the optimal Doxycycline induction concentration in the culture medium. The initial culture parameter set TPS was then updated to TP, where TPS = [tp1, tp2, ..., tp...]. n1 [D], where [D] is the Dox induction concentration; then, stem cells are cultured in the dynamic stem cell culture device to induce the synthesis of target miRNA; In this embodiment, it is important to note that doxycycline is a tetracycline antibiotic used here as an inducer. When doxycycline is added to the system, it binds to relevant regulatory proteins, thereby altering the conformation of these proteins so that they can bind to specific promoters such as TRE, initiating the transcription of downstream genes. It also induces the synthesis of miRNAs or target factors: through the above mechanism, once the promoter is activated, it initiates the transcription of downstream genes encoding miRNAs (such as miR-146a) or target factors (such as immune regulation-related factors), ultimately synthesizing the corresponding miRNAs or target factors. Finally, it updates the initial culture parameter set TPS to TP. Due to the increased Dox induction concentration, the number of culture parameter types is n1+1.

[0021] S2.3: First, the culture parameters, such as temperature, pH, and dissolved oxygen concentration, are monitored in real time using sensors in the stem cell dynamic culture equipment. Then, based on the cell synthesis throughput optimization theory, the monitoring results of the culture parameters are adjusted in real time to construct a mathematical model Y for dynamic regulation of culture parameters: Y = y(TP) + K × ((y opt (J))-y(TPS)), where y(TP) is the updated culture parameter vector, K is the control gain coefficient, and y opt (J) represents the optimal culture parameters corresponding to the synthetic flux J, where J = c1 × (v miR / v miR,max )+c2×(v exo / v exo,max )-c3×(v dfj / v dfj,max ), v miR v exo and v dfj These represent the miRNA synthesis rate, exosome secretion rate, and metabolic waste accumulation rate, respectively. miR,max v exo,max and v dfj,max c1, c2, and c3 represent the corresponding maximum values, and c1, c2, and c3 are the corresponding weights. These can be fitted using experimental data, for example, c1=0.6, c2=0.3, and c3=0.1. y opt n1 (J) represents the optimal value of the n1th culture parameter, y min n1 and y max n1 These are the minimum and maximum values ​​of the n1th culture parameter, respectively, K. J K is the flux sensitivity coefficient, with a value of [0,1]. Taking dissolved oxygen as an example, K J =0.5 represents the strength of the effect of flux change on dissolved oxygen demand, J0 is the critical value of synthesis flux, and K=k0×e -λ×t +0.5×ΔJ, where t is the incubation time, λ is the time decay factor (usually λ is 0.1), and ΔJ is the synthesis flux deviation, ΔJ=|JJ tar | / J tar J tar The target is to synthesize the throughput; finally, the optimal values ​​of n1+1 culture parameters are traversed, and the culture parameters are dynamically adjusted to the optimal values. This embodiment specifically explains that, based on the existing technology of cell synthesis flux optimization theory, a mathematical model is established by analyzing factors such as the synthesis pathways, reaction rates, and enzyme activities of intracellular metabolites to optimize cell synthesis flux and increase the yield of target products.

[0022] S2.4: Calculate the comprehensive coefficient η(E3) of the relative expression level of the target miRNA and cell viability. E miR and E miR,max V represents the expression level of the target miRNA under the current culture conditions and the maximum expression level of the target miRNA under all experimental conditions, respectively. cell and V cell,max These represent the maximum values ​​of stem cell viability under the current culture conditions and under all experimental conditions, respectively. Cell viability was detected using cell viability assays (such as the MTT assay, CCK-8 assay, etc.) under both current and all experimental conditions, expressed as absorbance value or relative viability percentage. b1 and b2 are corresponding weights, which can be adjusted according to the experimental objective and the degree of emphasis on miRNA expression levels and cell viability. For example, if more emphasis is placed on miRNA expression levels, b1 = 0.7 and b2 = 0.3. If η(E3) is less than the corresponding threshold, it indicates abnormal dynamic culture parameter regulation; the dynamic culture parameter regulation process should be repeated. Conversely, if it is greater than the threshold, it indicates normal dynamic culture parameter regulation. Controlled promoter system module: This module controls the expression of immune regulatory genes in stem cells through time and dosage control, and transmits the results to the synthetic gene loop module and the dynamic culture parameter regulation module. The steps include: S3.1: Based on a control promoter system (such as Tet-on, Hypoxia Response Element), the expression of immunomodulatory genes (such as IDO, TSG6, PGE2) in stem cells is controlled by adding and removing inducers (such as Dox pulsed treatment) at timed intervals, while simultaneously switching oxygen concentrations. This yields a control time window for miRNA expression level E(t). By adjusting the inducer concentration (such as 0.1-10 μg / mLDox) and oxygen concentration (1% vs. 5% O2), the miRNA expression dose E(D, O2) is quantified, allowing for time- and dose-controlled regulation, thereby indirectly enhancing exosome loading capacity. t0 and t1 represent the start and removal times of the inducer addition, t2 represents the end time of the experiment, O1 and O2 represent different oxygen concentrations, O1 < O2, for example, O1∈[1%,5%], O2∈[1%,5%], k1 is the linear rate of increase of miRNA expression, reflecting the efficiency of HIF-1α stabilization and binding to HRE to drive gene expression under Dox induction. The larger k1 is, the faster the gene expression increases during the induction phase. k2 is the exponential rate of decay of miRNA expression, reflecting the degradation rate of HIF-1α after Dox removal and the inhibitory efficiency of oxygen concentration recovery on gene expression. The larger k2 is, the faster the gene expression decreases after Dox removal. k1 is obtained by fitting a straight line with the time difference (t-t0) as the independent variable and the miRNA expression level E(t) as the dependent variable using the least squares method, and the slope of the straight line is obtained. k2 is obtained by fitting a straight line with the time difference (t to t1) as the independent variable and ln(E(t)) as the dependent variable using the least squares method, and the absolute value of the slope of the straight line is obtained. E miR,max [D] represents the maximum miRNA expression level, [D] represents the Dox concentration (e.g., 0.1-10 μg / mL), and K represents the maximum expression level. d Let E be the dissociation constant of Dox, i.e., E = E miR,max Dox concentration at / 2 The half-inhibition constant of the nutrient concentration, i.e., E = E miR,max The oxygen concentration at / 2, d1 and d2 are Hill coefficients, which are obtained by fitting the Hill equation and reflect the synergistic effect of Dox on oxygen regulation; It should be specifically noted in this embodiment that the control promoter system is an existing technology. In the fields of biomedical research and gene therapy, control promoter systems have been widely used, such as Tet-on and Hypoxia Response Element. The Tet-on system is a gene expression system based on tetracycline regulation. HRE is a promoter element that is sensitive to hypoxia. Under hypoxia conditions, HRE can be activated, thereby regulating the expression of downstream genes.

[0023] In this embodiment, it is important to note that the Hill equation is a mathematical model used in biochemistry and physiology to describe the synergistic effect of ligands (such as oxygen molecules, drugs, hormones, etc.) binding to receptors (such as proteins, nucleic acids, etc.). In oxygen concentration-dependent gene expression regulation, the Hill equation is often used to quantify the dose-response relationship between oxygen concentration and hypoxia response element (HRE) activity. Its core significance lies in explaining the nonlinearity, saturation, and synergistic nature of binding between biomolecules.

[0024] S3.2: The miRNA expression levels obtained through time-controlled and dose-controlled transfer are transferred to the synthetic gene loop module and the dynamic culture parameter regulation module; Exosome collection and purification module: This module collects stem cell exosomes obtained from the synthetic gene circuit module, dynamic parameter culture parameter regulation module, and control promoter system module. It then uses ultracentrifugation combined with membrane filtration and charge-selective purification of the stem cell exosomes, including the following steps: S4.1: Collect stem cells containing exosomes. First, perform a low-speed (v1) centrifugation (v1) at a speed lower than the specified speed (e.g., 300×g, 10 minutes) to remove cell pellet. Then, perform a second centrifugation at a speed (v2) (e.g., 500×g, 15 minutes) to remove cell debris. V1 is lower than V2. Then, transfer the exosome-containing cells after the second centrifugation to an ultracentrifuge tube and perform a third centrifugation. Next, filter the cells using a membrane filter with the target pore size (e.g., a 100nm pore size polycarbonate or polyethersulfone membrane) to further remove impurities smaller than exosomes, such as protein aggregates and small molecule metabolites, while also concentrating the exosomes. Then, determine the surface charge characteristics of the exosomes after membrane filtration, including positive and negative charges. Select an ion chromatography column with a charge characteristic matching that of the exosomes and purify the exosomes by binding them to oppositely charged groups on the chromatography medium to obtain purified stem cell exosomes. S4.2: Calculate the exosome recovery rate (RR) exo , C fin and C ini V represents the concentrations of exosomes in the initial exosome culture solution and the concentrations of purified exosomes, respectively, which can be determined by nanoparticle tracking analysis (NTA) or other exosome quantification methods. fin and V ini These represent the initial exosome culture solution volume and the purified exosome solution volume, respectively; if RR exo If the value is less than the corresponding threshold, it indicates abnormal exosome recovery. Repeat the exosome collection and purification module process. Otherwise, it indicates normal exosome recovery. Quality and Function Assay Module: Used to detect the quality and functional data of purified exosomes. Quality data includes exosome particle size distribution, marker protein and miRNA expression profiles. Functional data includes assessment of IL-10 upregulation and TNF-α downregulation rates using macrophage co-culture experiments, and assessment of exosome regulatory indicators of inflammatory responses using anti-inflammatory models, including the following steps: S5.1: First, use a camera to record the displacements of each exosome particle in the x and y directions at m different time points, respectively, Δx and Δy. Calculate the mean square displacement Δr of each particle at the m different time points. 2 (Δr) 2 =Δx 2 +Δy 2 The relationship between Δr and the diffusion coefficient D is:2 =4DΔt, based on the Stokes-Einstein equation, D=Δr 2 Substituting / 4Δt, we obtain the radius of each exosome particle. Then, a statistical analysis was performed on the radii of all exosome particles, including calculating the mean and standard deviation of the particle size, to obtain the distribution of exosome particle size. S5.2: First, Western blot was used to detect proteins in exosomes and obtain a protein type dataset PD, where PD = [P1, P2, ..., P...]. m ], where m represents the type of marker protein. The protein type dataset PD is divided into a marker protein type dataset PD1 (e.g., marker proteins are CD63 / CD9 / CD81) and a contaminant protein type dataset PD2 (e.g., serum proteins, cell debris proteins). Then, the exosome purity CR is calculated based on the marker protein type. exo , C mar，l The concentration of the first type of exosome marker protein, such as the CD63 protein concentration C. mar，1 =0.5 mg / mL, CD9 protein concentration C mar，2 =0.3 mg / mL, C imp，L The concentration of the first type of exosomal impurity protein, such as serum albumin concentration C. imp，1 =1 mg / mL, cell debris protein concentration C imp，2 =0.2 mg / mL, l∈m, L∈m, V ini and V fin The volumes are the purified exosome solution and the initial exosome culture solution, respectively. S5.3: Extract total RNA from exosomes and use high-throughput sequencing technology to obtain the relative expression levels (C) of various miRNAs (e.g., miR-146a, miR-21, let-7, etc.) in exosomes. Construct a miRNA expression profile by traversing the relative expression levels of each miRNA. Cou exo and Cou cun These represent the sequencing reads of each miRNA in the experimental group (exosome samples) and the sequencing reads in the control group (e.g., normal cell samples), respectively. c is a constant (usually a small value, such as 1), used to avoid the condition that Cou... exo and Cou cun When the value is 0, it can result in infinity; for example, in miRNA sequencing experiments, miR-146a's sequencing read Cou in the control group... cun =10, in the sequencing read Cou of the exosome experimental group exo =40, take c=1, then the relative expression level of miR-146a is... This indicates that the expression level of miR-146a in the exosome samples was approximately 2% lower than that in the control group. 1.89 An upward adjustment of approximately 3.66 times; S5.4: The levels of IL-10 and TNF-α in the culture supernatant were detected using enzyme-linked immunosorbent assay (ELISA) in macrophage co-culture experiments. The upregulation rate of IL-10 (η(IL-10)) and the downregulation rate of TNF-α (η(TNF-α)) by exosomes on macrophages were assessed by comparing the results before and after co-culture. C IL-10,exo The concentration of IL-10 in the supernatant after co-culturing exosomes and macrophages, C IL-10,cun The concentration of IL-10 in the culture supernatant of macrophages in the control group (without exosome treatment) C TNF-α,exo The concentration of TNF-α in the supernatant after co-culturing exosomes and macrophages, C TNF-α,cun The concentration of TNF-α in the culture supernatant of macrophages in the control group (without exosome treatment); S5.5: Using an anti-inflammatory model, such as a lipopolysaccharide (LPS)-induced macrophage inflammation model, exosomes were pretreated in inflammatory model cells, and the concentration C of the anti-inflammatory factor IL-10 in the exosomes was calculated. IL-10 With pro-inflammatory factors TNF-α and C IL-6 The concentration ratio of exosomes to inflammatory responses (CC) is a regulatory indicator of exosomes' role in inflammatory responses. C TNF-α and C IL-6 These are the pro-inflammatory factors TNF-α and C. IL-6 The concentration; This embodiment specifically describes the verification of the regulatory effect of exosomes on the inflammatory response using an anti-inflammatory model. The anti-inflammatory model involves selecting a macrophage cell line (such as RAW264.7 cells), lipopolysaccharide (LPS), exosomes, and cell culture medium. Using prepared experimental equipment such as a cell culture incubator, centrifuge, ELISA reader, and microscope, macrophages are seeded into culture dishes. The cells are divided into a control group, an LPS stimulation group, and an exosome treatment group. The control group receives no treatment; the LPS stimulation group receives LPS solution; and the exosome treatment group is pretreated with exosomes for a certain period before receiving LPS solution, thus constructing the anti-inflammatory model.

[0025] Stem cell exosome quality and function detection feedback module: This module compares the quality and function data of exosomes with corresponding thresholds, and feeds back any abnormal comparison results to the management terminal for human-computer interaction. The steps include: S6.1: If the exosome purity CR exoIf the value is less than the corresponding threshold, it indicates abnormal exosome purity, which is reported to the management terminal to optimize the exosome purification process; otherwise, it indicates normal exosome purity. If the relative expression level C of each miRNA is less than the corresponding threshold, it indicates abnormal relative expression level of each miRNA, which is reported to the management terminal to optimize the synthetic gene circuit module, dynamic parameter culture parameter regulation module, and control promoter system module; otherwise, it indicates normal relative expression level of each miRNA. S6.2: If the upregulation rate η(IL-10) of exosomes on macrophages is less than the corresponding threshold or the downregulation rate η(TNF-α) of exosomes on macrophages is less than the corresponding threshold, it indicates an abnormality in the macrophage co-culture experiment. This is reported to the management terminal to optimize the macrophage co-culture conditions, including exosome concentration quality checks, co-culture condition optimization, and macrophage status checks. Otherwise, it indicates normality. If the concentration ratio CC of anti-inflammatory factors to pro-inflammatory factors in exosomes is less than the corresponding threshold, it indicates an abnormality in the anti-inflammatory model. This is reported to the management terminal to optimize the anti-inflammatory model conditions, including exosome function verification and model establishment checks. For example, a more sensitive inflammation model (such as an LPS-induced acute pneumonia model) may be used, or the exosome dosage may be increased.

[0026] Secondly: The accompanying drawings of the embodiments disclosed in this invention only involve the structures involved in the embodiments disclosed in this invention. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of this invention can be combined with each other. In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A stem cell exosome immune regulation enhancement system based on synthetic biology regulation, characterized in that: include: Synthetic gene circuit module: Introduce an inducible non-coding RNA module in stem cells, design a synthetic promoter module expressing miR-146a to jointly regulate and transfect human umbilical cord mesenchymal stem cells with transcription factors, construct a synthetic gene circuit module containing transfected cell lines with proven stability, and transfer it to the dynamic culture parameter regulation module and the control promoter system module. The dynamic culture parameter regulation module seeds transfected cell lines containing stable and validated cells from the synthetic gene loop module into a dynamic stem cell culture device. It uses Doxycycline to activate the promoter expression system, induces the synthesis of target miRNA, and regulates environmental parameters during stem cell culture based on the cell synthesis throughput optimization theory, thus establishing a dynamic culture parameter regulation system. Controlled promoter system module: Through the controlled promoter system, the expression of immune regulatory genes in stem cells is controlled in terms of time and dosage, and the results of time and dosage control are transmitted to the synthetic gene loop module and the dynamic culture parameter regulation module; Exosome collection and purification module: Collects stem cell exosomes obtained from the synthetic gene circuit module, dynamic parameter culture parameter regulation module, and control promoter system module, and uses ultracentrifugation combined with membrane filtration process, combined with charge-selective purification of stem cell exosomes; Quality and Function Detection Module: Used to detect the quality and functional data of purified exosomes. Quality data includes exosome particle size distribution, marker protein and miRNA expression profiles. Functional data includes assessment of IL-10 upregulation rate and TNF-α downregulation rate through macrophage co-culture experiments, and assessment of exosome regulatory indicators of inflammatory response through anti-inflammatory models. Stem cell exosome quality and function detection feedback module: compares the quality and function data of exosomes with the corresponding thresholds, and feeds back the abnormal comparison results to the management terminal for human-computer interaction.

2. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The synthetic gene loop module is implemented as follows: S1.1: First, an inducible non-coding RNA module is introduced into stem cells to design a synthetic promoter module for expressing miR-146a; then, target transcription factors that bind to the designed synthetic promoter and regulate miR-146a expression are screened, and a gene encoding the target transcription factor is constructed; finally, the designed synthetic promoter module for expressing miR-146a, the transcription factor encoding gene, and the required regulatory elements are linked to the selected vector in the required order to construct a complete synthetic gene loop vector; simultaneously, the efficiency of miR-146a expression η(E1) is calculated, where η(E1) = C tar / C tot C tar For the expression level of target miR-146a, C tot The total miRNA expression level in stem cells is given. If the miR-146a expression efficiency η(E1) does not meet the expected value, the sequences of the designed miR-146a expression promoter module, transcription factor encoding gene, and required regulatory elements will be re-sequencing.

3. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The implementation of the synthetic gene circuit module further includes: S1.2: First, the constructed synthetic gene circuit vector is transfected into hUC-MSCs using transfection technology, and the stem cell transfection efficiency η(E2) is calculated simultaneously, where η(E2) = N. t / N0,N t To successfully transfect the synthetic promoter module expressing miR-146a, the number of stem cells N0 is the initial total number of stem cells used in the transfection experiment. If the transfection efficiency η(E) of some cells does not meet the expected value, the transfection process is repeated until the expected value is reached. Then, based on the selection marker gene in the synthetic gene loop vector, the transfected hUC-MSCs are screened and amplified using the corresponding selection agent. The expression levels C of miR-146a and the target transcription factor protein are recorded at n different passage numbers. miR and C TF The gene expression stability coefficient η(S) was calculated, and finally, a synthetic gene circuit module containing transfected cell lines with verified stability was constructed based on the gene expression stability coefficient.

4. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The dynamic culture parameter control module includes: S2.1: Initial culture condition setting: First, set the initial culture parameter type set TPS required by the stem cell dynamic culture device and the corresponding value of each parameter, TPS=[tp1,tp2,...,tp n1 ], tp n1 The n1st parameter value is used; then, the transfected cell line containing the stable cell line is seeded into the stem cell dynamic culture device. S2.2: Activating the promoter expression system using Doxycycline: The Dox induction concentration and induction time were obtained experimentally. The required Dox induction concentration of Dox solution was added to the dynamic stem cell culture device, and the initial culture parameter set TPS was updated to TP, where TPS = [tp1, tp2, ..., tp...]. n1 [D], where [D] is the Dox induction concentration; then, stem cells are cultured in the dynamic stem cell culture device to induce the synthesis of target miRNA.

5. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The aforementioned dynamic culture parameter control module further includes: S2.3: First, the culture parameters are monitored in real time using sensors in the stem cell dynamic culture device; then, the culture parameter monitoring results are adjusted in real time using the cell synthesis throughput optimization theory, and a dynamic control mathematical model Y for culture parameters is constructed, Y=y(TP)+K×((y opt (J))-y(TPS)), where y(TP) is the updated culture parameter vector, K is the control gain coefficient, and y opt (J) represents the optimal culture parameters corresponding to the synthetic flux J. y opt n1 (J) represents the optimal value of the n1th culture parameter, y min n1 and y max n1 These are the minimum and maximum values ​​of the n1th culture parameter, respectively, K. J J0 is the flux sensitivity coefficient, with a value of [0,1], and J0 is the critical value for synthesis flux. Finally, the optimal values ​​of n1+1 culture parameters are iterated and the culture parameters are dynamically adjusted to the optimal values. S2.4: Calculate the comprehensive coefficient η(E3) of the relative expression level of the target miRNA and cell viability. E miR and E miR,max V represents the expression level of the target miRNA under the current culture conditions and the maximum expression level of the target miRNA under all experimental conditions, respectively. cell and V cell,max b1 and b2 are the maximum values ​​of stem cell viability under the current culture conditions and under all experimental conditions, respectively, and the corresponding weights are b1 and b2. If η(E3) is less than the corresponding threshold, it indicates that the dynamic culture parameter regulation is abnormal and the dynamic culture parameter regulation process should be repeated. Otherwise, it indicates that the dynamic culture parameter regulation is normal.

6. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The controlled promoter subsystem module includes: S3.1: Based on the controlled promoter subsystem, the expression of immune regulatory genes in stem cells is controlled by adding and removing inducers at timed intervals, while switching oxygen concentrations, to obtain the miRNA expression level E(t) within a control time window. By adjusting the inducer concentration and oxygen concentration, the miRNA expression dose E(D, O2) is quantified, and time and dose control are performed respectively. t0 and t1 represent the start and removal times of the inducer addition, t2 represents the end time of the experiment, O1 and O2 represent different oxygen concentrations, O1 < O2, k1 represents the linear increase rate of miRNA expression, and k2 represents the exponential decay rate of miRNA expression. E miR,max [D] represents the maximum expression level of miRNA, [D] represents the Dox concentration, and K represents the maximum expression level of miRNA. d Let be the Dox dissociation constant. d1 and d2 are the half-inhibition constants of the nutrient concentration, and Hill's coefficients. S3.2: The miRNA expression levels obtained through time-controlled and dose-controlled transfer are transferred to the synthetic gene loop module and the dynamic culture parameter regulation module.

7. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The exosome collection and purification module includes the following steps: S4.1: Collecting stem cells containing exosomes, firstly centrifuging at a low speed (v1) determined to be less than a specified speed to remove cell pellets, then centrifuging at a speed (v2) to remove cell debris, where v1 is less than v2; then transferring the exosome-containing cells after the second centrifugation to an ultracentrifuge tube for a third centrifugation; next, filtering with a membrane filter of the target pore size, then determining the surface charge characteristics of the exosomes after membrane filtration, including positive and negative charges, selecting an ion chromatography column with a charge characteristic matching the exosomes, and purifying the exosomes by binding with oppositely charged groups on the chromatography medium to obtain purified stem cell exosomes; S4.2: Calculate the exosome recovery rate (RR) exo , C fin and C ini V represents the concentration of exosomes in the initial exosome culture solution and the concentration of exosomes after purification. fin and V ini These represent the initial exosome culture solution volume and the purified exosome solution volume, respectively; if RR exo If the value is below the corresponding threshold, it indicates abnormal exosome recovery, and the exosome collection and purification module process should be repeated; otherwise, it indicates normal exosome recovery.

8. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The quality and function detection module includes: S5.1: First, using a camera, record the displacements of each exosome particle in the x and y directions at m different time points, respectively, Δx and Δy, and calculate the mean square displacement Δr of each particle at the m different time points. 2 The relationship with the diffusion coefficient D is: Δr 2 =4DΔt, based on the Stokes-Einstein equation, D=Δr 2 Substituting / 4Δt, we obtain the radius of each exosome particle. Then, a statistical analysis was performed on the radii of all exosome particles, including calculating the mean and standard deviation of the particle size, to obtain the distribution of exosome particle size. S5.2: First, the Western blotting method is used to detect proteins in exosomes and obtain a protein type dataset PD, where PD = [P1, P2, ..., P...]. m [], where m represents the type of marker protein. The protein type dataset PD is divided into a marker protein type dataset PD1 and an impurity protein type dataset PD2; then, the exosome purity CR is calculated based on the marker protein type. exo ; S5.3: Extract total RNA from exosomes, obtain the relative expression level C of various miRNAs in exosomes using high-throughput sequencing technology, and construct a miRNA expression profile by iterating through the relative expression levels of each miRNA.

9. The stem cell exosome immune regulation enhancement system based on synthetic biology regulation according to claim 1, characterized in that: The quality and function detection module also includes: S5.4: Through macrophage co-culture experiment, enzyme-linked immunosorbent assay is used to detect the content of IL-10 and TNF-α in culture supernatant, and the upregulation rate η(IL-10) and downregulation rate η(TNF-α) of exosomes on macrophages are evaluated by comparing before and after co-culture. S5.5: Using an anti-inflammatory model, exosomes were pretreated with inflammatory model cells, and the concentrations of the anti-inflammatory factor IL-10 and the pro-inflammatory factors TNF-α and C in the exosomes were calculated. IL-6 The concentration ratio of exosomes to inflammatory responses is a key indicator of exosome regulation of inflammatory responses.

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