Disease model macrophage in-vivo programming method

Abstract

The invention relates to the technical field of immune research, in particular to a disease model macrophage in-vivo programming method which comprises the following steps: constructing a target transcription factor plasmid containing a peptide barcode protein sequence; wherein the target transcription factor plasmid contains the peptide barcode protein sequence and a tdTomato fluorescent protein sequence; forming a target transcription factor mRNA in vitro by taking the target transcription factor plasmid as a template; combining and embedding the target transcription factor mRNA into lipid nanoparticles; and injecting the lipid nanoparticles into the body of a mouse through intravenous injection, enabling the lipid nanoparticles to be stably expressed in the body of the mouse, taking the lipid nanoparticles as a disease animal model, and collecting a tissue sample. According to the invention, specific transcription factors and a digital coding technology are utilized, so that the efficiency of transforming the B cells into the macrophages is improved. In-vivo imaging and evaluation of transcription factors are realized through in-vivo delivery of lipid nanoparticles. And a new technical means is provided for process monitoring and mechanism research of a disease model.

Description

Technical Field

[0001] The present invention relates to the technical field of immune research, and in particular to a method for in vivo programming of disease model macrophages. Background Art

[0002] Aiming at the problem of autoreactive B cells and plasma cells overload and proliferation, which is the main pathological feature of the complex disease lupus, we use transcription factors that play an important role in the transformation of B cells into macrophages, design gene circuits, and combine lipid nanoparticle delivery in vivo, digital encoding of nucleic acid molecules, spatial omics imaging and other methods to complete the task of monitoring newly transformed macrophages in the evolution of lupus, draw a spatial omics map of macrophages in multiple tissue regions of disease animal models, and achieve the goal of extracting transcription factor features that drive the transformation of B cells into macrophages in multiple tissue regions such as bone marrow, spleen, kidney, heart and lungs, and improve prognosis. However, traditional gene editing has low efficiency and long cell engineering cycles, which makes it difficult to meet the needs of clinical transformation of macrophage programming. Summary of the invention

[0003] Based on this, the present invention proposes a method for in vivo programming of disease model macrophages to solve the problems of low efficiency and long technical cycle of macrophage programming in the prior art.

[0004] The core of the present invention is to utilize specific transcription factors, such as CEBPA, CEBPB, NRF2, etc., which play a key role in the transformation of B cells into macrophages. By designing a specific gene circuit, the transformation efficiency of directional macrophages is improved and the transformation cycle is shortened. The digital coding technology of nucleic acid molecules is used to precisely regulate the transcription factor gene circuit to achieve the regulation of the transformation process of B cells into macrophages.

[0005] According to a first aspect of the present invention, a method for in vivo programming of disease model macrophages is provided, comprising:

[0006] Constructing a target transcription factor plasmid comprising a peptide barcode protein sequence; wherein the target transcription factor plasmid comprises the peptide barcode protein sequence and a tdTomato fluorescent protein sequence;

[0007] by using the target transcription factor plasmid as a template to form the target transcription factor mRNA in vitro;

[0008] The target transcription factor mRNA is bound and embedded in lipid nanoparticles;

[0009] The lipid nanoparticles are intravenously injected into disease model mice to allow the lipid nanoparticles to be stably expressed in the mice, and then tissue samples are collected.

[0010] According to an embodiment of the present invention, the construction of a target transcription factor plasmid containing a peptide barcode comprises:

[0011] Designing a specific peptide barcode protein sequence for each mRNA encoded by a relevant transcription factor;

[0012] The specific barcode sequence is inserted into the plasmid vector by the Golden Gate cloning method; the coding sequence of each transcription factor is inserted into the plasmid vector, and each transcription factor uses a different BsaI restriction enzyme cutting site to form the target transcription factor plasmid.

[0013] According to an embodiment of the present invention, the target transcription factor mRNA is formed in vitro by using the target transcription factor plasmid as a template, comprising:

[0014] Transforming the target transcription factor plasmid into host cells, and culturing the host cells at 37° C. until OD600 is 0.6;

[0015] 5-10 mg / L of linearized plasmid DNA was screened and extracted on a medium containing corresponding antibiotics as a transcription template;

[0016] In a 50 mL reaction system containing 10 mL transcription buffer, add linearized plasmid DNA, T7 RNA polymerase, 2 mM ATP, 2 mM GTP, 2 mM CTP, 2 mM UTP, and 0.5 mM N1-Me-pUTP;

[0017] The reaction system was placed in a 37°C water bath and incubated for 4 hours to obtain mRNA;

[0018] The mRNA is purified by chloroform extraction and ethanol precipitation, and quantified by ultraviolet absorption spectroscopy to detect the purity and integrity of the mRNA, confirm that the 5' end of the mRNA has the correct cap structure, and obtain the target transcription factor mRNA.

[0019] According to an embodiment of the present invention, the step of embedding the target transcription factor mRNA in lipid nanoparticles comprises:

[0020] Dissolve the cationic lipid, cholesterol, DMG-PEG and auxiliary lipid in ethanol at a ratio of 5:5:10:1 to obtain a mixed solution;

[0021] Dissolving the target transcription factor mRNA in a citrate buffer having a pH value of 4.0 to obtain an mRNA solution;

[0022] The mixed solution and the mRNA solution are mixed to ensure that the ratio of water to ethanol is 3:1, so as to achieve a final weight ratio of lipid to RNA of 40:1; and stable lipid nanoparticles are formed.

[0023] According to an embodiment of the present invention, the present invention further includes performing imaging detection on tissue samples of the disease animal model; comprising:

[0024] performing spatial omics imaging on the tissue sample;

[0025] Imaging using confocal microscopy;

[0026] Perform quantitative analysis by liquid chromatography-mass spectrometry;

[0027] Monitoring and computational analysis of macrophage transformation progression were completed for each of the peptide barcoded protein sequences.

[0028] According to an embodiment of the present invention, performing spatial omics imaging on the tissue sample includes:

[0029] Collect peripheral blood and bone marrow, spleen, kidney, lung and other tissue samples from the disease model and make slices;

[0030] The fixed sections were permeabilized and spatial omics imaging was performed using STARmap technology;

[0031] Using a probe to perform in situ hybridization on the target transcription factor mRNA, labeling the target transcription factor mRNA, and converting it into cDNA with a fluorescent label through an enzymatic reaction;

[0032] While detecting the target transcription factor mRNA, immunofluorescence staining of tissue proteins is performed, using specific primary and secondary antibodies against disease marker proteins for fluorescent labeling;

[0033] Imaging the fluorescently labeled cDNA and protein signals through a confocal microscope to obtain multi-channel fluorescent images to provide raw data for subsequent data analysis;

[0034] The acquired fluorescence images were processed using image analysis software, including preprocessing steps such as denoising, contrast enhancement, and image registration;

[0035] The cDNA and protein signals in each cell are identified and located by using the set thresholds and algorithms, generating single-cell level gene expression and protein localization maps;

[0036] Spatial statistical methods are used to analyze the spatial distribution characteristics of cell subtypes and disease marker proteins in tissues to reveal their potential roles in the disease process.

[0037] According to an embodiment of the present invention, the imaging using a confocal microscope comprises:

[0038] Peripheral blood and tissue samples such as bone marrow, spleen, kidney and lung were collected from the disease model and made into 10 μm thick sections;

[0039] A laser scanning confocal microscope was used, and the excitation wavelength was selected as 554 nm and the emission wavelength was selected as 581 nm using a red channel filter;

[0040] Find the focal plane of the sample in brightfield mode, then switch to fluorescence mode;

[0041] Set the resolution to 1024x1024 pixels, the scan speed to 400Hz, and the pinhole size to 1 Airy unit;

[0042] Imaris was used for analysis to monitor and compute the macrophage transformation process.

[0043] According to an embodiment of the present invention, the liquid chromatography-mass spectrometry quantitative analysis comprises:

[0044] Purifying the protein with the peptide barcode protein sequence using affinity chromatography;

[0045] 1 μl of peptide sample was loaded directly onto one column;

[0046] A high performance liquid chromatograph was used, the initial mobile phase was a pure water phase doped with 0.1% formic acid, and the corresponding organic solvent phase was composed of pure acetonitrile, also containing 0.1% formic acid;

[0047] The elution program was designed as a gradient mode, with the following specific steps: initially, the proportion of the organic solvent phase was set at 1%, then gradually increased to 7% within 5 minutes, and then continued to increase to 25% and 36%. After reaching 36%, the proportion of the organic solvent phase in the mobile phase was rapidly increased to 80%, and maintained at this proportion for 4.5 minutes. After completing this step, the mobile phase was rapidly converted back to 99% of the aqueous phase and maintained at this proportion for 19.9 minutes to complete the thorough elution of the peptides;

[0048] The eluted peptides were introduced directly into the mass spectrometer by electrospray using a distal voltage of 2.2 kV;

[0049] The relative abundance of peptides was obtained, peptide barcodes were quantified, and the effects of different transcription factors on the transformation of B cells into macrophages were analyzed.

[0050] According to an embodiment of the present invention, the peptide barcode protein sequence includes a CEBPA barcode, a CEBPB barcode, an ACOD1 barcode, and an NRF2 barcode;

[0051] The CEBPA barcode is shown in SEQ ID NO.1;

[0052] The CEBPB barcode is shown in SEQ ID NO.2;

[0053] The ACOD1 barcode is shown in SEQ ID NO.3;

[0054] The NRF2 barcode is shown in SEQ ID NO.4.

[0055] The beneficial effects of the present invention are:

[0056] This paper provides a rapid and accurate method for in vivo programming of disease model macrophages. The efficiency of B cell to macrophage transformation is improved by using specific transcription factors and digital coding technology. In vivo imaging and evaluation of transcription factors are achieved through in vivo delivery of lipid nanoparticles. Spatial omics imaging technology is used to monitor the distribution and expression of newly transformed macrophages in real time. This paper provides a new technical means for the establishment of disease models and the monitoring of disease progression.

[0057] The protection scope of the present invention is not limited to the above-mentioned specific embodiments. Any equivalent changes made by those skilled in the art within the scope disclosed in the present invention should be included in the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A schematic diagram of the process of programming a carrier according to an embodiment of the present invention. DETAILED DESCRIPTION

[0059] According to a first aspect of the present invention, Figure 1 As shown, a method for in vivo programming of disease model macrophages is provided, comprising:

[0060] Constructing a target transcription factor plasmid containing a peptide barcode protein sequence; wherein the target transcription factor plasmid contains a peptide barcode protein sequence and a tdTomato fluorescent protein sequence;

[0061] Plasmid Construction Unique barcode sequences were designed for each mRNA encoding the relevant transcription factor and inserted into the plasmid vector by GoldenGate cloning method. A plasmid containing the peptide barcode protein sequence and the tdTomato fluorescent protein sequence was constructed to facilitate monitoring of transcription factor expression.

[0062] The target transcription factor mRNA is formed in vitro by using the target transcription factor plasmid as a template;

[0063] In vitro RNA generation: Host cells were cultured at 37°C until OD600 was about 0.6, and linearized plasmid DNA was extracted from the culture as a transcription template. Linearized plasmid DNA template, T7 RNA polymerase, nucleoside triphosphates, etc. were added to the reaction system containing transcription buffer for in vitro transcription to obtain the target mRNA.

[0064] The target transcription factor mRNA is bound and embedded in lipid nanoparticles;

[0065] Synthesis of Lipid Nanoparticles: Dissolve cationic lipids, cholesterol, DMG-PEG and auxiliary lipids in ethanol in proportion to obtain a mixed solution; dissolve the RNA obtained by in vitro transcription in a citrate buffer with a pH value of 4.0. The two solutions are quickly mixed to form stable lipid nanoparticles for in vivo delivery.

[0066] The lipid nanoparticles were injected intravenously into disease model mice, and tissue samples were collected after the lipid nanoparticles were stably expressed in the mice.

[0067] In vivo delivery and monitoring The lipid nanoparticles were injected intravenously into mice to stably express the lipid nanoparticles in the mice. Tissue samples of the disease animal model were collected and the tissues were lysed by freezing and grinding to obtain tissue lysates.

[0068] According to an embodiment of the present invention, constructing a target transcription factor plasmid containing a peptide barcode comprises:

[0069] Design specific peptide barcode protein sequences for each mRNA encoding relevant transcription factors;

[0070] The specific barcode sequence was inserted into the plasmid vector by the Golden Gate cloning method; the coding sequence of each transcription factor was inserted into the plasmid vector, and each transcription factor used a different BsaI restriction enzyme cutting site to form a target transcription factor plasmid.

[0071] According to an embodiment of the present invention, the target transcription factor mRNA is formed in vitro by using the target transcription factor plasmid as a template, comprising:

[0072] The target transcription factor plasmid was transformed into host cells, and the host cells were cultured at 37°C until OD600 reached 0.6;

[0073] 5-10 mg / L of linearized plasmid DNA was screened and extracted on a medium containing corresponding antibiotics as a transcription template;

[0074] In a 50 mL reaction system containing 10 mL transcription buffer, add linearized plasmid DNA, T7 RNA polymerase, 2 mM ATP, 2 mM GTP, 2 mM CTP, 2 mM UTP, and 0.5 mM N1-Me-pUTP;

[0075] The reaction system was placed in a 37°C water bath and incubated for 4 hours to obtain mRNA;

[0076] The mRNA was purified by chloroform extraction and ethanol precipitation, and quantified by ultraviolet absorption spectroscopy to detect the purity and integrity of the mRNA, confirming that the 5' end of the mRNA had the correct cap structure, and obtain the target transcription factor mRNA.

[0077] According to an embodiment of the present invention, the target transcription factor mRNA is embedded in lipid nanoparticles, comprising:

[0078] Dissolve the cationic lipid, cholesterol, DMG-PEG and auxiliary lipid in ethanol at a ratio of 5:5:10:1 to obtain a mixed solution;

[0079] dissolving the target transcription factor mRNA in a citrate buffer having a pH value of 4.0 to obtain an mRNA solution;

[0080] The mixture and the mRNA solution were mixed, ensuring that the ratio of water to ethanol was 3:1, so as to achieve a final weight ratio of lipid to RNA of 40:1; stable lipid nanoparticles were formed.

[0081] According to an embodiment of the present invention, the present invention further includes performing imaging detection on tissue samples of disease animal models; including:

[0082] Perform spatial omics imaging of tissue samples;

[0083] Imaging using confocal microscopy;

[0084] Perform quantitative analysis by liquid chromatography-mass spectrometry;

[0085] Complete monitoring of macrophage transformation progression and computational analysis for each peptide barcoded protein sequence.

[0086] Protein analysis uses affinity chromatography to obtain proteins containing peptide barcodes in tissue samples. The peptide barcodes of proteins are cut out using tobacco aphid virus protease to obtain peptide barcodes. The molecular weight of the peptide barcode and the relative abundance of the peptides are obtained by mass analysis of the peptide barcodes and their fragmented peptides.

[0087] Imaging analysis uses confocal microscopy to monitor and computationally analyze the macrophage transformation process in specific tissue samples.

[0088] Through the above steps, the present invention can provide a basis for the establishment of disease models and the monitoring of disease progression, and at the same time provide technical support for the realization of the immunotherapy goals of complex diseases. The embodiments of the present invention also describe in detail the key steps such as plasmid construction, in vitro RNA generation, lipid nanoparticle synthesis, and multimodal small animal imaging to ensure the operability and effectiveness of the present invention.

[0089] According to an embodiment of the present invention, spatial omics imaging is performed on a tissue sample, including:

[0090] Collect peripheral blood and bone marrow, spleen, kidney, lung and other tissue samples from the disease model and make slices;

[0091] The fixed sections were permeabilized and spatial omics imaging was performed using STARmap technology;

[0092] Using the probe to perform in situ hybridization on the target transcription factor mRNA, labeling the target transcription factor mRNA, and converting it into fluorescently labeled cDNA through an enzymatic reaction;

[0093] While detecting the target transcription factor mRNA, immunofluorescence staining of tissue proteins was performed using specific primary and secondary antibodies against disease marker proteins for fluorescent labeling;

[0094] The fluorescently labeled cDNA and protein signals are imaged through a confocal microscope to obtain multi-channel fluorescence images, providing raw data for subsequent data analysis;

[0095] The acquired fluorescence images were processed using image analysis software, including preprocessing steps such as denoising, contrast enhancement, and image registration;

[0096] The cDNA and protein signals in each cell are identified and located by using the set thresholds and algorithms, generating single-cell level gene expression and protein localization maps;

[0097] Spatial statistical methods are used to analyze the spatial distribution characteristics of cell subtypes and disease marker proteins in tissues to reveal their potential roles in the disease process.

[0098] According to an embodiment of the present invention, imaging using a confocal microscope includes:

[0099] Peripheral blood and tissue samples such as bone marrow, spleen, kidney and lung were collected from the disease model and made into 10 μm thick sections;

[0100] A laser scanning confocal microscope was used, and the excitation wavelength was selected as 554 nm and the emission wavelength was selected as 581 nm using a red channel filter;

[0101] Find the focal plane of the sample in brightfield mode, then switch to fluorescence mode;

[0102] Set the resolution to 1024x1024 pixels, the scan speed to 400Hz, and the pinhole size to 1 Airy unit;

[0103] Imaris was used for analysis to monitor and compute the macrophage transformation process.

[0104] According to an embodiment of the present invention, performing liquid chromatography-mass spectrometry quantitative analysis includes:

[0105] Proteins with peptide barcoded protein sequences were purified using affinity chromatography;

[0106] 1 μl of peptide sample was loaded directly onto one column;

[0107] A high performance liquid chromatograph was used, the initial mobile phase was a pure water phase doped with 0.1% formic acid, and the corresponding organic solvent phase was composed of pure acetonitrile, also containing 0.1% formic acid;

[0108] The elution program was designed as a gradient mode, with the following specific steps: initially, the proportion of the organic solvent phase was set at 1%, then gradually increased to 7% within 5 minutes, and then continued to increase to 25% and 36%. After reaching 36%, the proportion of the organic solvent phase in the mobile phase was rapidly increased to 80%, and maintained at this proportion for 4.5 minutes. After completing this step, the mobile phase was rapidly converted back to 99% of the aqueous phase and maintained at this proportion for 19.9 minutes to complete the thorough elution of the peptides;

[0109] The eluted peptides were introduced directly into the mass spectrometer by electrospray using a distal voltage of 2.2 kV;

[0110] The relative abundance of peptides was obtained, peptide barcodes were quantified, and the effects of different transcription factors on the transformation of B cells into macrophages were analyzed.

[0111] According to an embodiment of the present invention, the peptide barcode protein sequence includes a CEBPA barcode, a CEBPB barcode, an ACOD1 barcode, and an NRF2 barcode;

[0112] The CEBPA barcode is shown in SEQ ID NO.1;

[0113] The CEBPB barcode is shown in SEQ ID NO.2;

[0114] The ACOD1 barcode is shown in SEQ ID NO.3;

[0115] The NRF2 barcode is shown in SEQ ID NO.4.

[0116] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0117] Example 1: Gene circuit construction

[0118] S1.1 Plasmid construction

[0119] Design unique barcode sequences for each mRNA encoding the transcription factor of interest

[0120] The CEBPA barcode sequence is shown in SEQ ID NO. 1;

[0121] The CEBPB barcode sequence is shown in SEQ ID NO. 2;

[0122] The ACOD1 barcode sequence is shown in SEQ ID NO. 3;

[0123] The NRF2 barcode sequence is shown in SEQ ID NO. 4;

[0124] The sequence of the fluorescent tracer protein is shown in SEQ ID NO.5;

[0125] The CEBPA mRNA sequence is shown in SEQ ID NO.6;

[0126] The CEBPB mRNA sequence is shown in SEQ ID NO.7;

[0127] The ACOD1 mRNA sequence is shown in SEQ ID NO.8;

[0128] The NRF2 mRNA sequence is shown as SEQ ID NO.9.

[0129] The peptide barcode protein includes mSA carrier protein and barcode sequence and myc affinity tag. It is inserted into the plasmid vector by Golden Gate cloning method. A plasmid containing peptide barcode protein sequence and tdTomato fluorescent protein sequence is constructed. The coding sequence of each transcription factor is inserted into the plasmid vector, and each transcription factor uses a different BsaI restriction enzyme cutting site.

[0130] The plasmid vector was digested with restriction endonucleases to produce linearized vectors. The PCR products of CEBPA, CEBPB, NRF2 and tdTomato were digested with the same restriction endonucleases. The DNA fragments of CEBPA, CEBPB, NRF2 and tdTomato were connected to the linearized APOBEC3 vector pUC19 to form a recombinant plasmid. The recombinant plasmid was transformed into host cells and transformants were selected on a medium containing the corresponding antibiotics. Transcription factors such as CEBPA, CEBPB and NRF2 promote the transformation of B cells into macrophages by forming a gene circuit.

[0131] S1.2 In vitro RNA generation

[0132] Host cells were cultured at 37°C until OD600 was approximately 0.6. 5-10 mg / L of linearized plasmid DNA was extracted from the culture as a transcription template. In a 50 mL reaction system containing 10 mL of transcription buffer, linearized plasmid DNA template, 1 μL of T7 RNA polymerase, 2 mM ATP, 2 mM GTP, 2 mM CTP, 2 mM UTP, and 0.5 mM N1-Me-pUTP were added. The reaction system was placed in a 37°C water bath and incubated for 4 hours. The mRNA was purified by chloroform extraction and ethanol precipitation, and quantified using UV-UV absorption spectroscopy to detect the purity and integrity of the mRNA and confirm that the 5' end of the mRNA had the correct cap structure.

[0133] Example 2: Targeted in vivo editing of in vitro transcribed RNA

[0134] S2.1 Lipid Nanoparticle Synthesis

[0135] The cationic lipid, cholesterol, DMG-PEG and auxiliary lipid were dissolved in ethanol at a ratio of 5:5:10:1 to obtain a mixed solution; at the same time, the RNA obtained in Example 1.2 was dissolved in a citrate buffer with a pH value of 4.0. The two solutions were quickly mixed to ensure that the ratio of water to ethanol was 3:1, so as to achieve a final weight ratio of lipid to RNA of 40:1. The RNA solution was quickly mixed with the lipid solution in ethanol to form stable lipid nanoparticles. After the lipid nanoparticles were intravenously injected into mice, the mice were cultured for 3 weeks.

[0136] Example 3: Multimodal, cross-scale small animal imaging

[0137] S3.1 Spatial omics imaging

[0138] Peripheral blood and bone marrow, spleen, kidney, lung and other tissue samples were collected from the disease animal model and made into slices. The fixed slices were permeabilized and spatial omics imaging was performed using STARmap technology. The designed probes were used to perform in situ hybridization of transcription factors, label target mRNA molecules, and convert them into fluorescently labeled cDNA through enzymatic reactions. At the same time as mRNA detection, specific proteins were immunofluorescently stained and fluorescently labeled using specific primary and secondary antibodies against disease marker proteins. The fluorescently labeled cDNA and protein signals were imaged by confocal microscopy to obtain multi-channel fluorescence images to provide raw data for subsequent data analysis. The acquired fluorescence images were processed using image analysis software, including preprocessing steps such as denoising, contrast enhancement, and image registration. The cDNA and protein signals in each cell were identified and located by set thresholds and algorithms to generate gene expression and protein localization maps at the single-cell level. Spatial statistical methods were used to analyze the spatial distribution characteristics of cell subtypes and disease marker proteins in tissues to reveal their potential role in the disease process.

[0139] S3.2 Confocal microscopy imaging

[0140] For specific tissue samples, 10 μm thick sections were made. Using a laser scanning confocal microscope, using a red channel filter, select an excitation wavelength of 554 nm and an emission wavelength of 581 nm. Find the focal plane of the sample in bright field mode and then switch to fluorescence mode. Set the resolution to 1024x1024 pixels. Scan at 400 Hz. Set the pinhole size to 1 Airy unit. Use Imaris for analysis to complete the monitoring and computational analysis of the macrophage transformation process.

[0141] S 3.3 Quantification of peptide barcoded proteins by liquid chromatography-mass spectrometry

[0142] Purify peptide barcoded proteins using affinity chromatography. Load 1 μl of peptide sample directly onto a chromatography column.

[0143] A high performance liquid chromatograph was used. The initial mobile phase was a pure water phase, in which 0.1% formic acid was added. The corresponding organic solvent phase was composed of pure acetonitrile, which also contained 0.1% formic acid. The elution program was designed in gradient mode, and the specific steps were as follows:

[0144] Initially, the organic solvent ratio was set at 1%, and then gradually increased to 7% within 5 minutes. After that, the organic solvent ratio continued to increase, reaching 25% and 36% in sequence. After reaching 36% organic solvent, the organic solvent ratio in the mobile phase will be quickly increased to 80% and maintained at this ratio for 4.5 minutes. After completing this step, the mobile phase will be quickly converted back to 99% aqueous phase and maintained at this ratio for 19.9 minutes to complete the complete elution of peptides.

[0145] The eluted peptides were directly introduced into the mass spectrometer by electrospray using a distal voltage of 2.2 kV. The relative abundance of the peptides was obtained, the peptide barcodes were quantified, and the effects of different transcription factors on the transformation of B cells into macrophages were analyzed.

[0146] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific 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 in the scope of protection of the present invention.

Claims

1. A method for in vivo programming of disease model macrophages, characterized in that: include: Constructing a target transcription factor plasmid comprising a peptide barcode protein sequence; wherein the target transcription factor plasmid comprises the peptide barcode protein sequence and a tdTomato fluorescent protein sequence; forming target transcription factor mRNA in vitro by using the target transcription factor plasmid as a template; The target transcription factor mRNA is bound and embedded in lipid nanoparticles; The lipid nanoparticles are intravenously injected into disease model mice to allow the lipid nanoparticles to be stably expressed in the mice, and then tissue samples are collected.

2. The method for in vivo programming of disease model macrophages according to claim 1, characterized in that: The construction of a target transcription factor plasmid containing a peptide barcode comprises: Designing a specific peptide barcode protein sequence for each mRNA encoded by a relevant transcription factor; The specific barcode sequence is inserted into the plasmid vector by the Golden Gate cloning method; the coding sequence of each transcription factor is inserted into the plasmid vector, and each transcription factor uses a different BsaI restriction enzyme cutting site to form the target transcription factor plasmid.

3. The method for in vivo programming of disease model macrophages according to claim 1, characterized in that: The target transcription factor mRNA is formed in vitro by using the target transcription factor plasmid as a template, comprising: Transforming the target transcription factor plasmid into host cells, and culturing the host cells at 37°C until OD600 is 0.6; 5-10 mg / L of linearized plasmid DNA was screened and extracted on a medium containing the corresponding antibiotics as a transcription template; In a 50 mL reaction system containing 10 mL transcription buffer, add linearized plasmid DNA, T7 RNA polymerase, 2 mM ATP, 2 mM GTP, 2 mM CTP, 2 mM UTP, and 0.5 mM N1-Me-pUTP; The reaction system was placed in a 37°C water bath and incubated for 4 hours to obtain mRNA; The mRNA is purified by chloroform extraction and ethanol precipitation, and quantified by ultraviolet absorption spectroscopy to detect the purity and integrity of the mRNA, confirm that the 5' end of the mRNA has the correct cap structure, and obtain the target transcription factor mRNA.

4. The method for in vivo programming of disease model macrophages according to claim 1, characterized in that: The step of combining and embedding the target transcription factor mRNA in lipid nanoparticles comprises: Dissolve the cationic lipid, cholesterol, DMG-PEG and auxiliary lipid in ethanol at a ratio of 5:5:10:1 to obtain a mixed solution; Dissolving the target transcription factor mRNA in a citrate buffer having a pH value of 4.0 to obtain an mRNA solution; The mixed solution and the mRNA solution are mixed to ensure that the ratio of water to ethanol is 3:1, so as to achieve a final weight ratio of lipid to RNA of 40:1; and stable lipid nanoparticles are formed.

5. The method for in vivo programming of disease model macrophages according to claim 1, characterized in that: Also included is imaging detection of tissue samples of the disease animal model; including: performing spatial omics imaging on the tissue sample; Imaging using confocal microscopy; Perform quantitative analysis by liquid chromatography-mass spectrometry; Monitoring and computational analysis of macrophage transformation progression were completed for each of the peptide barcoded protein sequences.

6. The method for in vivo programming of disease model macrophages according to claim 5, characterized in that: Performing spatial omics imaging on the tissue sample, including: Collect peripheral blood and bone marrow, spleen, kidney, lung and other tissue samples from the disease model and make slices; The fixed sections were permeabilized and spatial omics imaging was performed using STARmap technology; Using a probe to perform in situ hybridization on the target transcription factor mRNA, labeling the target transcription factor mRNA, and converting it into cDNA with a fluorescent label through an enzymatic reaction; While detecting the target transcription factor mRNA, immunofluorescence staining of tissue proteins is performed, using specific primary and secondary antibodies against disease marker proteins for fluorescent labeling; Imaging the fluorescently labeled cDNA and protein signals through a confocal microscope to obtain multi-channel fluorescent images to provide raw data for subsequent data analysis; The acquired fluorescence images were processed using image analysis software, including preprocessing steps such as denoising, contrast enhancement, and image registration; The cDNA and protein signals in each cell are identified and located by using the set thresholds and algorithms, generating single-cell level gene expression and protein localization maps; Spatial statistical methods are used to analyze the spatial distribution characteristics of cell subtypes and disease marker proteins in tissues to reveal their potential roles in the disease process.

7. The method for in vivo programming of disease model macrophages according to claim 5, characterized in that: The imaging using a confocal microscope comprises: Peripheral blood and tissue samples such as bone marrow, spleen, kidney and lung were collected from the disease model and made into 10 μm thick sections; A laser scanning confocal microscope was used, and the excitation wavelength was selected as 554 nm and the emission wavelength was selected as 581 nm using a red channel filter; Find the focal plane of the sample in brightfield mode, then switch to fluorescence mode; Set the resolution to 1024x1024 pixels, the scan speed to 400Hz, and the pinhole size to 1 Airy unit; Imaris was used for analysis to monitor and compute the macrophage transformation process.

8. The method for in vivo programming of disease model macrophages according to claim 5, characterized in that: The liquid chromatography-mass spectrometry quantitative analysis comprises: Purifying the protein with the peptide barcode protein sequence using affinity chromatography; 1 μl of peptide sample was loaded directly onto one column; A high performance liquid chromatograph was used, with the initial mobile phase being a pure water phase doped with 0.1% formic acid, and the corresponding organic solvent phase being composed of pure acetonitrile, also containing 0.1% formic acid; The elution program was designed in gradient mode, with the following specific steps: initially, the proportion of the organic solvent phase was set at 1%, then gradually increased to 7% within 5 minutes, and then continued to increase to 25% and 36%. After reaching 36%, the proportion of the organic solvent phase in the mobile phase was rapidly increased to 80%, and maintained at this proportion for 4.5 minutes. After completing this step, the mobile phase was rapidly converted back to 99% of the aqueous phase and maintained at this proportion for 19.9 minutes to complete the thorough elution of the peptides; The eluted peptides were introduced directly into the mass spectrometer by electrospray using a distal voltage of 2.2 kV; The relative abundance of peptides was obtained, peptide barcodes were quantified, and the effects of different transcription factors on the transformation of B cells into macrophages were analyzed.

9. The method for in vivo programming of disease model macrophages according to claim 1, characterized in that: The peptide barcode protein sequence includes CEBPA barcode, CEBPB barcode, ACOD1 barcode, and NRF2 barcode; The CEBPA barcode is shown in SEQ ID NO.1; The CEBPB barcode is shown in SEQ ID NO.2; The ACOD1 barcode is shown in SEQ ID NO.3; The NRF2 barcode is shown in SEQ ID NO.4.

Images