Preparation method of fusion cell as well as product and application thereof

Abstract

The invention discloses a preparation method of a fusion cell as well as a product and application thereof, and relates to the technical field of biology. The preparation method comprises the following steps: transfecting mesenchymal stem cells by using a construction vector of human ACE2 protein, transfecting macrophages by using a construction vector of new coronavirus S protein, and mixing the two transfected cells to obtain the fusion cell. The fusion cell has a good removal effect on IgG, has good wettability on inflammatory injury and IgG deposition kidney tissues, and can promote the recovery of the injured kidney cells; a good cell burial effect is achieved on kidney cell apoptotic bodies; under the stimulation of endocytosis of IgG, phagocytosis of kidney cell apoptotic bodies and proinflammatory factors, relatively high anti-inflammatory, anti-oxidation and repair-promoting factor expression states can be maintained; for mature dendritic cells and fusion cells, the PDL-1 level can be up-regulated, and an immunosuppression effect is achieved. The fusion cell provided by the invention has better potential for treating nephropathy, and provides a new direction for clinical application and treatment strategies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing fusion cells, a product thereof and an application thereof. Background Art

[0002] Immune nephropathy is a chronic glomerular disease with the same immunopathological characteristics caused by a variety of causes, including purpuric nephritis, lupus nephritis and IgA nephropathy. Due to the dysfunction of the patient's immune system, immune cells are activated, and antibody immune complexes are produced and deposited in the kidneys, causing damage to the kidney's intrinsic cells, triggering an inflammatory response, and destroying the normal function of the kidney's intrinsic cells. Patients develop proteinuria, hematuria, edema and other kidney disease symptoms and become ill. Among them, some patients will eventually develop uremia. The current treatment methods mainly include plasma exchange, immunosuppressants, hormones and antiplatelet aggregation, all of which aim to relieve symptoms and slow down the deterioration of the disease, and the treatment effect is very limited.

[0003] Macrophages (abbreviated as ) is a type of immune cell located in tissues that differentiates from monocytes in the blood after passing through blood vessels. It participates in nonspecific defense (innate immunity) and specific defense (cellular immunity) in the human body. Its main function is to phagocytose cell debris and pathogens, while presenting antigens and activating other immune cells to respond to pathogens. Macrophages usually exist in two different subgroups: (1) classically activated / M1 macrophages, which are proinflammatory cells, polarized by lipopolysaccharide (LPS) alone or in combination with Th1 cytokines (such as IFN-γ, GM-CSF), and produce proinflammatory cytokines, and (2) alternatively activated / M2 macrophages, which have anti-inflammatory and immunomodulatory effects, are polarized by Th2 cytokines IL-4 and IL-13, and produce anti-inflammatory cytokines such as IL-10 and TGF-β. In the renal microenvironment of immune nephropathy, there will be extensive macrophage infiltration, among which M1 type will secrete pro-inflammatory factors, aggravating the condition. In the later stage, although M2 anti-inflammatory type is also activated, it will secrete pro-fibrotic factors to promote renal fibrosis. At the same time, the deposition of antibody immune complexes will also mediate the ADCC effect of macrophages and damage the kidneys. In recent years, many scholars have also focused on using M2 macrophages to treat immune nephropathy.

[0004] Bone marrow mesenchymal stem cells (BMSC) are a type of adult stem cells originating from the mesoderm, with self-renewal and multidirectional differentiation potential, and can differentiate into a variety of interstitial tissues, such as bone, cartilage, fat, bone marrow hematopoietic tissue, etc., and are now widely used in experimental and clinical research and genetic engineering regeneration in the fields of bone, heart, cartilage, central nervous system, skin, etc., and also have great application prospects in the field of tissue and organ repair. There are also many clinical studies showing that exogenous BMSC administration can prevent kidney damage and promote kidney recovery through immune regulation of the immune system and the release of paracrine factors and microbubbles.

[0005] Several studies have shown that BMSCs and macrophages can interact with each other. BMSCs can restore the slit diaphragm damage between podocyte foot processes by reducing macrophage infiltration and polarizing them to M2 type. They can also promote the release of anti-inflammatory factors, reduce the release of pro-inflammatory factors, and enhance the phagocytic ability of macrophages. Macrophages can also improve the anti-inflammatory, pro-repair, pro-angiogenesis and immunosuppressive functions of BMSCs.

[0006] Cell fusion refers to the process of merging two or more cells into one cell under natural conditions or by artificial induction. It includes the connection and fusion of plasma membranes, cytoplasmic fusion, and the formation of a mixed system of nuclei, organelles, and enzymes. Cell fusion has been proven to occur frequently in tumor microenvironments and inflammatory immune microenvironments. The existing induction methods have relatively low fusion efficiency, which limits their clinical applicability.

[0007] In this study, we transiently transfected human ACE2 protein and SARS-CoV-2 S protein plasmids to induce macrophage-BMSC cell fusion in vitro and The fusion cells are purified to explore their immunomodulatory effects, and their respective advantages are used to amplify the anti-inflammatory and immunosuppressive effects. Studies have shown that IL-6 in immune nephropathy aggravates the course of the disease by promoting inflammatory responses, the formation of immune complexes, and activating immune cells. We prepared the fusion cells into bionic nanovesicles, and used the specific proteins expressed on their surface as nanobait for the inflammatory factor IL-6, targeting the antibody deposition site, adsorbing IL-6 in the microenvironment, and proposed a strategy of combined treatment with fusion cells and their bionic nanovesicles. The above-mentioned fusion cells and derived nanovesicles can be used as a new means of treating immune nephropathy and promote the development of new clinical immune nephropathy treatments.

[0008] The existing commonly used fusion methods include: PEG method, electric fusion method and Sendai virus induction method (poor specificity, cell damage), co-incubation method (low specificity and fusion rate), and the existing methods all require a large amount of screening work on the cells after fusion treatment to select heterotypic fusion cells. The present invention uses transient transfection of human ACE2 protein and new coronavirus S protein to efficiently induce the generation of heterotypic fusion cells. Summary of the invention

[0009] The purpose of the present invention is to provide a method for preparing fused cells and a product and application thereof.

[0010] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0011] In one aspect, the present invention provides a method for preparing fusion cells, comprising the following steps: transfecting mesenchymal stem cells with a construct vector of human ACE2 protein, transfecting macrophages with a construct vector of novel coronavirus S protein, and mixing the transfected mesenchymal stem cells and macrophages to obtain fusion cells;

[0012] The construction vector of human ACE2 protein is pHG-CMV-hACE2, and the NCBI sequence number of hACE2 is NM_021804; the construction vector of the new coronavirus S protein is pcDNA3.1-2019-HnCoV-S, and the NCBI sequence number of 2019-HnCoV-S is NC_045512.2.

[0013] Specifically, the mesenchymal stem cells include human primary mesenchymal stem cells, mouse primary mesenchymal stem cells, rat primary mesenchymal stem cells, hamster primary mesenchymal stem cells, experimental miniature pig primary mesenchymal stem cells, experimental dog primary mesenchymal stem cells, experimental monkey primary mesenchymal stem cells and immortalized mesenchymal stem cell lines derived from the above various experimental animals.

[0014] Furthermore, the mesenchymal stem cells include human mesenchymal stem cells and mouse mesenchymal stem cells.

[0015] Furthermore, the mesenchymal stem cells are mouse mesenchymal stem cells.

[0016] Furthermore, the mesenchymal stem cells include embryonic mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp mesenchymal stem cells, and various mesenchymal stem cells induced and differentiated by iPSC (induced pluripotent stem cells).

[0017] Furthermore, the mesenchymal stem cells are mouse bone marrow mesenchymal stem cells.

[0018] Furthermore, the mesenchymal stem cells are immortalized mouse bone marrow mesenchymal stem cell lines.

[0019] Specifically, the macrophages include human primary macrophages, mouse primary macrophages, rat primary macrophages, hamster primary macrophages, experimental miniature pig primary macrophages, experimental dog primary macrophages, experimental monkey primary macrophages and immortalized macrophage cell lines directly derived from or induced by the above various experimental animals.

[0020] Further, the macrophages are selected from RAW264.7 (mouse mononuclear macrophage leukemia cells), J774A.1 (mouse mononuclear macrophages), MH-S (mouse alveolar macrophages), Ana-1 (mouse macrophages), WEHI3B (mouse myeloid mononuclear leukemia cells), iBMDM (mouse immortalized bone marrow-derived macrophages), mouse primary macrophages (sources include bone marrow / bone, abdominal cavity, spleen, brain, adipose tissue, liver, lung, kidney, heart, muscle tissue and blood lymphatic system), THP-1 (human mononuclear leukemia cells), U937 (human histiocytic lymphoma cells), Mono Mac6 / Mono Mac1 (human acute mononuclear leukemia cells), human primary macrophages (sources include bone marrow / bone, abdominal cavity, spleen, brain, adipose tissue, liver, lung, kidney, heart, muscle tissue and blood lymphatic system), and one or more of various macrophages induced and differentiated from monocytes or iPSCs.

[0021] Furthermore, the macrophages are RAW264.7.

[0022] Furthermore, the ratio of mesenchymal stem cells and macrophages mixed is (1-5):1;

[0023] Furthermore, the ratio of mesenchymal stem cells and macrophages mixed was 1:1.

[0024] Specifically, the construction vector of human ACE2 protein transfects mesenchymal stem cells including the following steps:

[0025] (1) Plasmid transfection was performed when mesenchymal stem cells were cultured to a confluence of 60-80%;

[0026] (2) Prepare transfection mixture: the ratio of OPTI reagent, plasmid and transfection reagent is (80-100) μl: 1 μg: (3-8) μL, incubate for 30-60 min;

[0027] (3) Add the transfection mixture into the mesenchymal stem cell culture medium and culture.

[0028] Furthermore, in step (1), plasmid transfection is performed when the mesenchymal stem cells are cultured to a confluence of 70%.

[0029] Furthermore, in step (2), the ratio of OPTI reagent: plasmid: transfection reagent is 80 μl: 1 μg: 3 μL.

[0030] Furthermore, the incubation condition in step (2) is incubation at room temperature in the dark for 30 minutes.

[0031] Furthermore, the plasmid described in step (2) is pHG-CMV-hACE2.

[0032] Furthermore, step (3) specifically includes discarding the mesenchymal stem cell culture medium, washing once with 500 μL PBS, adding 500 μL culture medium and the prepared transfection mixture, placing in an incubator (37° C.), and replacing with fresh culture medium after 12 hours.

[0033] Specifically, the construction vector of the novel coronavirus S protein transfects macrophages, comprising the following steps:

[0034] (1) Plasmid transfection was performed when macrophages were cultured to 60-80% confluence;

[0035] (2) Prepare transfection mixture: OPTI reagent: plasmid: transfection reagent ratio is (80-100) μl: 1 μg: (1-5) μL, incubate for 30-60 min;

[0036] (3) Add the transfection mixture into the macrophage culture medium and culture.

[0037] Furthermore, in step (1), plasmid transfection is performed when the macrophages are cultured to a confluence of 70%.

[0038] Furthermore, in step (2), the ratio of OPTI reagent: plasmid: transfection reagent is 80 μl: 1 μg: 2 μL.

[0039] Furthermore, the incubation condition in step (2) is incubation at room temperature in the dark for 30 minutes.

[0040] Furthermore, the plasmid described in step (2) is pcDNA3.1-2019-HnCoV-S.

[0041] Furthermore, step (3) specifically includes discarding the macrophage culture medium, washing once with 500 μL PBS, adding 500 μL culture medium and the prepared transfection mixture, placing in an incubator (37° C.), and replacing with fresh culture medium after 12 hours.

[0042] On the other hand, the present invention provides a method for purifying the above-mentioned fusion cells, comprising co-incubating the adherent fusion cells with magnetic nanoparticles, resuspending the cells, centrifuging, resuspending the cells and continuing to culture them adherently, and removing the non-adherent cells to obtain purified fusion cells.

[0043] Specifically, the ratio of the fused cells to the magnetic nanoparticles is (5-10)×10 5 Cells: (5-30) μg magnetic nanoparticles.

[0044] Furthermore, the ratio of the fused cells to the magnetic nanoparticles is 5×10 5 cells: 25 μg magnetic nanoparticles.

[0045] Specifically, the co-incubation time of the fused cells and the magnetic nanoparticles is 12-24 hours;

[0046] Furthermore, the co-incubation time of the fused cells and the magnetic nanoparticles is 12 hours.

[0047] Specifically, the time for continuing adherent culture is 1-5 hours.

[0048] According to some embodiments of the present invention, the method for purifying the fused cells is as follows: the cell mixture before purification (5×10 5 The cells were incubated with 25ug magnetic nanoparticles for 12h, the cell mixture was centrifuged, washed to remove excess nanoparticles, and added to a well plate. A directional magnetic field was applied above and below the well plate to make the cells move rapidly toward the bottom of the plate. The cells that preferentially adhered to the wall and were obviously elliptical were fused cells. After 3h, the non-adherent cells were discarded and the culture was continued to obtain high-purity fused cells.

[0049] In another aspect, the present invention provides a fusion cell prepared according to the above preparation method or the above purification method.

[0050] In another aspect, the present invention provides a method for preparing fusion cell-derived nanovesicles, comprising the following steps:

[0051] (1) Centrifuge the above fused cells to obtain cell pellets, and resuspend the cells in PBS;

[0052] (2) Push the cells 40-60 times, the pore size of the polycarbonate membrane is 200 nm, centrifuge at 800-1200g for 10-30 min, take the supernatant, place it in a 100KD ultrafiltration tube, and centrifuge at 2000-3000g for 10-30 min.

[0053] Specifically, the centrifugation condition in step (1) is 800-1200 rpm, and the centrifugation time is 5-10 min;

[0054] Furthermore, the centrifugal condition in step (1) is 1000 rpm, and the centrifugal time is 5 min.

[0055] Specifically, the cells were pushed 50 times as described in step (2).

[0056] Specifically, step (2) is to use a liposome extruder to push the cells 50 times, centrifuge at 1000g for 10 minutes, take the supernatant, and centrifuge the supernatant at 3000g for 10 minutes to obtain nanovesicles.

[0057] In another aspect, the present invention provides fusion cell-derived nanovesicles prepared by the above preparation method.

[0058] In yet another aspect, the present invention provides use of the above-mentioned fusion cells or fusion cell-derived nanovesicles in the preparation of products for treating kidney disease.

[0059] Specifically, the kidney disease is immune kidney disease.

[0060] Furthermore, the immune kidney disease includes purpuric nephritis, lupus nephritis, IgA nephropathy, membranous nephropathy and transplant nephropathy.

[0061] Specifically, the product is a medicine.

[0062] Specifically, the transplant nephropathy is a kidney disease caused by immune reasons after kidney transplantation.

[0063] In yet another aspect, the present invention provides a product for treating kidney disease, wherein the product comprises the above-mentioned fusion cells and fusion cell-derived nanovesicles.

[0064] Specifically, the product is a medicine.

[0065] Furthermore, the medicine also includes a pharmaceutically acceptable carrier.

[0066] The beneficial effects of the present invention are:

[0067] 1. The vector constructed by transiently transfecting human ACE2 protein and SARS-CoV-2 S protein in the present invention can induce the fusion of BMSCs and RAW264.7 in a short time, and continue to culture for 24 hours after purification. Immunofluorescence and qRT-PCR detection show that human ACE2 protein and SARS-CoV-2 S protein are not expressed at the protein and mRNA levels, which will not affect the subsequent functional detection.

[0068] 2. Use superparamagnetic nanoparticles for purification. These nanoparticles have the characteristics of low toxicity, low immunogenicity, and easy absorption. They can promote the anti-inflammatory effect of BMSCs and macrophages. Purification using a directional magnetic field will not cause damage to the fused cell mixture, and is simple, economical, and efficient.

[0069] 3. The purified fusion cells have a good scavenging effect on IgG, and maintain a high expression of anti-inflammatory, antioxidant and pro-repair factors; they have good infiltration into inflammatory damaged renal tissues with IgG deposition (kidney organoids), and can promote the recovery of damaged renal cells; at the same time, they have a good burial effect on apoptotic bodies of renal cells; under the conditions of pro-inflammatory macrophage induction, they still have a high expression of anti-inflammatory, antioxidant and pro-repair factors. For mature DC cells, fusion cells significantly upregulate their PDL-1 levels, exerting an immunosuppressive effect, and clinically used immunosuppressants after renal transplantation also work through this pathway (inhibiting DC cell maturation and activation). Fusion cells inherit the characteristics of macrophages internalizing IgG and phagocytosing apoptotic bodies, and also inherit the antioxidant, anti-inflammatory and pro-repair effects of BMSC in a damaged and highly inflammatory environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Fluorescence detection of fusion cell purification efficiency.

[0071] Figure 2 qRT-PCR was used to detect the expression of FcγR-related factors in RAW264.7, BMSC and fusion cells (Hybird). Compared with RAW264.7, *p<0.05, ***p<0.001; compared with the BMSC group, ###p<0.001.

[0072] Figure 3 qRT-PCR was used to detect the expression levels of anti-inflammatory and antioxidant related factors in RAW264.7, BMSC and fusion cells. Compared with RAW264.7, *p<0.05, **p<0.01, ***p<0.001; compared with the BMSC group, #p<0.05, ##p<0.01, ###p<0.001.

[0073] Figure 4 qRT-PCR was used to detect the expression of adhesion and chemotaxis related factors in RAW264.7, BMSC and fusion cells. Compared with RAW264.7, ***p<0.001; compared with BMSC group, ###p<0.001.

[0074] Figure 5 To detect the ink phagocytosis ability of RAW264.7, BMSC and fusion cells.

[0075] Figure 6 Fluorescence detection of IgG internalization by fusion cells.

[0076] Figure 7 qRT-PCR was used to detect the changes in the expression of anti-inflammatory and antioxidant factors after the fusion cells phagocytosed IgG; compared with the Hybrid group, *p<0.05, ***p<0.001.

[0077] Figure 8 The infiltration of fused cells into MPC5 3D microspheres and the clearance of IgG antibodies after 6 hours of DOX treatment were detected; in the figure, A: MPC5 without DOX treatment + Hybird, B: MPC5 DOX treatment + Hybird, C: MPC5 DOX treatment primary antibody and secondary antibody incubation + Hybird, D: MPC5 DOX treatment primary antibody and secondary antibody incubation + Hybird pre-activated by inflammatory factors, E: MPC5 DOX treatment primary antibody and secondary antibody incubation.

[0078] Fig. 9 The infiltration of fused cells into MPC5 3D microspheres and the clearance of IgG antibodies after 24 hours of DOX treatment were detected; in the figure, A: MPC5 without DOX treatment + Hybird, B: MPC5 DOX treatment + Hybird, C: MPC5 DOX treatment primary antibody and secondary antibody incubation + Hybird, D: MPC5 DOX treatment primary antibody and secondary antibody incubation + Hybird pre-activated by inflammatory factors, E: MPC5 DOX treatment primary antibody and secondary antibody incubation.

[0079] Fig.10 The level of JC-1 (mitochondrial membrane potential) was detected by flow cytometry in the MPC5+DOX group, compared with the MPC5 group, ***p<0.001.

[0080] Fig.11 The ROS level in MPC5+DOX group was detected by flow cytometry. Compared with the MPC5 group, ***p<0.001.

[0081] Fig.12 The expression level of CD80 in MPC5+DOX group was detected by flow cytometry. Compared with the MPC5 group, *p<0.05.

[0082] Fig.13 qRT-PCR was used to detect the changes in the expression of inflammatory factors after DOX treatment of MPC5. Compared with the MPC5 group, ***p<0.001.

[0083] Fig.14 The morphological changes of MPC5 cells were observed under a microscope after 24 hours of co-culture of fusion cells with inflammatory damage and IgG deposition MPC5.

[0084] Fig.15 To detect ADCC effect by flow cytometry (MPC5 apoptosis before and after 24h of co-culture of fusion cells with immune-damaged MPC5 with IgG deposition, the ratio shown in the figure is the proportion of apoptotic MPC5 cells to total MPC5 cells).

[0085] Fig.16 Fluorescence microscopy was used to observe the phagocytosis of ACs by fusion cells.

[0086] Fig.17 qRT-PCR was used to detect the changes in the expression of anti-inflammatory factors after the fusion cells phagocytosed ACs. Compared with the Hybrid group, *p<0.05, **p<0.01, ***p<0.001.

[0087] Fig.18 The morphological changes of RAW264.7 and fusion cells after stimulation by inflammatory factors.

[0088] Figure 19-20 The expression changes of anti-inflammatory and pro-inflammatory factors in RAW264.7 and fusion cells after stimulation by inflammatory factors, (-) indicates the group without LPS / IFNγ, (+) indicates the group pretreated with LPS / IFNγ), where Fig.19 Middle, **p<0.01, ***p<0.001 compared with (-)LPS / IFNγ group; Fig. 20 **p<0.01, ***p<0.001 compared with the (+)LPS / IFNγ RAW264.7 group.

[0089] Fig.21 The expression of PDL-1 in DC cells was detected after co-culture of fusion cells and DC cells. Compared with the DC group, ***p<0.001.

[0090] Fig. 22 Immunofluorescence was used to detect IgG targeting of NV.

[0091] Fig.23 ELISA was used to detect the clearance ability of NV on free IL-6. Compared with the PC3+IL6 group, ***p<0.001; compared with the PC3+NV+IL6 group, ###p<0.001.

[0092] In the above figures, RAW refers to RAW264.7, and BM refers to BMSC. DETAILED DESCRIPTION

[0093] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0094] Human ACE2 protein, the constructed vector is pHG-CMV-hACE2, hACE2 NCBI sequence number: NM_021804.

[0095] The SARS-CoV-2 S protein was constructed using the pcDNA3.1-2019-HnCoV-S vector, and the 2019-HnCoV-S NCBI sequence number was NC_045512.2.

[0096] Both of the above vectors were constructed by Shanghai Heyuan Biotechnology Co., Ltd.

[0097] Example 1 Preparation of fusion cells

[0098] 1.1 Preparation of cell mixture

[0099] pHG-CMV-hACE2 was transfected with BMSC (mouse bone marrow mesenchymal stem cells), and pcDNA3.1-2019-HnCoV-S was transfected with RAW264.7 (mouse macrophage-tumor-derived cell line). The transfection reagent used was TransIT-X2 transfection (Cat. No. MIR6000) from Mirus. The specific steps of transfection were as follows: 24 hours after transfection, superparamagnetic nanoparticles were incubated. After 12 hours, the cells were digested. BMSC and RAW264.7 were mixed in a 1:1 ratio. After 24 hours, it was seen that all cells were attached to the wall. After trypsin digestion again, the cells were induced to adhere quickly with a static magnetic field, and the non-adherent cells were removed. The purification efficiency was detected by immunofluorescence, and the purity of the fused cells was found to be above 90%.

[0100] RAW264.7 transfection method:

[0101] When the RAW264.7 cells were cultured to 70% confluence, plasmid transfection was performed. Prepare transfection mixture (12-well plate): OPTI reagent: plasmid: transfection reagent = 80 μl: 1 μg: 2 μL. Incubate the prepared mixed reagent at room temperature in the dark for 30 minutes. Discard the RAW264.7 cell culture medium, wash once with 500 μL PBS, then add 500 μl culture medium and the prepared transfection mixture, place in an incubator (37°C), and replace with fresh culture medium after 12 hours.

[0102] BMSC transfection method:

[0103] When the BMSC cells are cultured to 70% confluence, plasmid transfection is performed. Prepare the transfection mixture (12-well plate): OPTI reagent: plasmid: transfection reagent = 80μl: 1μg: 3μL. Incubate the prepared mixed reagent at room temperature in the dark for 30 minutes. Discard the BMSC cell culture medium, wash once with 500μL PBS, then add 500μl culture medium and the prepared transfection mixture, place in an incubator (37°C), and replace with fresh culture medium after 12 hours.

[0104] 1.2 Purification using superparamagnetic nanoparticles includes the following steps:

[0105] The cell mixture before purification (5×10 5 The cells were incubated with 25ug nanoparticles (purchased from Xianfeng Nano, Cat. No. 100966) for 12h, the cell mixture was centrifuged, washed to remove excess nanoparticles, and added to the well plate. Magnets were added above and below the well plate to allow the cells to move rapidly toward the bottom of the plate. The cells that preferentially adhered to the wall and were obviously elliptical were fused cells. After 3h, the non-adherent cells were discarded and the culture was continued to obtain high-purity fused cells.

[0106] 1.3 Fluorescence detection

[0107] Detection method: After transfecting BMSC and RAW264.7 cells respectively according to the above method, BMSC were labeled with CFSE dye (purchased from Maokang Biotechnology, product number: MX3009, the labeling steps were to dilute the probe with HBSS solution 1:2000, incubate in the incubator away from light for 30 minutes, and wash twice with PBS), and RAW264.7 cells were labeled with Cell Tracker Red dye (purchased from Maokang Biotechnology, product number MX4109, the staining method was to dilute with complete culture medium 1:500, incubate in the incubator away from light for 30 minutes, and wash twice with PBS), and fused according to the above 1.1 operation steps, and then purified according to the above 1.2 purification steps, and observed under a fluorescence microscope to observe the proportion of red and green dual fluorescence positive cells.

[0108] Test results: Fluorescence detection found that the purity of fusion cells was greater than 90% ( Figure 1 ).

[0109] Comparative Example 1

[0110] The purified fusion cells (denoted as Hybrid) were compared with RAW264.7 and BMSC alone:

[0111] 1. qRT-PCR detection found that compared with the other two cells, the fusion cells highly expressed anti-inflammatory factors, antioxidant factors and complement inhibitory factors.

[0112] The qRT-PCR detection method is as follows:

[0113] RNA extraction: remove the cell supernatant, add 1ml Trizol to each well, gently blow and mix, and let stand at room temperature for 10 minutes. Transfer the suspension into an EP tube (1.5m), add 0.2ml chloroform to each tube, shake violently first, and then let stand at room temperature for 5 minutes. Then centrifuge at 4°C and 12000rpm for 15 minutes, and carefully transfer the upper colorless and transparent aqueous phase (approximately 400μl) to a new EP tube. Add an equal amount of isopropanol (IPA), mix thoroughly by turning upside down, and centrifuge at 12000rpm and 4°C for 15 minutes. White precipitates can be observed at the bottom of the tube. The supernatant was carefully removed, 1 ml of ethanol (75%) was added to each tube, and the tubes were thoroughly mixed by inversion. The tubes were centrifuged at 12000 rpm and 4°C for 10 min, the supernatant was carefully removed, and the tubes were dried at room temperature for 5-15 min. 0.02-0.03 ml of DPEC water was added, and the tubes were allowed to stand at room temperature for 5-15 min to fully dissolve the precipitate. The RNA concentration was measured and the samples were stored at -80°C.

[0114] qRT-PCR fluorescence quantitative analysis:

[0115] (1) Genomic DNA removal

[0116] The reaction system is as follows:

[0117] Table 1

[0118]

[0119]

[0120] Mix by gently pipetting. Incubate at 42°C for 2 min.

[0121] (2) Reverse transcription reaction

[0122] The reaction system is as follows:

[0123] Table 2

[0124] The reaction solution in step (1) 16μl 5x HiScriptⅡqRT SuperMixⅡ 4μl Total 20μl

[0125] The reaction conditions are as follows:

[0126] Table 3

[0127] temperature time Number of cycles 37℃ 15min 1 85℃ 5sec 1 4℃ - -

[0128] (3) PCR reaction

[0129] The reaction system is as follows:

[0130] Table 4

[0131] 2x ChamQ SYBR qPCR Master Mix(Low ROX Premixed) 10.0μl Primer1 (10 μl) 0.4μl Primer2 (10 μl) 0.4μl Template DNA / cDNA 1μl <![CDATA[ddH2O]]> 8.2μl Total 20μl

[0132] The primer sequences are as follows:

[0133] Table 5

[0134]

[0135]

[0136]

[0137] Test results:

[0138] FcγR receptors: Compared with RAW264.7, CD16 and CD32 in the Hybrid group were significantly upregulated; compared with BMSC, CD16, CD32, CD64, CD45 and Itgam in the Hybrid group were significantly upregulated ( Figure 2 ). This indicates that Hybrid inherited the ability of RAW264.7 cells to bind antibodies.

[0139] Anti-inflammatory and antioxidant part: Compared with RAW264.7, TSG-6, TSG101, SOD2, IDO, PGE-2, CD206, Arg1, IL-10, CD274 and TGFβ in the Hybrid group were significantly upregulated; compared with the BMSC group, SOD2, PGE-2, CD206, Arg1, IL-10, CD274 and TGFβ in the Hybrid group were significantly upregulated, while TSG-6 and IDO were significantly decreased ( Figure 3 ). This indicates that Hybrid has inherited the powerful anti-inflammatory and antioxidant capabilities of BMSC.

[0140] Adhesion factors: Compared with the RAW264.7 group, the ICAM-1 in the Hybrid group increased significantly; compared with the BMSC group, the ICAM-1 in the Hybrid group increased significantly, while the CXCR4 decreased significantly ( Figure 4 ).

[0141] 2. Phagocytic ability test: ink phagocytosis test

[0142] Detection method and results: The same number of BMSCs, RAW264.7 and Hybrid were cultured in the same concentration of ink dye (the ink is ordinary Idege ink, which can be used after filtering out large impurities with filter paper, and 10ul of ink is added to 1ml of culture medium and mixed well) for 4 hours. It was found that the ability of the fused cells to phagocytize carbon particles was stronger than that of BMSC, but weaker than that of RAW264.7, indicating that Hybrid partially inherited the phagocytic ability of RAW264.7 ( Figure 5 ).

[0143] Experimental Example 1: Clearance Efficacy of Fusion Cells on Free IgG

[0144] Experimental methods:

[0145] 20ug IgG-FITC (labeled with green fluorescence) and Hybrid (cell volume 3-6×10 5 ) were co-incubated for 4 h, and the co-localization of IgG and Hybrid cells was detected by fluorescence microscopy. The experimental results are shown in Figure 6 , it was found that after 4 hours, a large number of green fluorescent IgG antibodies were gathered inside the cells, indicating that Hybrid cells can internalize IgG very well. Then qRT-PCR was used to detect (the qRT-PCR detection method is the same as that of comparative example 1) the expression of anti-inflammatory and antioxidant factors after Hybrid absorbed IgG for 4 hours. The results showed that Hybrid can maintain a high expression of anti-inflammatory and antioxidant factors ( Figure 7 ).

[0146] Experimental Example 2 Effects of Inflammatory Damage + IgG Deposition on Kidney Podocyte Organoids

[0147] Experimental methods:

[0148] After treating mouse renal podocytes (MPC5) with 1 μM DOX (adriamycin) for 24 h, 0.2 ul rabbit anti-mouse CD80 antibody / sample (antibody purchased from eBioscience, catalog number 16-10A1) was added, the antibody was diluted with complete medium, incubated in a 37°C incubator in the dark for 30 min, washed with PBS, and 1 ul mouse anti-rabbit green fluorescent secondary antibody / sample (antibody purchased from Proteintech, catalog number SA00014-4) was added, also diluted with complete medium, incubated in a 37°C incubator in the dark for 30 min), washed with PBS, and the cells were scraped with a cell scraper. The cell suspension was made into 3D microsphere organoids in vitro using DNA origami technology (3D microsphere organoids were made by Puheng Biotechnology Co., Ltd.), and then co-incubated with Hybrid (Hybird was labeled with Cell-tracker-Deep Red, the dye was purchased from Shanghai Maokang Biological Co., Ltd., catalog number MX4110, and the labeling steps were referred to the instruction manual), and detected at 6 h and 24 h, respectively.

[0149] The experimental results are as follows Figure 8 As shown, fluorescence microscopy revealed that after 6 h of co-culture, compared with the groups not treated with DOX and not loaded with IgG, Hybrid had stronger infiltration into the microspheres treated with DOX and combined with IgG deposition and could effectively clear IgG (green fluorescence was basically invisible, while MPC5 cells treated with only IgG and DOX still had obvious green fluorescence).

[0150] The experimental results are as follows Fig. 9As shown, fluorescence microscopy observation after 24 hours revealed that compared with the group not treated with DOX, Hybrid had stronger infiltration into the DOX-only treated group and the DOX-combined IgG-loaded group.

[0151] The above results suggest that Hybird has a strong infiltration capacity into inflammatory damaged IgG-deposited renal organoids, and the degree of infiltration increases significantly over time, and it has an effective and rapid ability to clear IgG.

[0152] The basis for the successful modeling of IgG deposition in inflammatory injury:

[0153] After treating mouse renal podocytes with 1 μM DOX (adriamycin) for 24 h, apoptosis, mitochondrial membrane potential, ROS level, and CD80 expression were detected by flow cytometry, and the expression of inflammatory factors was detected by qRT-PCR.

[0154] Detection method:

[0155] Apoptosis detection: The above cells (DOX-treated cells) and supernatant were collected into flow tubes, centrifuged at 1200rpm for 5min, the supernatant was discarded, 1ml PBS buffer was added to each tube and centrifuged at 1200rpm for 5min, then the 5x buffer and ddH2O in the apoptosis kit were mixed at a ratio of 1:4 to prepare the working buffer, 250μl was added to each tube, and then 2.5μl Annexin V-FITC and 5μl PI were added in sequence, blown evenly, incubated in the dark for 5min, and detected by flow cytometer. The apoptosis kit was purchased from Beyotime Biotechnology Co., Ltd., item number C1062S.

[0156] Mitochondrial membrane potential detection: The above cells (DOX-treated cells) were washed once with PBS buffer, and the prepared JC-1 working solution (ddH2O: JC-1: 5x buffer = 1ml: 6.25μl: 250μl, 1ml / well) was added and placed in a 37°C incubator for 30min. Washed twice with PBS, and detected by upflow cytometry after digestion. The mitochondrial membrane potential kit was purchased from Bio-Tech Biotechnology Co., Ltd., item number C2006.

[0157] ROS detection: discard the supernatant of the above cells (DOX-treated cells), add the prepared ROS dye solution (pure DMEM / F12 medium 1:1000 dilution of ROS probe), incubate at 37°C in the dark for 30 minutes, discard the supernatant, wash twice with PBS buffer, add trypsin digestion and stop digestion with complete medium, then draw the cell suspension into the flow tube for detection on the flow cytometer. ROS detection kit was purchased from Bio-Tech Biotechnology Co., Ltd., catalog number: S0033S.

[0158] Detection of CD80 expression: The above cells (DOX-treated cells) were digested, centrifuged, and resuspended. Then, 0.2ul rabbit anti-mouse CD80 antibody / sample (the antibody was purchased from eBioscience, catalog number 16-10A1) was used. The antibody was diluted with PBS, incubated at room temperature in the dark for 30min, washed with PBS, and 1ul mouse anti-rabbit green fluorescent secondary antibody / sample (the antibody was purchased from Proteintech, catalog number SA00014-4) was added. The antibody was diluted with PBS, incubated at room temperature in the dark for 30min, washed with PBS, and resuspended in 500ul PBS and detected by flow cytometer.

[0159] The qRT-PCR detection method was the same as that of Comparative Example 1.

[0160] The primer sequences are as follows:

[0161] Table 6

[0162]

[0163] Experimental results: After MPC5 was treated with 1 μM DOX, apoptosis was significantly increased and mitochondrial membrane potential was significantly downregulated ( Fig.10 ), ROS levels increased significantly ( Fig.11 ), IL-6, TNF-α, IL-1β and IFNγ expression levels were significantly increased ( Fig.13 ), CD80 expression was significantly upregulated ( Fig.12 ), indicating that MPC5 showed inflammatory damage after treatment with DOX, and the highly expressed CD80 could bind to CD80 antibody, so the IgG antibody deposition could be simulated after labeling with rabbit anti-mouse CD80 primary antibody and mouse anti-rabbit IgG-488 fluorescent secondary antibody ( Figure 8 ).

[0164] Experimental Example 3 Effect on in vitro 2D culture of inflammatory injury + IgG deposition in renal podocytes

[0165] In vitro 2D culture inflammatory IgG deposition injury model + Hybird co-culture, observe the effect of Hybird on damaged kidney cells. MPC5 was treated with 1μM DOX and labeled with CD80 primary antibody / secondary antibody (add 0.2ul rabbit anti-mouse CD80 antibody / sample, the antibody was diluted with complete medium, incubated at 37℃, incubator away from light for 30min, washed with PBS, add 1ul mouse anti-rabbit green fluorescent secondary antibody / sample, the antibody was diluted with complete medium, incubated at 37℃, incubator away from light for 30min), co-cultured with fusion cells, and observed cell morphology under a microscope after 24h.

[0166] The experimental results are as follows Fig.14As shown, it was found that there was no significant difference in cell density and morphology in the MPC5 inflammatory injury group before and after 24 hours of cell culture. The cells were swollen and the nuclei were enlarged, as shown in the figure. Compared with the MPC5 inflammatory injury group, the Hybrid co-culture group showed a significant increase in the cell gap. Light microscopic morphological observation found that the damaged kidney cells tended to return to normal, as shown in the figure.

[0167] Flow cytometry was further used to detect the apoptosis of MPC5 cells after the fusion cells were co-cultured with inflammatory-damaged MPC5 cells.

[0168] Detection steps: MPC5 was treated with 1 μM DOX for 24 h, then stained with Cell Tracker Deep Red (purchased from Shanghai Maokang Biological Company, catalog number MX4110) (diluted 1:500 with complete culture medium), and labeled with CD80 primary antibody / secondary antibody (same method as before), co-cultured with Hybrid, and apoptosis was detected by flow cytometry after 24 h.

[0169] The steps of apoptosis detection are as follows:

[0170] The above cells (DOX treated + antibody labeled cells) and supernatant were collected into flow tubes, centrifuged at 1200rpm for 5min, supernatant was discarded, 1ml PBS buffer was added to each tube and centrifuged at 1200rpm for 5min, then 5xbuffer and ddH2O in the apoptosis kit were mixed at a ratio of 1:4 to prepare working buffer, 250μl was added to each tube, and then 5μl PI was added, blown evenly, incubated in the dark for 5min, and detected by flow cytometer. The apoptosis kit was purchased from Biotech Biotechnology Co., Ltd., item number C1062S.

[0171] Test results: Flow cytometry showed that the apoptosis rate of MPC5 in the group without co-culture with Hybrid increased significantly after 24 hours. Compared with the group with inflammatory injury of MPC5 alone, the apoptosis rate of the group with co-culture with Hybrid decreased significantly. No obvious ADCC effect (i.e. antibody-mediated cytotoxicity, macrophages will kill damaged cells expressing specific antigens in the presence of IgG antibodies, i.e. ADCC effect) was observed. Fig.15 .

[0172] The above results showed that in the presence of IgG antibodies, Hybrid did not produce ADCC effect on inflammatory damaged MPC5 cells, and the cell morphology and activity improved.

[0173] Experimental Example 4 Phagocytosis of apoptotic bodies in kidneys by fusion cells

[0174] Mouse kidney podocytes were prepared into apoptotic bodies (ACs) by high temperature boiling method. The specific method was that the cells were resuspended in 1 ml PBS, treated at 100℃ for 10-15 minutes, centrifuged at 3000rpm, the precipitate was taken, resuspended in complete medium, and then incubated with Hybrid for 2 hours (ACs: fusion cells = 5:1). Fluorescence microscopy observation showed that Hybrid phagocytosis of ACs ( Fig.16 ), 24 hours later, qRT-PCR was used to detect the expression of anti-apoptotic and antioxidant factors. Compared with the group without ACs engulfment, the expression of SOD2, Arg1, IL-10, CD274 and TGF-β1 in the group with ACs engulfment was significantly increased, while IDO and PGE-2 were slightly decreased ( Fig.17 ).

[0175] The qRT-PCR detection method was the same as that of Comparative Example 1.

[0176] Primer sequences:

[0177] Table 7

[0178]

[0179] Experimental Example 5 Adaptability of fused cells to the M1 macrophage-inducing environment

[0180] RAW264.7 and Hybrid were treated with inflammatory factors (LPS 5 ng / ml and IFNγ 20 ng / ml) to induce the differentiation of RAW264.7 into M1 (pro-inflammatory macrophage phenotype) for 24 h. The cell morphology was observed under a microscope, and the expression levels of anti-inflammatory and pro-inflammatory factors were detected by qRT-PCR.

[0181] The experimental results are as follows Fig.18 It can be seen that RAW264.7 can be polarized to M1 phenotype under LPS / IFNγ stimulation. After Hybrid was treated with the above inflammatory factors, Hybrid did not show obvious morphological changes of M1 macrophages.

[0182] The qRT-PCR detection method was the same as that of Comparative Example 1.

[0183] Primer sequences:

[0184] Table 8

[0185]

[0186] The qRT-PCR results of anti-inflammatory factors are as follows Fig.19As shown in the figure, it can be seen that compared with the normal RAW264.7 group, the expression of CD206, Arg1 and IL-10 in the RAW264.7 group after LPS / IFNγ treatment decreased; compared with the RAW264.7 group after LPS / IFNγ treatment, the expression of CD206, Arg1 and IL-10 in the Hybrid group after LPS / IFNγ treatment was significantly upregulated ( Fig. 20 ).

[0187] Pro-inflammatory factors: Compared with the normal RAW264.7 group, the expressions of iNOS, TNF-α, and IL-6 in the RAW264.7 group and the fusion cell group treated with LPS / IFNγ were significantly increased ( Fig.19 ); Compared with the RAW264.7 group treated with LPS / IFNγ, the TNF-α and IL-6 levels in the fusion cell group treated with LPS / IFNγ were significantly decreased ( Fig. 20 ).

[0188] The above results suggest that the fusion cells can maintain a certain anti-inflammatory phenotype under LPS / IFNγ stimulation, and no changes in M1 pro-inflammatory macrophages occur.

[0189] Experimental Example 6 Effect of fused cells on dendritic cells (DC cells)

[0190] DC cells were first pretreated with 5 μg / ml LPS (purchased from Solebow Biotech, catalog number L8880) for 24 h to induce DC cell maturation, and then co-cultured with Hybrid (fusion cells and DC cells were mixed in a 1:1 ratio) in a 37°C incubator for 24 h, and the expression of PDL1 in DC cells was detected by flow cytometry.

[0191] Test results: After Hybrid and DC cells were co-cultured for 24 hours, flow cytometry revealed that the expression of PDL-1 was significantly upregulated ( Fig.21 ), and this phenomenon was more significant after pre-stimulation of fusion cells with IFNγ / IL-1β, suggesting that Hybrid has a significant immunosuppressive effect on DC cells.

[0192] Experimental Example 7

[0193] The fusion cells showed strong expression of anti-inflammatory and antioxidant factors. IL-6 has been shown to play an important role in promoting damage in immune nephropathy. If the high IL-6 factor in the renal microenvironment can be adsorbed, the damage of IL-6 to normal renal cells can be inhibited. Bionic nano-bait (i.e., nanovesicles derived from cells expressing inflammatory factor receptors) can be well used as adsorption carriers of inflammatory factors. We detected that the nanovesicles derived from fusion cells expressed CD16 / CD64, which can target and bind to the IgG-Fc segment; expressed PDL1, which can inhibit the activity of immune cells by binding to PD1 on the surface of immune cells and activating the PD-1-PDL1 pathway; expressed IL-6R, which can adsorb IL-6 and inhibit the damage of IL-6.

[0194] The present invention uses PC3 cells (human prostate cancer cells), which are fixed with 4% paraformaldehyde for 15 minutes in vitro, and then treated with immunofluorescence permeabilization solution for 1 hour, combined with mouse anti-human PSA antibody (purchased from Abcom, product number ab140337), which has a mouse IgG-Fc segment, and uses human cells and fixation treatment to minimize the influence of human IL-6R and cell-secreted IL-6 on the experimental results.

[0195] Specific experimental steps:

[0196] The fused cells were stained with DIO dye (purchased from Biotime Biotechnology Co., Ltd., product number C1038, green fluorescence), and the specific steps were as follows: cells were inoculated into a 12-well plate at an appropriate density, cells with a growth density of 80% were selected, the supernatant was discarded, and the cells were washed once with PBS buffer, and dye: enhancer: culture medium = 2.5ul: 5ul: 1ml was prepared and added to the wells, incubated in a 37°C incubator, the supernatant was discarded after 30 minutes, and PBS buffer was added to wash 3 times.

[0197] After the staining, nanovesicles (NV) were prepared. The specific steps were as follows: scrape the cells with a cell scraper, precipitate the cells at 1000rpm for 5min, resuspend the cells with 1ml PBS, and use a liposome extruder (purchased from Nazhida Company, with an extrusion membrane of 200nm) to suck into a screw-mouth syringe, and connect it to another screw-mouth syringe through a threaded connector. Push the cells in the syringe back and forth quickly for about 50 times, then transfer them to a sterile tube, use a centrifuge to centrifuge at 1000g for 10min to remove impurities, and then take the supernatant and transfer it to a sterile 100KD ultrafiltration tube, centrifuge at 3000g for 10min to obtain nanovesicles.

[0198] PC3 cells were seeded onto 12-well plates. When they grew to an appropriate density, they were fixed with immunostaining fixative for 15 min, washed three times with PBS, then permeabilized with immunostaining permeabilization solution for 60 min, washed three times with PBS, blocked with 2% BSA for 30 min, and then divided into 4 groups: ① PC3+PSA antibody group, ② PC3+NV group, and ③ PC3+PSA antibody+NV group. PSA primary antibody was added to ① and ③ for incubation for 30 min, washed three times with PBS, and secondary antibody (purchased from Abcom, product number ab150116, diluted 1:200 with PBS containing 2% BSA) was added to group ① for incubation for 30 min. After the incubation, NV was added to ② and ③ for incubation for 2 h, washed three times with PBS, and observed and photographed under a fluorescence microscope.

[0199] The experiment found that PC3 cells highly expressed PSA (indicated by red fluorescence of the secondary antibody), and the fused cell nanovesicles could be enriched on cells labeled with PSA antibodies (indicated by green fluorescence), indicating that the fused cell-derived nanovesicles could target and bind to the IgG deposition area ( Fig. 22 ).

[0200] Then, the present invention used 1000 pg / ml mouse IL-6 factor (purchased from Nearshore Protein Biotechnology, catalog number C43G) to co-incubate the fixed PC3 cells and divided them into the following four groups: ① PC3+IL-6; ② PC3+PSA+IL-6; ③ PC3 (1-5×10 6 cells)+NV(1-5×10 10 ④PC3+PSA+NV+IL-6, ELISA was used to detect the residual IL-6 level in the supernatant, and it was found that compared with group ③, the IL-6 level in group ④ was significantly decreased ( Fig.23 ). The above results suggest that NV targeting the IgG site can be used as a nanobait to effectively adsorb IL-6 and prevent its inflammatory damage to cells.

[0201] Specific experimental steps:

[0202] The cells were divided into 4 groups: ① PC3 + ​​IL-6; ② PC3 + ​​PSA + IL-6; ③ PC3 + ​​NV + IL-6; ④ PC3 + ​​PSA + NV + IL-6. After adding IL-6 antibody and incubating for 1 hour, the supernatant was collected. After 1000rpm for 10 minutes, the supernatant was taken for ELISA-detection (ELISA kit purchased from Lianke Bio, item number E-AB-F1207A). The method is as follows: dilute the antigen with coating solution, 10ug / ml, add 100ul to each well, incubate at 37℃ for 1h, and place in a refrigerator at 4℃ overnight. Pour out the liquid in the wells, add 200ul washing solution, let it stand for three minutes, repeat three times, and finally invert the reaction plate on absorbent paper to drain the washing solution in the wells and dry it. Add 200ul blocking solution and incubate at 37℃ for 1h. Wash 3 times in the same steps. Add the collected supernatant: dilute it with diluent at 1:1000, 1:2000, 1:4000, 1:6000, 1:8000, 1:10000, 1:20000, 1:40000, 100ul per well. At the same time, dilute the diluent control, and dilute the negative control group at 1:1000. Place at 37℃ for 2h. Wash as before. Add antibody, 100ul per well, 37℃ for 1h. Wash as before, and then wash twice with ddH2O. Add 100ul TMB application solution to each well, react in the dark room for 10min, and stop when blue color appears. Add 50ul stop solution to each well and react for 3-5min. Record the reading at 490nm with an enzyme marker.

[0203] The fixed permeabilized PSA antibody labeling method for PC3 cells and the extraction method of nanovesicles were the same as before.

[0204] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing fusion cells, characterized in that: The method comprises the following steps: using a construct vector of human ACE2 protein to transfect mesenchymal stem cells, using a construct vector of novel coronavirus S protein to transfect macrophages, and mixing the transfected mesenchymal stem cells and macrophages to obtain fusion cells; The construction vector of human ACE2 protein is pHG-CMV-hACE2, and the NCBI sequence number of hACE2 is NM_021804; the construction vector of the new coronavirus S protein is pcDNA3.1-2019-HnCoV-S, and the NCBI sequence number of 2019-HnCoV-S is NC_045512.

2.

2. The preparation method according to claim 1, characterized in that: The mixing ratio of mesenchymal stem cells and macrophages is (1-5):

1.

3. The preparation method according to claim 1, characterized in that: The construction vector of human ACE2 protein and transfection of mesenchymal stem cells comprises the following steps: (1) Plasmid transfection was performed when mesenchymal stem cells were cultured to a confluence of 60-80%; (2) Prepare transfection mixture: the ratio of OPTI reagent, plasmid and transfection reagent is (80-100) μl: 1 μg: (3-8) μL, incubate for 30-60 min; (3) Add the transfection mixture into the mesenchymal stem cell culture medium and culture.

4. The preparation method according to claim 1, characterized in that: The construction vector of the novel coronavirus S protein and transfection of macrophages comprises the following steps: (1) Plasmid transfection was performed when macrophages were cultured to 60-80% confluence; (2) Prepare transfection mixture: OPTI reagent: plasmid: transfection reagent ratio is (80-100) μl: 1 μg: (1-5) μL, incubate for 30-60 min; (3) Add the transfection mixture into the macrophage culture medium and culture.

5. The fused cell prepared by the preparation method according to any one of claims 1 to 4.

6. The method for purifying fused cells according to claim 5, characterized in that: The method comprises co-incubating the adherent fusion cells and magnetic nanoparticles, resuspending the cells, centrifuging the cells, resuspending the cells and continuing to culture the cells adherently, and removing the non-adherent cells to obtain purified fusion cells.

7. The purification method according to claim 6, characterized in that The ratio of the fused cells to the magnetic nanoparticles is (5-10)×10 5 Cells: (5-30) μg magnetic nanoparticles.

8. The purification method according to claim 6, characterized in that The co-incubation time of the fusion cells and the magnetic nanoparticles is 12-24 hours, and the time of the continued adherent culture is 1-5 hours.

9. A method for preparing fusion cell-derived nanovesicles, characterized in that: The following steps are involved: (1) centrifuging the fused cells according to claim 5 to obtain a cell pellet, and resuspending the cells in PBS; (2) Push the cells 40-60 times, the pore size of the polycarbonate membrane is 200 nm, centrifuge at 800-1200g for 10-30 min, take the supernatant, place it in a 100KD ultrafiltration tube, and centrifuge at 2000-3000g for 10-30 min.

10. The fused cell-derived nanovesicles prepared by the preparation method according to claim 9.

11. Use of the fusion cell according to claim 5 or the fusion cell-derived nanovesicle according to claim 10 in preparing a product for treating kidney disease.

12. A product for treating kidney disease, characterized in that: The product comprises the fusion cells described in claim 5 or the fusion cell-derived nanovesicles described in claim 10.

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