Composition, enzymatic solution, extraction method and application for single cell extraction of neuroimmune niche

Abstract

The present invention belongs to the field of single cell extraction technology, and specifically relates to a composition, an enzymatic solution, an extraction method and an application for extracting single cells of a neuroimmune niche, wherein the composition comprises actinomycin D, triptolide and / or anisomycin. The number and types of single cells of a neuroimmune niche extracted by enzymatic hydrolysis from the brain or skull based on the composition are larger than those extracted by the existing enzymatic hydrolysis method or cold grinding method, and the artifact effect of single cell sequencing is significantly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of single cell extraction, and in particular relates to a composition, an enzymatic hydrolysate, an extraction method and an application thereof for extracting single cells of a neuroimmune niche. Background Art

[0002] Neuroimmune niche single-cell sequencing is an important means to deeply explore the complex interactions between neural and immune cells in the brain's neuroimmune microenvironment. For example, sequencing can reveal the heterogeneity of gene expression in different cell types, analyze the fine interaction network between cells, and may discover new cell subpopulations or types; understanding the above information will help us better understand the normal function of the nervous system, and can also provide new clues and basis for the pathogenesis and treatment strategies of neuroimmune-related diseases such as neurodegenerative diseases and autoimmune encephalitis. Extraction of neuroimmune niche single cells is a necessary means before neuroimmune niche single-cell sequencing. However, the number and types of single cells extracted using the existing neuroimmune niche single-cell method are small, and artifact effects will occur during sequencing, affecting the sequencing results.

[0003] For example, cold mechanical grinding, one of the single-cell extraction methods, extracts single cells by physically decomposing tissue (extrusion, friction), and is generally only used for relatively loose tissue structures such as lymph nodes, spleen, etc.; since brain tissue is rich in myelin, brain cells are difficult to dissociate, resulting in a low cell yield and difficulty in preparing single-cell sequencing samples of sufficient quantity; therefore, it is generally necessary to increase the number of tissue samples in large quantities to make up for the lack of cell number, which is not conducive to corresponding research on samples that are difficult to obtain; in addition, although the cold mechanical method is often used for single-cell preparation of bone marrow, it cannot separate stromal cells and mesenchymal cells in the bone cavity. Although the cell yield is not low, the cell diversity is low, and the single-cell sequencing results cannot truly reflect the interaction between various cell groups in the bone marrow microenvironment. Although the enzymatic hydrolysis method can improve the yield of tissue structural cells and increase single-cell diversity by enzymatic hydrolysis, the temperature stimulation during the enzymatic hydrolysis process (generally 37°C heat shock) will generally cause heat stimulation to the cells, triggering the cell heat shock response, activating the heat shock transcription factor (HSF1) and promoting the rapid expression of heat shock or heat shock proteins (HSPs) to relieve stress. It will also lead to the activation of related early stress genes, causing the transcriptional characteristics of some heat shock sensitive cells in the subsequent single-cell sequencing results to produce artifact effects, resulting in distortion of single-cell analysis results.

[0004] Therefore, it is necessary to develop a method for extracting single cells from the neuroimmune niche that can increase cell yield and diversity while eliminating artifact effects caused by heat shock. Summary of the invention

[0005] The present invention improves upon the existing enzymatic hydrolysis method to obtain a new extraction method. The extraction method of the present invention uses actinomycin D and triptolide and / or anisomycin on the basis of enzymatic hydrolysis during the extraction process. The extraction method can not only increase the cell yield and diversity, but also effectively reduce the number of tissue samples required for single cell extraction of the neuroimmune niche, and can also eliminate the artifact effect caused by heat shock, which can effectively reduce the number of tissue samples required for single cell extraction of the neuroimmune niche.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a composition for extracting single cells of a neuroimmune niche, comprising actinomycin D and triptolide.

[0008] Preferably, in the above composition, the mass ratio of actinomycin D to triptolide is (4-6):(2-4).

[0009] Preferably, the above composition further comprises anisomycin.

[0010] More preferably, the mass ratio of the above-mentioned actinomycin D, triptolide and anisomycin is (4.5-5.5):(3-3.5):(25-30).

[0011] Another aspect of the present invention provides an enzymatic hydrolysis solution for extracting single cells of the neuroimmune niche, which comprises the composition of the present invention.

[0012] Preferably, in the enzymatic hydrolysate, the concentration of actinomycin D is 4 μg / ml-6 μg / ml, the concentration of triptolide is 2 μg / ml-4 μg / ml, and the concentration of anisomycin is 25 μg / ml-30 μg / ml.

[0013] On the other hand, the present invention provides a method for extracting single cells of a neuroimmune niche, comprising: extracting a tissue using an enzymatic method to obtain single cells of a neuroimmune niche, wherein the tissue is the brain or the skull; and the enzymatic hydrolysis solution used in the enzymatic method is the enzymatic hydrolysis solution mentioned above in the present invention.

[0014] Preferably, in the above extraction method, when the tissue is the brain, the neuroimmune niche single cells include one or more of microglia, monocytes / macrophages or neutrophils in the brain; when the tissue is the skull, the neuroimmune niche single cells include bone marrow immune cells.

[0015] More preferably, the above-mentioned bone marrow immune cells include one or more of granulocytes and neutrophils.

[0016] Preferably, in the above extraction method, when the tissue is the brain, the enzymatic hydrolysis solution also includes papain; when the tissue is the skull, the enzymatic hydrolysis solution also includes collagenase.

[0017] Preferably, the above extraction method is selected from one of the following methods:

[0018] When the tissue is brain, extraction methods include:

[0019] (1) mixing the brain with the enzymatic hydrolysis solution, performing enzymatic hydrolysis, and centrifuging and discarding the supernatant to obtain a cell tissue mixture;

[0020] (2) removing tissue fragments from the cell-tissue mixture, centrifuging and discarding the supernatant, and removing myelin to obtain single cells of the neuroimmune niche;

[0021] When the tissue is skull bone, extraction methods include:

[0022] (a) mixing the skull with an enzymatic hydrolysis solution, performing an enzymatic hydrolysis reaction, and separating the liquid from the solid to obtain a liquid;

[0023] (b) The liquid is centrifuged and the supernatant is discarded, and red blood cells are removed to obtain single cells of the neuroimmune niche.

[0024] More preferably, the conditions of the above enzymatic hydrolysis reaction include: the reaction time is 10 min-30 min.

[0025] More preferably, in step (a) of the above extraction method, one or more of the following conditions are met: (i) after the reaction, the skull bone flap is crushed and clamped before liquid-solid separation is performed to obtain a liquid; (ii) after the reaction, a reaction stopping liquid is added to terminate the reaction before liquid-solid separation is performed to obtain a liquid.

[0026] More preferably, the above-mentioned crushing and clamping of the skull bone flap is performed in an environment of 0°C-4°C.

[0027] Preferably, the above extraction method also satisfies one or more of the following conditions: (A) before the tissue is extracted by enzymatic hydrolysis to obtain neuroimmune niche single cells, the tissue is processed into fragments; (B) before the tissue is extracted by enzymatic hydrolysis to obtain neuroimmune niche single cells, the tissue is infiltrated with a solution at 0°C-4°C, and the solution contains the composition of the present invention (including actinomycin D and triptolide); if the tissue is the brain, the solution is a PBS solution; if the tissue is the skull, the solution is an RPMI solution; (C) before the tissue is extracted by enzymatic hydrolysis to obtain neuroimmune niche single cells, the body is perfused, and the perfusion solution is a 0°C to 6°C PBS solution, and the PBS solution contains actinomycin D and triptolide; (D) after the tissue is extracted by enzymatic hydrolysis, dead cells are removed to obtain neuroimmune niche single cells.

[0028] More preferably, in the above method, dead cells are removed using a MS column in combination with a magnetic field.

[0029] More preferably, the MS column is rinsed with Binding Buffer before use.

[0030] In another aspect, the present invention provides a use of the composition for extracting single cells of the neuroimmune niche in the present invention in extracting single cells of the neuroimmune niche.

[0031] In yet another aspect, the present invention provides a method for single-cell sequencing of a neuroimmune niche, comprising: extracting a neuroimmune niche single cell from a tissue using the extraction method of the present invention.

[0032] The beneficial effects of the present invention include at least:

[0033] (1) The number and types of neuroimmune niche single cells extracted from the brain or skull by enzymatic hydrolysis of the composition for extracting neuroimmune niche single cells provided by the present invention are larger than those extracted by the existing cold grinding method.

[0034] (2) The artifact effects of neuroimmune niche single cells extracted from the brain or skull by enzymatic hydrolysis using the composition for extracting neuroimmune niche single cells provided by the present invention are significantly reduced when subsequent sequencing is performed compared to single cells extracted by existing enzymatic hydrolysis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1a is the normalized cell viability of different treatment groups;

[0036] Figure 1b The expression of heat shock pathway gene HSF1a in different treatment groups;

[0037] Figure 1c The expression of artifact marker gene FOS in different treatment groups;

[0038] Figure 1d The expression of Dusp1 gene, an artifact marker gene, in different treatment groups;

[0039] Figure 1e The expression of IL1b, a marker gene of the inflammatory pathway, in different treatment groups;

[0040] Figure 1f The expression of IL6, a marker gene of the inflammatory pathway, in different treatment groups;

[0041] Figure 2a The cell yield of a single skull bone marrow sample by conventional enzymatic hydrolysis (SBM-CTL) and mechanical cold grinding (SBM-Dounce);

[0042] Figure 2bThe number of non-immune cells in a single skull bone marrow sample obtained by conventional enzymatic hydrolysis (SBM-CTL) and mechanical cold grinding (SBM-Dounce);

[0043] Figure 3a UMAP images of the cell clustering integration of skull bone marrow single cell sequencing by the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention;

[0044] Figure 3b This is a UMAP graph comparing cell clustering of skull bone marrow single-cell sequencing using the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention.

[0045] Figure 4a It is a highlighting image of neutrophil cell markers and pseudo-activation phenotype artifact markers in the mouse skull bone marrow;

[0046] Figure 4b It is a ratio diagram of neutrophil cell markers and pseudo-activation phenotype artifact markers in mouse skull bone marrow;

[0047] Figure 5a This is a highlight image of pseudo-granulocyte cell markers and pseudo-activated phenotype artifact markers in mouse skull bone marrow;

[0048] Figure 5b It is a ratio diagram of pseudo-granulocyte (Granulocyte) cell markers and pseudo-activation phenotype artifact markers in mouse skull bone marrow;

[0049] Figure 6a Cell clustering UMAP test chart (neutrophils) for cells extracted by conventional enzymatic hydrolysis method (SBM-CTL) and optimized enzymatic hydrolysis method (SBM-OPT) of the present invention;

[0050] Figure 6b The volcano plot of differentially expressed genes between pseudoactivated cells and normal cells of mouse skull bone marrow (neutrophils);

[0051] Figure 7a Cell clustering UMAP test chart (granulocytes) for cells extracted by conventional enzymatic hydrolysis method (SBM-CTL) and optimized enzymatic hydrolysis method (SBM-OPT) of the present invention;

[0052] Figure 7b Volcano plot of differentially expressed genes between pseudo-activated cells and normal cells of mouse skull bone marrow (granulocytes);

[0053] Figure 8aThe total number of microglia in brain tissue obtained by cold grinding (brain-Dounce), conventional enzymatic hydrolysis (brain-CTL) and optimized enzymatic hydrolysis (brain-OPT) of the present invention;

[0054] Figure 8b is the percentage of pseudo-activated microglia in microglia obtained by conventional enzymatic hydrolysis (brain-CTL) and optimized enzymatic hydrolysis (brain-OPT) of the present invention;

[0055] Figure 8c Comparison of UMAP images of microglial populations for single-cell sequencing of mouse brain;

[0056] Figure 9a It is a UMAP diagram comparing cell clustering of brain single-cell sequencing using the common enzymatic hydrolysis method (brain-CTL) and the optimized enzymatic hydrolysis method (brain-OPT) of the present invention;

[0057] Figure 9b The gene expressions of microglial cell markers (Tmem119, P2ry12, Cx3cr1) and pseudo-activation phenotype artifact markers (Fos, Cccl4, Junb, Jun, Zfp36);

[0058] Fig.9c It is the classification diagram of exAM (pseudo-activated) and homeostatic (steady state) microglia;

[0059] Figure 9d It is the volcano plot of differential gene expression between exAM (pseudo-activated) and homeostatic (steady state) microglia;

[0060] Fig.10a This is the UMAP image of monocyte / macrophage populations in mouse brain;

[0061] Fig.10b The volcano map of differentially expressed genes between pseudo-activated cells (exA-Mono / mac) and normal cells (Mono / Mac) in the mouse brain;

[0062] Fig.11a This is the UMAP image of neutrophil populations in the mouse brain;

[0063] Fig.11b This is a volcano map of differentially expressed genes between pseudo-activated neutrophils (exA-Neutrophil) and normal cells (Neutrophil) in the mouse brain. DETAILED DESCRIPTION

[0064] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0065] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0066] An embodiment of the present invention provides a composition for extracting single cells of a neuroimmune niche, comprising actinomycin D and triptolide.

[0067] It should be noted that actinomycin D has excellent ability to inhibit RNA polymerase synthesis, and has a lower working concentration than other RNA polymerase inhibitors (such as alpha-Amanitin (inhibits RNA polymerase II and III), Rifampicin (inhibits RNA synthesis) or DRB (inhibits RNA polymerase II), etc.), and can completely block transcription; in addition, actinomycin D can widely inhibit all types of RNA polymerases (including RNA polymerases I, I and III). At the same time, actinomycin D can insert between double-stranded DNA to act, and has stronger stability.

[0068] It should also be noted that triptolide can inhibit HSF1 expression and has more inhibitory targets than other HSF1 inhibitors (such as KRIBB11, Quercetin, Pifithrin-μ or KNK437), such as inhibiting NF-κB, RNA polymerase II and other key cell signaling pathways; in addition, triptolide can exhibit potent biological activity at low concentrations, reducing drug dosage and side effects.

[0069] In some specific examples, in the above composition, the mass ratio of actinomycin D to triptolide is (4-6): (2-4), such as 4.5:2.6, 4.7:2.8, 5:3.2, 5:3.064 or 5.5:3.7, etc., preferably 5:3.064, at which the extraction effect is better.

[0070] In some specific examples, the above composition further includes anisomycin, that is, the composition includes actinomycin D, triptolide and anisomycin.

[0071] It should be noted that the selection of triptolide echoes anisomycin, which can inhibit the pro-inflammatory effect of anisomycin, while other HSF1 inhibitors do not have this effect, which will cause the pro-inflammatory effect of anisomycin to take effect, affecting the effect of the present invention. In addition, while anisomycin inhibits protein synthesis, it will activate JNK, MAPK and other pathways, but because the combined triptolide itself inhibits NF-κB and weakens the activation of JNK and p38MAPK induced by the inflammatory factor TNF-α, the inflammatory activation effect of anisomycin is not obvious; in addition, if anisomycin is not added, protein synthesis cannot be inhibited; if other protein synthesis inhibitors are added, the anti-inflammatory pathway of triptolide cannot be compensated; at the same time, triptolide can inhibit the expression of cell cycle proteins (such as cyclin D1) and block the cell cycle in the G1 / S phase; anisomycin can block the cell cycle in the G1 and G2 / M phases by inhibiting protein synthesis. The combination of the two can basically cover the cell cycle and eliminate the influence of the cell cycle. Cancelling any one of them will cause cell loss in the analysis results.

[0072] It should also be noted that the combined use of actinomycin D, triptolide and anisomycin can comprehensively inhibit abnormal cell activation from the three levels of RNA transcription, DNA synthesis, and protein synthesis, so that the single-cell RNA level can accurately reflect its physiological state level; it can better play the role of inhibiting thermal stimulation during enzymatic hydrolysis and better extract single cells of the neuroimmune niche that reflect the in situ state.

[0073] In some specific examples, the mass ratio of the above-mentioned actinomycin D, triptolide and anisomycin is (4.5-5.5): (3-3.5): (25-30), such as 4.7:3.3:26, 4.9:3.2:27, 5.3:3.6:28 or 5:3.064:27.1, etc., preferably 5:3.064:27.1, and the extraction effect is better under this ratio.

[0074] The embodiment of the present invention also provides an enzymatic hydrolysis solution for extracting single cells of the neuroimmune niche, which includes the composition of the present invention comprising actinomycin D, triptolide and anisomycin.

[0075] It should be noted that the composition containing actinomycin D, triptolide and anisomycin can be added with different buffer substances or additives to prepare an enzymatic solution for enzymatic hydrolysis of tissues, and then the neuroimmune niche single cells are extracted; the neuroimmune niche single cells extracted based on the enzymatic solution significantly reduce the artifact effects of subsequent single-cell sequencing.

[0076] It should also be noted that when the tissue is skull, the enzymatic hydrolysis method of the present invention can reduce the artifact effects of single-cell sequencing results of myeloid immune cells in the skull bone marrow and repair pseudo-activated inflammatory pathways; in addition, the single-cell preparation process of skull bone marrow is different from the preparation method of long bone bone marrow. Due to its unique tissue structure, skull bone marrow is not suitable for mechanical flushing and extrusion by syringe; and the use of fine forceps to squeeze skull bone marrow cells faces problems such as insufficient cell number or lack of adherent cells in the bone cavity; the enzymatic hydrolysis method of the present invention avoids the above problems.

[0077] In some specific examples, in the enzymatic hydrolyzate, the concentration of actinomycin D is 4μg / ml-6μg / ml, such as 4.5μg / ml, 5μg / ml or 5.5μg / ml; the concentration of triptolide is 2μg / ml-4μg / ml, such as 2.758μg / ml-3.371μg / ml, 2.758μg / ml, 3.064μg / ml or 3.371μg / ml; anisomycin is 25μg / ml-30μg / ml, such as 26.5μg / ml-27.5μg / ml, 26.8μg / ml, 27.1μg / ml or 27.3μg / ml; preferably, the concentration of actinomycin D is 5μg / ml, the concentration of triptolide is 3.064μg / ml, and the concentration of anisomycin is 27.1μg / ml.

[0078] It should be noted that during the enzymatic hydrolysis process, if anisomycin is not added, then under the influence of triptolide, the expression of NF-κB pathway-related genes TNF-α, IL-1β, IL-6, COX-2, iNOS, etc. is downregulated; the expression of AP-1 pathway-related genes MMPs is downregulated; the genes related to the STAT3 pathway are downregulated; anisomycin can inhibit the nonspecific inhibitory effect of triptolide through the stress kinase pathway; in addition, if the dose of anisomycin used is greater than the dose used in the experiment, the genes related to the pro-inflammatory pathway are highly expressed, and cell apoptosis is induced due to the lack of intracellular proteins; when it is lower than the dose used, it cannot make up for the nonspecific inhibitory effect of triptolide.

[0079] An embodiment of the present invention also provides a method for extracting single cells of a neuroimmune niche, comprising: extracting a tissue using an enzymatic method to obtain single cells of a neuroimmune niche, wherein the tissue is the brain or the skull; and the enzymatic hydrolysis solution used in the enzymatic hydrolysis method is the enzymatic hydrolysis solution described above in the present invention.

[0080] It should be noted that the enzymatic hydrolysis method is a method for extracting single cells that is well known in the art, and different proteins in the tissue are digested by enzymatic hydrolysis; it should be understood that different tissues have different applicable enzymes, but they are all enzymes well known in the art.

[0081] In some specific examples, in the above extraction method, when the tissue is the brain, the neuroimmune niche single cells include one or more of microglia, monocytes / macrophages or neutrophils in the brain; when the tissue is the skull, the neuroimmune niche single cells include bone marrow immune cells.

[0082] It should be noted that bone marrow immune cells refer to one or more types of immune cells in the bone marrow.

[0083] In some specific examples, the bone marrow immune cells include one or more of granulocytes and neutrophils.

[0084] In some specific examples, in the above extraction method, when the tissue is brain, the enzymatic hydrolysis solution also includes papain; when the tissue is skull, the enzymatic hydrolysis solution also includes collagenase.

[0085] It should be noted that, as mentioned above, different tissues are suitable for different proteases; for example, when the tissue is skull, collagenase (such as collagenase VIII) is preferred; specifically, the collagenase type has been screened to meet the specificity and relative activity required for skull tissue digestion, and the collagenase concentration has been verified to ensure digestion efficiency while minimizing the damage of collagenase to cell membranes and the effects of surface receptors and proteins, thereby ensuring the number of living cells; for another example, when the tissue is brain, the enzyme in the enzymatic hydrolysis solution may preferably be papain, which is more suitable for enzymatic hydrolysis of the brain than other enzymes.

[0086] It should also be noted that, in addition to the enzyme and the composition of the present invention, the above-mentioned enzymatic hydrolysis solution also includes a buffer substance or a culture medium or other enzymes (such as deoxyribonuclease DNase I (DNase I)). For example, the enzymatic hydrolysis solution for enzymatic hydrolysis of the brain generally also includes a phosphate buffer substance (i.e., a PBS system); for example, the enzymatic hydrolysis solution for enzymatic hydrolysis of the skull generally also includes a culture medium material, such as RPMI culture medium. In addition, for the enzymatic hydrolysis solution for enzymatic hydrolysis of the skull, unlike the DMEM used in the prior art, RPMI has a lower glucose concentration (2g / L) and a higher amino acid richness. The low sugar concentration can reduce the stress response of the cells. At the same time, during the enzymatic hydrolysis process, RPMI is more stable than DMEM in a low CO2 environment.

[0087] In some specific examples, when the tissue is brain, the extraction method includes:

[0088] (1) The brain is mixed with an enzymatic hydrolysis solution, subjected to enzymatic hydrolysis, and centrifuged to discard the supernatant to obtain a cell-tissue mixture; (2) Tissue fragments are removed from the cell-tissue mixture, centrifuged to discard the supernatant, and myelin is removed to obtain a single cell of the neuroimmune niche.

[0089] It should be noted that in step (1), the brain is mixed with the enzymatic solution and then incubated at 37°C on a rocker platform for 20 minutes, and then the tissue mixture is ground by blowing up and down to allow the undissociated tissue fragments to settle to the bottom of the test tube; the traditional digestion time (30 minutes) can be shortened, thereby reducing the heat shock stimulation time and reducing the effect of inhibitor toxicity on cell activity; in addition, in steps (1) and (2), grinding, centrifugation and removal of myelin are all carried out under low temperature conditions, which can avoid the effect of heat shock, maintain cell activity and reduce the RNA degradation rate.

[0090] In some specific examples, when the tissue is skull, the extraction method includes: (a) mixing the skull with an enzymatic hydrolysis solution, performing an enzymatic hydrolysis reaction, and separating the liquid from the solid to obtain a liquid; and (b) centrifuging the liquid to discard the supernatant and remove the red blood cells to obtain a neuroimmune niche single cell.

[0091] It should be noted that it should be understood that before enzymatic hydrolysis, the meninges and the connective tissue and muscle tissue remaining on the surface of the skull can be completely peeled off using pre-cooled precision forceps under a dissecting microscope (operation on ice); adding this step can remove possible tissue contamination during the preparation of skull bone marrow single cells. After this step, use fine scissors to cut the skull bone flap into small pieces with a length and width of less than 2mm X2mm; adding this step can increase the contact area between the tissue and the enzymatic hydrolysis solution during the enzymatic hydrolysis process, so that the inside of the bone cavity is fully mixed with the enzymatic hydrolysis solution, increase the enzymatic hydrolysis efficiency, reduce the enzymatic hydrolysis time, and increase the yield of matrix cells.

[0092] In some specific examples, the conditions of the enzymatic hydrolysis reaction may include: the reaction time is 10 min-30 min.

[0093] It should also be noted that it should be understood that the enzymatic hydrolysis process in the extraction method of the present invention reacts best at an appropriate temperature, such as 37°C-40°C, and the enzymatic hydrolysis time is 10min-30min, such as 15min-30min or 20min-25min; specifically, the enzymatic hydrolysis reaction time determines the reaction time of anisomycin. If the reaction time of anisomycin is too long, the genes related to the pro-inflammatory pathway are highly expressed, and cell apoptosis is induced due to the lack of intracellular proteins; if the reaction time of anisomycin is too short, anisomycin does not begin to take effect.

[0094] The above enzymatic hydrolysis is preferably carried out at 37°C for 20 min.

[0095] In some specific examples, in step (a) of the above extraction method, one or more of the following conditions are met:

[0096] (i) After the reaction, the skull bone flap is crushed and clamped before liquid-solid separation to obtain liquid; specifically, by adopting the cold mechanical extrusion (crushing and clamping) method, the problem of insufficient cell yield caused by shortening the digestion time is compensated, and the cell dissociation in the bone cavity is further promoted, and the yield of matrix cells, endothelial cells, mesenchymal cells and other cells is increased, and the diversity of cell types is increased; in addition, the number of times of squeezing with fine forceps has been verified, and the selection of crushing and clamping 100 times can maximize the balance between the time required for operation and the number of cells obtained;

[0097] (ii) adding a reaction termination solution after the reaction to terminate the reaction and then performing liquid-solid separation to obtain a liquid; specifically, by adding the reaction termination solution, not only the digestion of collagenase is terminated, but also the inhibitor is diluted, thereby further reducing its toxicity to cells.

[0098] In some specific examples, the above-mentioned crushing and clamping of the skull bone flap is performed in an environment of 0°C-4°C.

[0099] It should be noted that the above-mentioned crushing and clamping of the skull bone flap is performed on ice, thereby avoiding possible thermal stimulation.

[0100] In some specific examples, the above extraction method also satisfies one or more of the following conditions:

[0101] (A) Before extracting the neuroimmune niche single cells using the enzymatic method, the tissue is processed into pieces; specifically, processing the tissue into pieces increases the contact area between the tissue and the enzymatic solution, which is more conducive to enzymatic hydrolysis;

[0102] (B) Before the tissue is extracted by enzymatic hydrolysis to obtain a single cell of the neuroimmune niche, the tissue is infiltrated with a solution at 0°C-4°C, wherein the solution comprises the composition of the present invention (including actinomycin D and triptolide); the tissue is the brain, and the solution is a PBS solution; the tissue is the skull, and the solution is an RPMI solution; specifically, when the tissue is mechanically sheared before enzymatic hydrolysis, low-temperature infiltration can reduce the RNA degradation rate and maintain cell activity, and the inhibitor can inhibit the RNA expression of the cell ex vivo. In addition, when the tissue is the skull, the use of the culture medium RPMI can maintain the cell activity to the greatest extent and reduce the abnormal cell metabolism in vitro;

[0103] (C) Before the tissue is extracted by enzymatic hydrolysis to obtain single cells of the neuroimmune niche, the body is perfused with a 0°C to 6°C PBS solution containing actinomycin D and triptolide. Specifically, before the enzymatically hydrolyzed tissue is removed, the body is perfused (usually cardiac perfusion) to maintain the in situ expression level of cellular RNA using inhibitors while removing blood. The pre-cooled perfusion solution can also reduce the degradation rate of cellular proteins and RNA. In addition, during perfusion, only actinomycin D and triptolide are used in combination, and anisomycin is added during the subsequent enzymatic hydrolysis process because anisomycin can inhibit protein synthesis but can also activate JNK / SAPK, MAP kinase and other signaling pathways, which activate related stress pathways and affect the single-cell sequencing results; in addition, if actinomycin D and triptolide are not added during the perfusion process, the cell artifact effect cannot be suppressed (the inhibitor does not contact the cells for long enough, resulting in insufficient inhibitor efficacy; at the same time, in the early stage of mouse death, the cells themselves have begun to be stressed due to the death of the body, so they must be added as soon as possible). However, if anisomycin is added to the perfusion fluid at the same time, the activation effect of anisomycin on related stress pathways will dominate, and at the same time, it will lead to reduced cell activity and ultimately fewer live cells (anisomycin has a stronger ability to induce cell apoptosis than the first two inhibitors, and too long contact time with tissue cells will upregulate pro-inflammatory pathway genes, so the contact time with cells must be minimized); the combination of actinomycin D and triptolide during the perfusion process doubles the inhibition of RNA synthesis. If either of them is used alone, the working concentration needs to be increased, resulting in cell apoptosis (reduction in the number of live cells);

[0104] (D) After the tissue is extracted by enzymatic hydrolysis, dead cells are removed to obtain neuroimmune niche single cells; specifically, in order to improve the purity and yield of neuroimmune niche single cells, dead cells are removed from the cells after enzymatic hydrolysis and extraction, and the removal method is a method well known in the art.

[0105] In some specific examples, in the above method, dead cells are removed using an MS column in combination with a magnetic field.

[0106] It should be noted that the use of MS columns in conjunction with magnetic fields to remove dead cells in the present invention does not require flow cytometry, takes a shorter time, and compared with the effect of voltage on cells during flow cytometry, the cells are less stimulated and have higher activity.

[0107] In some specific examples, the MS column is rinsed with Binding Buffer before use.

[0108] It should be noted that rinsing the MS column with Binding Buffer before use can increase the cell yield.

[0109] In some specific examples, in the above extraction method, when the tissue is brain, the extraction includes: incubating at 37°C, 50RPM for 20 minutes; when the tissue is skull, the extraction includes: incubating at 37°C on a rocker platform for 20 minutes.

[0110] It should be noted that triptolide can induce cell apoptosis through various pathways, thereby affecting the preparation of single-cell samples. The present invention verifies the optimal digestion time through experiments combined with the enzymatic hydrolysis rate, balances the relationship between cell number, cell viability and cell diversity, and ensures high-quality single-cell sample preparation results.

[0111] It should also be noted that in the extraction method of the present invention, after enzymatic hydrolysis, except for the addition of inhibitors during room temperature operation, the inhibitors need to be removed during ice operation. The reason is still because the inhibitors themselves have cytotoxicity, and it is necessary to minimize the contact time with cells while ensuring the inhibitory effect.

[0112] An embodiment of the present invention also provides a use of the composition for extracting single cells of the neuroimmune niche in the present invention in extracting single cells of the neuroimmune niche.

[0113] It should be noted that in single-cell sequencing, cell cycle-related genes dominate clustering, interfere with clustering results and affect subsequent analysis; although cell cycle-free related analysis algorithms can be used, it will still affect cell clustering; in the composition of the present invention, triptolide can inhibit the expression of cell cycle proteins (such as cyclin D1) and block the cell cycle in the G1 / S phase; anisomycin can block the cell cycle in the G1 and G2 / M phases by inhibiting protein synthesis; through the combined use of inhibitors, cell cycle-related gene proteins can be effectively inhibited, eliminating the influence of cell cycle-related genes on cell clustering in the analysis of single-cell sequencing results.

[0114] It has been verified that based on the effects of the composition of the present invention (actinomycin D and triptolide and / or anisomycin), its application in the single-cell extraction of the neuroimmune niche can reduce the artifact effects of subsequent single-cell sequencing.

[0115] An embodiment of the present invention also provides a method for sequencing a neuroimmune niche single cell, which comprises: extracting a neuroimmune niche single cell from a tissue using the extraction method of the present invention.

[0116] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0117] In the following example, the single-cell sequencing method follows these steps:

[0118] (1) Preparation of cell suspension: The obtained single cell samples are quality inspected and counted, and the cell activity is required to be at least greater than 80%. The qualified cells are washed and resuspended to an appropriate cell concentration of 700 cells / μl to 1200 cells / μl for machine operation;

[0119] (2) GEMs generation and labeling: Using the 10X Genomics Chromium™ system, the single cell suspension and Mix that passed the quality inspection, Single Cell 5'GEM Gel Bead with sequence tag barcode, and oil droplet were added to the injection channel respectively;

[0120] (3) Running the Chromium TM system, a "single cross" cross system is formed through a microfluidic channel network, and finally a single-cell micro-reaction system GEMs (Gel Bead in Emulsion) encapsulated by oil droplets is formed; along with the encapsulation, the cells in the GEMs are lysed, and the mRNA in the cells is released. The mRNA combines with the labeled molecules on the gel microbeads and is reverse transcribed into cDNA under the action of reverse transcriptase;

[0121] (4) cDNA amplification: Recovery Agent is added to mix with GEM to break the oil and release cDNA. The cDNA is then amplified, purified, and enriched. The cDNA concentration is detected and quantified using Qubit quantitative reagent to meet the library construction requirements.

[0122] (5) Construction of 5-terminal transcriptome library: After fragmentation, end modification, ligation of sequencing adapter Read2, sample index and other experimental processes, the library is finally quantitatively inspected to obtain the 5-terminal transcriptome gene expression data; the sequencing data mainly includes Read 1 and Read 2, where Read 1 contains 16bp 10x Barcode and 12bp UMI sequence;

[0123] (6) Sequencing: The NovaSeq X Plus sequencing platform was used with the PE150 sequencing mode. The single-cell sequencing volume reached 50k read pairs / cell, and the 5-terminal transcriptome data of each cell was obtained.

[0124] 1. Inhibitor concentration, combination and condition screening

[0125] Mouse lung fibroblast cells were used as an experimental model, and heat stimulation at 42°C for 30 minutes was used in vitro to simulate the heat shock response during enzymatic hydrolysis. The specific method is as follows:

[0126] (1) Prepare ice-cold PBS solution in a 50 mL tube and place on ice until ready for use;

[0127] (2) Take 3-month-old C57 mice, anesthetize them and perform cardiac perfusion with 30 ml of ice-cold PBS; immediately remove the lungs after perfusion and place them in PBS prepared on ice;

[0128] (3) Place the excised lung tissue in a 10 cm culture dish (on ice) and mince it with a sterile scalpel (usually use two blades to mince the tissue completely into a paste);

[0129] (4) The minced tissue was suspended in 10 ml of digestion solution and incubated at 37°C for 2 hours; wherein the digestion solution formula is: DMEM / F12 medium + 1% L-glutamine (original concentration 200 mM, final concentration after addition 2 mM) + 2.5 μg / ml amphotericin B + 1% P / S (original solution concentration: penicillin 10000 U / mL, streptomycin 10 mg / mL, final concentration after addition: penicillin 100 U / mL, streptomycin 100 μg / mL) + collagenase VIII (1 mg / mL);

[0130] (5) After the incubation is completed (observe the cell status in the tube, there should be no tissue blocks, and the solution is uniformly turbid), add culture medium to terminate digestion; wherein, the culture medium formula is: DMEM / F12 medium + 1% L-glutamine (original concentration 200mM, final concentration after addition 2mM) + 2.5μg / ml amphotericin B + 1% P / S (original solution concentration: penicillin 10000U / mL, streptomycin 10mg / mL, final concentration after addition: penicillin 100U / mL, streptomycin 100μg / mL) + 15% FBS;

[0131] (5) Centrifuge at 500 g for 5 minutes and discard the supernatant.

[0132] (6) Resuspend the cell pellet in fresh culture medium and culture the cells at 3×10 6 Inoculate the target culture plate. Then, cover with MilicellCM membrane; culture the cells in a 37°C, 5% CO2 incubator for 1 day without disturbing them;

[0133] (7) After one day, carefully remove the covering membrane and replace with fresh culture medium. At this point, the cells should have attached to the wall. Careful observation should be made during the operation. If the cells are not attached to the wall, the culture can be continued for another day. Pay attention to the operation skills when changing or adding liquid to avoid washing the cells away.

[0134] (8) Wait until the cell number reaches 70% enrichment (change the culture medium every 4 days);

[0135] (9) When the cell quantity is sufficient, digestion is performed using a standard trypsin digestion procedure;

[0136] (10) Fibroblasts were seeded into 12-well plates (seeding density was 0.1×10 6 , 2 ml of culture medium per well);

[0137] (11) Wait until the cell confluence reaches >90% before starting the experiment;

[0138] (12) Add corresponding different inhibitors and perform different treatments (see Table 1 for details); then collect the cells.

[0139] In addition, a control group CTL1-RT in which single cells were extracted without enzymatic hydrolysis was set up, and a control group CTL2-Heat in which single cells were extracted by enzymatic hydrolysis but without using inhibitors was set up.

[0140] The details of the different treatment groups and the control group are shown in Table 1 below.

[0141] Table 1 Different experimental parameters of heat shock response

[0142]

[0143] After the heat shock reaction was carried out according to the above different treatments, the cell activity was verified by flow cytometry, and the expression of the marker genes Fos and Dusp1 of the gene artifact effect, the marker gene HSF1a of the heat shock effect, and the marker genes IL1b and IL6 of the inflammatory pathway were detected by qPCR technology. The results are as follows: Figures 1a to 1f As shown, the results show:

[0144] Through cell activity detection, D2, T2 and A2 can be obtained as the lowest working concentrations. (Although there is a difference in the activity of A1 and A2, the replenishment effect of A1 on the relevant pathway is lower, so the A2 concentration is selected); when only actinomycin and triptolide are included, all pathways are inhibited, but compared with CTL1-RT, the inhibitory effect has seriously affected the normal expression of pathway-related genes and needs to be replenished, so anisomycin is needed; if the method of further reducing the concentration of actinomycin and triptolide without anisomycin is considered, it is not feasible, because anisomycin is the only inhibitor that can be used for protein translation and is indispensable; in the 1H, 30Min and 15min experiments of D2+T2+A2, it can be seen that the cell activity decreases with the incubation time, and when the three inhibitors are used in combination, the longer the incubation time, the higher the expression of genes related to the inflammatory pathway, proving that anisomycin is dominant in the combined process, so the incubation time of anisomycin should be reduced. Therefore, by comparing the (D2+T2)+A2 and (D2+A2)+T2 experiments, it can be seen that during the heat stimulation process, anisomycin should be added later. This method is used for single-cell preparation experiments, in which the perfusion solution contains only actinomycin and triptolide, and the enzymatic solution contains three inhibitors; the final comparison shows that the intracellular gene expression of cells extracted from the (D2+T2)+A2 group is closest to that of normal cells extracted from the CTL1-RT group, indicating that this combination has the smallest difference in gene expression caused by inhibiting the heat shock effect, is more conducive to subsequent cell sequencing, and has the best effect; the details are as follows:

[0145] (1) Actinomycin D is relatively mild in cytotoxicity and has a broad-spectrum and strong inhibitory effect on mRNA transcription; in addition, when the concentration of actinomycin D is 10ug / ml, the cytotoxicity is relatively low and the inhibitory effect is relatively obvious, which is suitable for optimizing the enzymatic hydrolysis method; in addition, actinomycin D has a strong inhibitory effect on the artifact effect marker genes Fos and Dusp1, has a certain inhibitory effect on the heat shock effect marker gene HSF1a, and has a strong inhibitory effect on the inflammatory pathway marker genes IL1b and IL6;

[0146] (2) Triptolide has general cytotoxicity, and its lowest working concentration is T2. When used alone, it exhibits strong biological activity at low concentrations and has a strong inhibitory effect on thermal stimulation pathways represented by HSF1a. It also has a strong inhibitory effect on artifact effects and inflammation-related pathways.

[0147] (3) When triptolide was used in combination with actinomycin D, no enhanced cytotoxicity was found; in addition, when triptolide was used in combination with actinomycin D, it was found that the strong inhibitory effect of triptolide on artifact effect marker genes and heat shock effect marker genes was reversed and restored to normal levels; in addition, when triptolide was used in combination with actinomycin D, the inhibitory function of triptolide would affect the experimental results; and, when actinomycin D was used in combination with triptolide, it was found that due to their different mechanisms of action, the inhibitory effect on artifact marker genes (Fos and Dusp1) and heat shock pathway genes (HSF1a) could make cells closer to the actual situation at normal temperature; however, the inhibitory effect of the combination of dual inhibitors on inflammation-related gene pathways still distorted the gene expression detection;

[0148] (4) As a protein synthesis inhibitor, anisomycin has a stimulatory effect on core marker genes. However, as a protein synthesis inhibitor, anisomycin is indispensable for its inhibitory effect at multiple levels in single cells. It has general cytotoxicity and can stimulate the upregulation of the expression levels of artifact effect marker gene Dusp1, heat shock effect marker gene HSF1a, and inflammatory pathway marker genes (IL1b and IL6).

[0149] (5) The combination of anisomycin and actinomycin D or triptolide can eliminate this pseudo-activation effect to a certain extent; in addition, when anisomycin was used in combination with the other two inhibitors, it was found that the combination of actinomycin D and anisomycin could not inhibit the activation of inflammation-related pathways; the combination of triptolide and anisomycin could not inhibit the activation of artifact effect marker gene pathways; in comparison, the combination of the three inhibitors had a relatively balanced and potent effect;

[0150] (5) The combination of the three inhibitors has a relatively balanced and potent effect, but the toxicity of the three drugs combined to cells is still relatively high, and the inhibitory effect on related pathway genes is not satisfactory. Therefore, the drug combination time and incubation sequence will be further verified. The results show that the incubation time of the three inhibitors combined is positively correlated with cytotoxicity;

[0151] (6) By verifying the effect of the order of inhibitor addition, it can be seen that the longer the incubation time of anisomycin, the greater the cytotoxicity; at the same time, the longer the incubation time of anisomycin, the more obvious the promotion effect on artifact effects and inflammation-related pathways; from the experimental results, it can be seen that anisomycin should be added last to reduce its cytotoxicity and stimulation of various pathways.

[0152] 2. Preparation and verification of mouse skull bone marrow single cells

[0153] (I) Preparation of single cells from mouse skull bone marrow

[0154] Example 1 Preparation of Mouse Skull Bone Marrow Single Cells

[0155] (1) After the mice were anesthetized, 20 ml of ice-cold perfusion buffer containing inhibitors (perfusion buffer: PBS, inhibitor: actinomycin D 5 μg / ml + triptolide 3.064 μg / ml) was used for whole-body perfusion through the heart. After the perfusion, the corresponding tissues were immediately separated (with a time interval of less than 2 minutes) and perfused (from the heart to the whole body), and then the mouse skull was surgically separated;

[0156] (2) The stripped skull was immersed in complete RPMI solution (2% (V / V) FBS, 1% L-glutamine (stock concentration 200 mM, concentration after addition 2 mM), 1% penicillin-streptomycin (stock concentration penicillin 10000 U / mL, streptomycin 10 mg / mL, concentration after addition: penicillin 100 U / mL, streptomycin 100 μg / mL), 1% sodium pyruvate (stock concentration: 100 mM, concentration after addition 1 mM), 1% non-essential amino acids (stock concentration: 1 mM, concentration after addition 0.1 mM), 1.5% HEPES (stock concentration: 1 M, final concentration after addition 15 mM) containing inhibitors (6 cm culture dish, placed on ice);

[0157] (3) After infiltration, use pre-cooled fine forceps under a dissecting microscope to peel off all the meninges and the connective tissue and muscle tissue remaining on the surface of the skull (operate on ice);

[0158] (4) Transfer the cleaned skull to complete RPMI solution (same as the above formula) containing collagenase VIII (1 mg / ml), DNaseI (50 U / ml), inhibitors (5 μg / ml actinomycin D + triptolide 3.064 μg / ml + anisomycin 27.1 μg / ml) (place a 6 cm culture dish on ice) for infiltration; after infiltration, use pre-cooled fine scissors to cut the skull into pieces (length and width less than 2 mm X2 mm); transfer the culture dish (containing the cut skull) to a 37°C incubator and incubate at 50 RPM (round per minute, 50 RPM is 50 revolutions per minute) for 20 minutes;

[0159] (5) After digestion, add an equal volume of pre-cooled complete RPMI solution containing 10% FBS (same as the above recipe) to terminate digestion, and place the culture dish on ice; use pre-cooled fine forceps to crush and clamp the skull flap in the culture dish 100 times (operate on ice);

[0160] (6) Filter through a 70 μm cell strainer and centrifuge at 4°C, 500 g for 5 min, discard the supernatant; add 1 ml of ACK red blood cell lysis buffer to resuspend the cells and incubate on ice for 5 min to remove red blood cells;

[0161] (7) Add 5 ml of FACS solution (PBS buffer containing 2% FBS) to stop red cell lysis, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, and resuspend in PBS for subsequent dead cell removal.

[0162] (8) The cells resuspended in PBS were centrifuged at 300 g for 10 min at 4°C, the supernatant was discarded, and the cells were resuspended in 100 μl DeadCellRemoval Microbeads (Miltenyi Biotec, the number of cells per reaction should be 10^7) (after discarding the supernatant, the tube contains solid cell blocks). The resuspension solution contained 5 μg / ml of inhibitor actinomycin D, 3.064 μg / ml of triptolide, and 27.1 μg / ml of anisomycin, and incubated at room temperature for 15 min.

[0163] (9) Using an MS column in combination with a magnetic field to remove dead cells (Miltenyi Biotec), specifically comprising: placing the MS column in a magnetic field, and rinsing the column with 500ul 1X Binding Buffer; then mixing the cell suspension after the above incubation with 1ml Binding Buffer (containing inhibitors 5μg / ml actinomycin D, 3.064μg / ml triptolide, and 27.1μg / ml anisomycin), passing through the MS column, and collecting the filtered cells to obtain mouse skull bone marrow single cells;

[0164] (10) The obtained mouse skull bone marrow single cells were centrifuged at 4°C, 500 g for 5 minutes, the supernatant was removed, and the cells were resuspended in PBS for subsequent single cell library construction.

[0165] Comparative Example 1

[0166] The conventional enzymatic hydrolysis method (SBM-CTL) was used to extract single cells from mouse skull bone marrow. The difference between the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention (Example 1) is that the perfusion solution and the enzymatic hydrolysis solution of the conventional enzymatic hydrolysis method (SBM-CTL) do not contain inhibitors; and the conventional enzymatic hydrolysis method does not include the step of using a clamp to crush 100 times after digestion; the other steps are the same as the optimized enzymatic hydrolysis method in Example 1.

[0167] Comparative Example 2

[0168] Comparative Example 2 refers to "Kolabas, ZI et al. Distinct molecular profiles of skullbone marrow in health and neurological disorders. Cell 186, 3706-3725. e29 (2023)" and uses a cold mechanical grinding method to extract mouse skull bone marrow single cells, including: after extracting the mouse skull bone cover, placing it in a DEME solution, using fine scissors to cut the bone cover into small pieces, after brief shaking, filtering with a 70 μm cell strainer, and centrifuging at 4°C, 500g for 5 minutes, discarding the supernatant to obtain cells.

[0169] (II) Comparison of single-cell sequencing of mouse skull bone marrow prepared by different preparation methods

[0170] (1) Conventional enzymatic hydrolysis (SBM-CTL) versus mechanical cold grinding (SBM-Dounce)

[0171] Comparison of the cell yield and non-immune cell number of skull bone marrow samples extracted by conventional enzymatic hydrolysis (SBM-CTL) (Comparative Example 1) and mechanical cold grinding (SBM-Dounce) (Comparative Example 2) showed that Figure 2a and 2b As shown, the results show that the conventional enzymatic hydrolysis method (SBM-CTL) can increase the cell yield of a single skull bone marrow sample (which can save mouse samples) compared to cold grinding (SBM-Dounce), and increase the number of non-immune cells (the increase in the number of non-immune cells mainly increases the endothelial, epithelial and stromal cells, which is very important for studying tissue microenvironment, that is, in terms of cell types, the enzymatic hydrolysis method is better than the cold grinding method); therefore, the present invention is improved on the basis of the conventional enzymatic hydrolysis method.

[0172] (2) Conventional enzymatic hydrolysis method (SBM-CTL) versus optimized enzymatic hydrolysis method (SBM-OPT)

[0173] The skull bone marrow single cells extracted by the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention (Example 1) were sequenced and grouped according to the above-mentioned single-cell sequencing method (i.e., using the NovaSeq XPlus sequencing platform, adopting the PE150 sequencing mode, the single-cell sequencing amount reached 50kreadpairs / cell, and the 5-terminal transcriptome data of each cell was obtained, and the cells were grouped using the UMAP method), as shown below:

[0174] The single cell sequencing clustering diagram extracted by SBM-CTL and SBM-OPT is shown in Figure 3a and 3bAs shown in the figure, 3a is the cell clustering diagram of the SBM-CTL and SBM-OPT sequencing results after UMAP dimensionality reduction and fusion display, from which it can be seen that the SBM-CTL group has an extra group of cells compared with the SBM-OPT group (the circled cells, this group of cells is unknown at this time); then on this basis, SBM-CTL and SBM-OPT are displayed separately, and cell clustering is performed, as shown in 3b, the circled part in 3b represents the non-overlapping cells between the two, mainly located in Neutrophil and Granulocyte, revealing that pseudo-activated cells mainly exist in neutrophils and granulocytes; the above results show that there is an artifact effect in the SBM-CTL group, and SBM-OPT can effectively reduce the artifact effect; the artifact effect mainly affects the myeloid cells in the skull bone marrow, such as Neutrophil (neutrophil) and Granulocyte (granulocyte).

[0175] In addition, the Neutrophil cell marker and the pseudo-activation phenotype artifact marker light up the picture as shown in Figure 4a As shown, the proportion of pseudo-activated Neutrophil is shown in Figure 4b As shown in the figure, the results show that the conventional enzymatic hydrolysis method increases the amount of Neutrophil pseudo-activated neutrophils (exA-Neutrophil), and the optimized enzymatic hydrolysis method of the present invention can effectively inhibit the artifact effect caused by heat shock; the light-up diagram of Granulocyte cell markers and pseudo-activated phenotype artifact markers is shown in Figure 5a As shown, the proportion of pseudo-activated Granulocytes is shown in Figure 5b As shown, the results show that the conventional enzymatic hydrolysis method increases the number of pseudo-activated granulocytes (exA-Granulocytes), and the optimized enzymatic hydrolysis method of the present invention can effectively inhibit the artifact effect caused by heat shock;

[0176] In addition, the cells extracted by the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention were subjected to the cell clustering UMAP test. Figure 6a As shown, the gene expression difference between pseudo-activated cells and normal cells in the cell population (Neutrophil) was analyzed using the differential gene volcano plot. The results are shown in Figure 6b As shown; the results showed that neutrophils were pseudo-activated, the differentially expressed genes were significantly up-regulated and down-regulated, and the overall expression was pro-inflammatory; the cells extracted by the conventional enzymatic hydrolysis method (SBM-CTL) and the optimized enzymatic hydrolysis method (SBM-OPT) of the present invention were subjected to cell clustering UMAP test, and the results were as follows Figure 7a As shown, the gene expression difference between pseudo-activated cells and normal cells in the cell group (Granulocyte) was analyzed using the differential gene volcano plot. The results are as follows Figure 7bAs shown; the results showed that granulocytes were pseudo-activated, differentially expressed genes were significantly up- and down-regulated, and the overall pattern was inflammatory.

[0177] 3. Single-cell preparation and verification of mouse brain

[0178] (I) Single-cell preparation of mouse brain

[0179] In the following examples, EBSS (sterile Earle's balanced salt solution) (ThermoScientific; Cat. No.: 24010043): contains calcium, magnesium, bicarbonate and phenol red, and should be refrigerated after each use; papain solution: contains L-cysteine, and is prepared with 5 ml of EBSS to obtain a solution with a final concentration of 20 units of papain, 1 mM L-cysteine ​​and 0.5 mM EDTA per ml; the solution needs to be incubated briefly at 37°C to ensure complete dissolution and activity; deoxyribonuclease I (DNase) solution: is prepared with 0.5 ml of EBSS to obtain a solution with 2000 units of DNase per ml; to ensure enzyme activity, avoid vigorous mixing; ovalbumin inhibitor solution: ovomucoidprotease inhibitor and bovine serum albumin are dissolved in 32 ml of EBSS to obtain a solution containing 10 mg of ovomucoidprotease inhibitor and 10 mg of bovine serum albumin per ml. Use an aliquot from this bottle for each dissociation; refrigerate after each use. Stable at 2-8°C after reconstitution.

[0180] In the following example, the papain digestion buffer is prepared as follows:

[0181] (1) Add 32 ml of EBSS (bottle 1) to the ovalbumin inhibitor solution and allow to dissolve while the other ingredients are prepared; mix before use; reconstitute for the first use, then store and reuse;

[0182] (2) Add 5 ml of EBSS to the papain solution; then place in a 37°C water bath until the papain is completely dissolved and the solution is clear; the solution should be used immediately but can be kept at room temperature during the dissection. Provide a separate bottle of papain for each dissociation.

[0183] (3) Add 500 μl of EBSS to the DNase and mix gently—DNase is sensitive to shear denaturation; add 250 μl of this solution to the papain-containing solution; this preparation contains a final concentration of approximately 20 units / ml of papain and 0.005% of DNase.

[0184] Example 2 Single cell preparation of mouse brain

[0185] (1) After the mice were anesthetized, they were perfused with 20 ml of ice-cold perfusion buffer containing inhibitors (perfusion buffer: PBS, inhibitor: actinomycin D 5 μg / ml + triptolide 3.064 μg / ml) (systemic perfusion through the heart);

[0186] (2) As soon as possible after perfusion, the mouse brain was dissected and removed, immersed in 10 ml PBS solution containing inhibitors (actinomycin D 5 μg / ml + triptolide 3.064 μg / ml), protected from light and placed on ice;

[0187] (3) Transfer the brain tissue to a culture dish (on ice) containing a digestion buffer containing papain and DNase solution (containing inhibitors 5 μg / ml actinomycin D, 3.064 μg / ml triptolide, and 27.1 μg / ml anisomycin); mince the brain using a sterile pre-cooled scalpel (the pieces should be less than or equal to 10×10×10 mm), and transfer the tissue fragments and digestion buffer to a 50 ml centrifuge tube; incubate the 50 ml tube containing the tissue at 37°C on a rocker platform for 20 minutes;

[0188] (3) After the incubation, take out the ground tissue mixture by blowing up and down with a 10 ml pipette (use a pipette to suck up and then eject the mixture so that the mixture is ground into smaller pieces when passing through the tube mouth and the tube) to allow the undissociated tissue fragments to settle to the bottom of the test tube; use a pipette to carefully transfer the cell suspension to a new 15 ml tube, centrifuge at 4°C, 500g for 5 minutes; prepare the solution to resuspend the cells: mix 2.7 ml EBSS with 300 μl recombinant albumin-follicle-like protein inhibitor solution in a sterile tube, DNase solution (150 μl, 2000 U / ml), and add inhibitors (actinomycin D with a final concentration of 5 μg / ml, triptolide 3.064 μg / ml, and anisomycin 27.1 μg / ml);

[0189] (4) After centrifugation at 4°C, 500 g for 5 minutes, discard the supernatant, resuspend the cells with the above solution, and filter with a 70 μm cell strainer to remove tissue debris to obtain a cell suspension; transfer the cell suspension to a 15 ml tube, centrifuge at 4°C, 500 g for 5 minutes, and discard the supernatant; resuspend the cells with 30% Percoll solution (30% Percoll solution formula: 9 ml Percoll + 1 ml 10X PBS + 23 ml 1XPBS) (pre-cooled at 4°C) to obtain a Percoll cell solution;

[0190] (5) Centrifuge the Percoll cell solution at 4°C, 700 g for 15 min (the centrifuge speed is set to 0), carefully remove the supernatant containing myelin, wash the cells with pre-cooled PBS solution (500 g, 5 min), discard the supernatant, resuspend the cells with PBS, and adjust the cell concentration to less than 1×10^9 for subsequent dead cell removal operations;

[0191] (6) Resuspend the cells in the above PBS, centrifuge at 300g for 10 minutes at 4°C, discard the supernatant, and resuspend them in 100 μl DeadCellRemoval Microbeads (Miltenyi Biotec, the number of cells per reaction should be 10^7) containing 5 μg / ml of inhibitor actinomycin D, 3.064 μg / ml of triptolide, and 27.1 μg / ml of anisomycin, and incubate at room temperature for 15 minutes;

[0192] (7) Using MS column in combination with magnetic field to remove dead cells (Miltenyi Biotec), specifically including: placing the MS column in a magnetic field, rinsing the column with 500 μl 1X Binding Buffer; then mixing the cell suspension after the above incubation with 1 ml Binding Buffer (containing inhibitors 5 μg / ml actinomycin D, 3.064 μg / ml triptolide and 27.1 μg / ml anisomycin), passing through the MS column, and collecting the filtered cells to obtain single cells of the mouse brain;

[0193] (8) The obtained mouse brain single cells were centrifuged at 4°C, 500 g for 5 minutes, the supernatant was removed, and the cells were resuspended in PBS for subsequent single cell library construction.

[0194] Comparative Example 3

[0195] The difference between Comparative Example 3 (conventional enzymatic hydrolysis (brain-CTL)) and the optimized enzymatic hydrolysis (brain-OPT) of the present invention (Example 2) is that the perfusion solution and the enzymatic hydrolysis solution of the conventional enzymatic hydrolysis (brain-CTL) do not contain inhibitors; the other steps are the same as the optimized enzymatic hydrolysis of Example 2.

[0196] Comparative Example 4

[0197] Comparative Example 4: A mechanical cold grinding method (brain-Dounce) was used to extract single cells from the mouse brain, specifically comprising: after the mouse was anesthetized, the heart was perfused with a pre-cooled 30 ml PBS solution, and then the brain was quickly removed; the brain was placed in a mortar pre-cooled on ice for 30 minutes, and 10 ml PBS solution was added, and the brain was ground with a pre-cooled pestle (the whole process was performed on ice); after the tissue was homogenized, it was filtered using a 70 μm cell strainer to collect the filtrate; the filtrate was centrifuged at 4°C and 500 g for 5 minutes; a 30% Percoll solution (30% Percoll solution formula: 9 ml Percoll + 1 ml 10X PBS + 23 ml 1XPBS) (precooled at 4℃) to resuspend the cells to obtain Percoll cell solution; the Percoll cell solution is centrifuged at 4℃, 700g for 15 minutes (the centrifuge lifting speed is adjusted to 0), the supernatant containing myelin is carefully aspirated, the cells are washed with precooled PBS solution (500g, 5 minutes), the supernatant is discarded, and the cells are resuspended with PBS to obtain.

[0198] (II) Comparison of brain single-cell sequencing prepared by different preparation methods

[0199] (1) Comparison of conventional enzymatic hydrolysis, the optimized enzymatic hydrolysis of the present invention, and mechanical cold grinding

[0200] The total number of microglia obtained by the conventional enzymatic hydrolysis method, the optimized enzymatic hydrolysis method of the present invention and the mechanical cold grinding method (the total number of microglia was obtained by flow cytometry: the method is that after obtaining the cell suspension, the total number of cells is first counted under a microscope using a cell counting plate; then, some cells are taken out for cell flow analysis, and after obtaining the proportion of microglia, the total number of cells is multiplied by the corresponding percentage to obtain the number of microglia) Figure 8a As shown, the results show that the total number of microglia obtained by the common enzymatic hydrolysis method (brain-CTL) and the optimized enzymatic hydrolysis method (brain-OPT) of the present invention is much greater than that by cold grinding (brain-Dounce), and the optimized enzymatic hydrolysis method (brain-OPT) of the present invention is slightly better than the common enzymatic hydrolysis method (brain-CTL);

[0201] In addition, if Figure 8b As shown, the conventional enzymatic hydrolysis method (brain-CTL) increases the number of pseudo-activated microglia (exAM) in microglia, and the optimized enzymatic hydrolysis method (brain-OPT) of the present invention can effectively inhibit the exAM caused by the heat shock effect of enzymatic hydrolysis;

[0202] In addition, if Figure 8c As shown, single-cell sequencing microglial population analysis showed that most microglial cells were sensitive to heat shock, and the optimized enzymatic hydrolysis method (brain-OPT) effectively eliminated artifact effects.

[0203] (2) Single-cell sequencing experiments using conventional enzymatic hydrolysis and the optimized enzymatic hydrolysis method of the present invention

[0204] Single cells of the mouse brain obtained by the conventional enzymatic hydrolysis method (brain-CTL) and the optimized enzymatic hydrolysis method (brain-OPT) of the present invention were sequenced and grouped according to the above-mentioned single-cell sequencing method (i.e., using the NovaSeq X Plus sequencing platform, adopting the PE150 sequencing mode, the single-cell sequencing amount reached 50kreadpairs / cell, obtaining the 5-terminal transcriptome data of each cell, and using the UMAP method to group the cells), as shown below:

[0205] (a) For mouse brain microglia

[0206] For the classification of microglia, Figure 9a As shown, the conventional enzymatic hydrolysis method (brain-CTL) has an artifact effect compared to the optimized enzymatic hydrolysis method (brain-OPT) of the present invention. The conventional enzymatic hydrolysis method (CTL) will form a group of pseudo-activated microglia (ex vivo activated microglia, exAM) that highly express early-stress genes in the microglial cell group; and the exAM cell group will not appear in the sample prepared by the mechanical cold grinding method (Dounce), indicating that the heat effect of enzymatic hydrolysis will cause the artifact effect of pseudo-activation of microglia; and in the sample prepared by the optimized enzymatic hydrolysis method (OPT) of the present invention, the exAM group did not appear;

[0207] In addition, the gene expression of microglial cell markers (Tmem119, P2ry12, Cx3cr1) and pseudo-activation phenotype artifact markers (Fos, Cccl4, Junb, Jun, Zfp36) was shown in Figure 2 Figure 9b As shown, the results show that the optimized enzymatic hydrolysis method in the present invention can eliminate the artifact effect in brain microglia, and the abnormal upregulation of inflammation-related pathways shown by exAM itself: TNF signaling pathway, inflammatory response, response to heat and IL1betaproduction and other related pathways are falsely activated, which has a serious impact on the identification of different states of microglial cell groups; the abnormal upregulation of these pathways can be inhibited in the OPT method of the present invention, thereby accurately restoring the true transcriptional phenotype and state of microglia.

[0208] In addition, if Fig.9c and 9d As shown, exAM cells showed significant differences from homeostatic microglia, with multiple inflammation-related gene pathways upregulated.

[0209] (b) For mouse brain monocytes / macrophages

[0210] Mouse brain monocytes / macrophages Fig.10a As shown in the figure, the dark purple cell group in the lower left corner shows the cell group after heat shock in Example 3 without adding inhibitors (exAmonomac, exA: EX vivoActivated, in vitro activated monocytes / macrophages), the light purple cell group in the upper right corner is the cell group to which inhibitors are added in Example 2 (Mono / Mac, monocytes / macrophages), and the yellow cell group is other cell group Endothelial (endothelial cells); the results show that the obvious degree of interval between different cell groups shows that they are sensitive to pseudo-activation. The ordinary enzymatic hydrolysis method (Comparative Example 3) not only causes pseudo-activation of microglia, but also causes artifact effects and pseudo-activation of limbic-related macrophages in the brain, indicating that the extraction method of Example 1 can reduce the cell group produced after heat shock and produce more bone marrow immune cells.

[0211] In addition, the gene expression difference between pseudo-activated cells and normal cells in the cell population (exAmonomac) of comparative example 3 (generated using Metascape), that is, the volcano map of differentially expressed genes between pseudo-activated cells and normal cells is shown in Fig.10b As shown, the results show that the relevant immune cells are more sensitive to the heat shock effect, and more differential gene expressions appear; specifically, the monocytes / macrophages in the mouse brain are pseudo-activated by the heat shock effect, and the heat shock effect causes the upregulation of programmed cell death signals (programmed cell death), the increase of negative genes in the phosphate metabolic pathway (phosphate metabolic process), and the upregulation of TNF signaling pathway related pathways; at the same time, unlike microglia, the pseudo-activated monocyte / macrophage population is affected by heat shock, and cell proliferation and normal cell metabolic pathways are downregulated. The present invention can avoid pseudo-activation / pseudo-inhibition of related pathways.

[0212] (c) Neutrophils in mouse brain

[0213] Neutrophils in mouse brain Fig.11aAs shown in the figure, the light green cell group in the lower left corner shows the cell group after heat shock in Example 3 without adding inhibitors (exAneutro, exA: EX vivoActivated, in vitro activated neutrophils), and the purple cell group in the upper right corner is the cell group (Neutrophil, neutrophils) to which inhibitors are added in Example 1; the results show that the obvious degree of interval between different cell groups shows that they are sensitive to pseudo-activation. The ordinary enzymatic hydrolysis method (Comparative Example 3) not only causes pseudo-activation of microglia, but also causes artifact effects and pseudo-activation of neutrophils in the brain; it shows that the extraction method of Example 2 can reduce the cell group produced after heat shock and produce more bone marrow immune cells.

[0214] In addition, the gene expression difference between the pseudo-activated cells and normal cells in the cell population (exAneutro) in Comparative Example 3 (generated using Metascape), that is, the volcano map of differentially expressed genes between pseudo-activated cells and normal cells is shown in Fig.11b As shown, the results show that the relevant immune cells are more sensitive to the heat shock effect, and more differential gene expressions appear; specifically, the pseudo-activation phenomenon caused by the heat shock effect of the relevant immune cells is more complicated, and different gene pathways are upregulated and downregulated; that is, the neutrophils in the brain are also affected by the heat shock effect, and the artifact effect is obvious; at the same time, multiple inflammation-related gene pathways such as pseudo-activated neutrophil leukocyte activation, response to virus, and inflammatory response are significantly upregulated, and the pseudo-activation of related pathways can be repaired by the method of the present invention.

[0215] The above description shows that the present invention has found through single-cell sequencing results that brain cells are sensitive to heat shock effects, and produce obvious artifact effects during the enzymatic hydrolysis process. By further analyzing the results of brain single-cell clustering, it was found that myeloid cells such as neutrophils and granulocytes in the brain are more sensitive to heat shock effects, and have obvious clustering effects with normal cells in physiological states in the analysis of single-cell sequencing results. Among them, neutrophils and granulocytes are affected by pseudo-activation stimulation and show obvious pro-inflammatory characteristics, and TNF and myeloid cell migration-related pathways are significantly upregulated.

[0216] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A composition for extracting single cells from a neuroimmune niche, characterized in that: Including actinomycin D and triptolide.

2. The composition for extracting single cells from the neuroimmune niche according to claim 1, characterized in that: The mass ratio of actinomycin D to triptolide is (4-6):(2-4).

3. The composition according to claim 1, characterized in that The composition also includes anisomycin.

4. The composition according to claim 3, characterized in that The mass ratio of actinomycin D, triptolide and anisomycin is (4.5-5.5): (3-3.5)：(25-30)。 5. An enzymatic hydrolysate for extracting single cells from a neuroimmune niche, characterized in that: The composition comprises the composition according to any one of claims 3 to 4.

6. The enzymatic hydrolysate according to claim 5, characterized in that In the enzymatic hydrolysate, the concentration of actinomycin D is 4μg / ml-6μg / ml, the concentration of triptolide is 2μg / ml-4μg / ml, and the concentration of anisomycin is 25μg / ml-30μg / ml.

7. A method for extracting single cells of a neuroimmune niche, characterized in that: include: The tissue is extracted by enzymatic hydrolysis to obtain single cells of the neuroimmune niche, and the tissue is the brain or the skull; the enzymatic hydrolysis solution used in the enzymatic hydrolysis method is the enzymatic hydrolysis solution described in claim 5 or 6.

8. The extraction method according to claim 7, characterized in that When the tissue is the brain, the neuroimmune niche single cells include one or more of microglia, monocytes / macrophages in the brain, or neutrophils; When the tissue is cranial, the neuroimmune niche single cells include myeloid immune cells.

9. The extraction method according to claim 8, characterized in that Bone marrow immune cells include one or more of granulocytes and neutrophils.

10. The extraction method according to any one of claims 7 to 9, characterized in that: When the tissue is brain, the enzymatic solution also includes papain; When the tissue is skull bone, the enzymatic solution further includes collagenase.

11. The extraction method according to any one of claims 7 to 9, characterized in that Choose one of the following methods: When the tissue is brain, extraction methods include: (1) mixing the brain with the enzymatic hydrolysis solution, performing enzymatic hydrolysis, and centrifuging and discarding the supernatant to obtain a cell tissue mixture; (2) removing tissue fragments from the cell-tissue mixture, centrifuging and discarding the supernatant, and removing myelin to obtain single cells of the neuroimmune niche; When the tissue is skull bone, extraction methods include: (a) mixing the skull with an enzymatic hydrolysis solution, performing an enzymatic hydrolysis reaction, and separating the liquid from the solid to obtain a liquid; (b) The liquid is centrifuged and the supernatant is discarded, and red blood cells are removed to obtain single cells of the neuroimmune niche.

12. The extraction method according to claim 11, characterized in that The conditions of the enzymatic hydrolysis reaction include: the reaction time is 10min-30min.

13. The extraction method according to claim 11 or 12, characterized in that The tissue is skull, and in step (a) of the extraction method, one or more of the following conditions are met: (i) After the reaction, the skull bone flap is crushed and clamped and then liquid-solid separation is performed to obtain a liquid; (ii) After the reaction, a reaction termination liquid is added to terminate the reaction and then liquid-solid separation is performed to obtain a liquid.

14. The extraction method according to claim 13, characterized in that The skull flap was crushed and clamped in an environment of 0℃-4℃.

15. The extraction method according to any one of claims 7, 8, 9, 12 or 14, characterized in that: The extraction method also meets one or more of the following conditions: (A) Before the tissue was extracted by enzymatic method to obtain single cells of the neuroimmune niche, the tissue was processed into pieces; (B) before extracting the tissue using an enzymatic method to obtain a single cell of the neuroimmune niche, the tissue is infiltrated with a solution at 0°C-4°C, the solution comprising the composition of claim 1 or 2; The tissue was brain, and the solution was PBS solution; The tissue was skull, and the solution was RPMI solution; (C) before extracting the tissue by enzymatic method to obtain the neuroimmune niche single cells, the body is perfused, and the perfusion fluid is a 0°C to 6°C PBS solution, and the PBS solution contains the composition of claim 1 or 2; (D) The tissue was extracted using enzymatic hydrolysis and dead cells were removed to obtain single cells of the neuroimmune niche.

16. The extraction method according to claim 15, characterized in that Dead cells were removed using a MS column in combination with a magnetic field.

17. The extraction method according to claim 16, characterized in that Before using the MS column, rinse it with Binding Buffer.

18. Use of the composition according to any one of claims 1 to 4 in extracting single cells from the neuroimmune niche.

19. A single-cell sequencing method for a neuroimmune niche, characterized in that: include: Neuroimmune niche single cells are extracted from tissues using the extraction method described in any one of claims 7 to 17.

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