Preparation method and application of immortalized stem cell-derived apoptotic extracellular vesicles

Abstract

The invention discloses a construction method of an immortalized stem cell-derived apoptotic extracellular vesicle. The immortalized stem cell-derived apoptotic extracellular vesicle is obtained by inducing an immortalized mesenchymal stem cell line to derive through IFN-gamma and TNF-alpha. Wherein the immortalized mesenchymal stem cells are obtained by infecting mesenchymal stem cells with lentiviruses carrying hTERT (human telomerase reverse transcriptase) genes and SV40LT (SV40LT) genes. According to the invention, the human umbilical cord mesenchymal stem cell (IMSC) system which is infinitely proliferated and stable is successfully established by transfecting SV40LT and hTERT, and the human umbilical cord mesenchymal stem cell (IMSC) system has relatively high proliferation capacity and can provide a large number of mesenchymal stem cells. Meanwhile, it is verified and found that the synergistic effect of TNF-alpha and IFN-gamma can enhance the expression of IMSCs immunomodulatory molecules and enhance the immunomodulatory activity of the IMSCs immunomodulatory molecules; and the I < + > T-IMSCs-apoEVs can be used for effectively inducing the M1 macrophages to be converted into the M2 macrophages. Meanwhile, the I + T-IMSCs-apoEVs disclosed by the invention is injected into a CIA mouse, so that inflammation can be effectively inhibited, tissue repair is promoted, and joints are protected from being injured.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a preparation method and application of immortalized stem cell-derived apoptotic extracellular vesicles. Background Art

[0002] Mesenchymal stem cells (MSCs) from various sources have attracted much attention due to their ability to reduce or prevent inflammation, and have shown strong efficacy in treating various inflammatory and autoimmune diseases. However, more and more studies have shown that MSCs-derived exosomes (EVs) are safer and more stable than MSCs-based cell therapies; they have the advantages of low immunogenicity, barrier penetration, and degradability, showing great potential in disease treatment.

[0003] In recent years, many studies have shown that apoEVs derived from mesenchymal stem cells have great potential in the treatment of inflammation, immunomodulation, tissue regeneration and targeted therapy. It is worth noting that apoEVs derived from mesenchymal stem cells can actively target macrophages at the site of inflammation and regulate macrophage phenotype, thereby alleviating inflammation and promoting the transition from an inflammatory state to tissue repair and regeneration. Due to its many advantages, mesenchymal stem cell-derived apoEV cell therapy is generally favored, but there are also some limitations. First, the current methods for apoEV production still have problems such as low yield, practicality, and economic burden. In addition, changes in the source of mesenchymal stem cells may change the properties and functions of EVs. For example, as cells age, MSCs change the properties of their derived EVs.

[0004] The methods for constructing immortalized stem cells that have been reported so far mainly use SV40L or hTERT transduction alone for construction. However, the immortalized cells prepared by transduction alone have poor stability, proliferation, and poor cell stemness maintenance effects. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a preparation method and application of immortalized stem cell-derived apoptotic extracellular vesicles;

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A method for constructing an immortalized mesenchymal stem cell line comprises infecting mesenchymal stem cells with a lentivirus carrying hTERT gene and SV40LT gene to obtain an immortalized mesenchymal stem cell line.

[0008] In certain specific embodiments, the immortalized mesenchymal stem cells are immortalized human umbilical cord mesenchymal stem cells.

[0009] In certain specific embodiments, the immortalization genes are hTERT gene and SV40LT gene.

[0010] Furthermore, the method for constructing the immortalized mesenchymal stem cells is specifically as follows:

[0011] This example provides a method for constructing immortalized mesenchymal stem cells, which specifically includes: plasmid extraction according to the instructions (Tiangen Biotechnology), wherein the lentiviral vector is packaged by this laboratory, and the specific steps are:

[0012] 1. Reagents:

[0013] 1) HEK 293T cells (purchased from ATCC);

[0014] 2) Transfection reagent Lipofectamine TM LTX Reagent with PLUS TM Reagent (purchased from Invitrogen);

[0015] 3) Opti-MEM serum-free and dual-antibody-free medium, DMEM medium, and fetal bovine serum (all purchased from gbico);

[0016] 2. Lentivirus packaging steps

[0017] Day 0 (8:30) Plate HEK 293T cells

[0018] 293T cells were inoculated into a six-well plate at 830,000 / well.

[0019] Day 1 (8:30) Transfection

[0020] Culture in the incubator for about 24 hours, then transfection can be performed. Specific experimental steps:

[0021] 1) Take two 1.5 ml aliquots and centrifuge them, and add 150 μL Opti-MEM to each aliquot;

[0022] 2) Take one of the tubes and add psPAX2: pMD2.G: vector plasmid with target gene (hTERT gene or SV40LT gene) at a mass ratio of (1.5μg: 0.5μg: 2μg), then add 4μL PLUS TM Reagent (included in the transfection reagent), mix well and mark as tube A;

[0023] 3) Take one of the tubes and add 12 μL of transfection reagent, mix well and mark it as tube B;

[0024] 4) Add tube B to tube A, mix well and let stand at room temperature for 5 minutes;

[0025] 5) Slowly add the rested transfection reagent (A+B) into the HEK 293T cells.

[0026] Day 1(17:00)

[0027] Fluid exchange

[0028] After transfection for more than 6 h, replace the HEK 293T supernatant containing the transfection reagent with 2 mL of DMEM complete medium (containing 10% fetal bovine serum);

[0029] Day 3(8:30)

[0030] Collecting lentivirus

[0031] 48 hours after transfection, the virus was collected in a 15 mL centrifuge tube and centrifuged at 2000 g for 5 minutes to remove cell debris and impurities to obtain the lentivirus (hTERT lentivirus or SV40LT lentivirus), which was then aliquoted and stored at -80°C for later use.

[0032] 3. Lentivirus titer determination

[0033] 4. Lentiviral infection of MSCs

[0034] Day 0(8:30)

[0035] 1) Mesenchymal stem cells were seeded into a 12-well plate at a density of 5 cells per well, with 1 ml of stem cell culture medium per well.

[0036] Day 0(15:00)

[0037] 2) After MSCs adhered to the wall for 6 hours, SV40LT lentivirus was added at an MOI of 10, and 40 μl of lentivirus enhanced infection solution P (Hitrans GP) (Genechem) was immediately added.

[0038] Day 0 (21:00);

[0039] 3) After 6 hours, hTERT lentivirus was added at an MOI of 10.

[0040] Day 1(8:30)

[0041] 4) Replace with fresh culture medium and continue subculturing for four days.

[0042] Day 5(8:30)

[0043] 5) Replace the culture medium with fresh one and add 50 μg / ml neomycin (calculated in activity units), and continue culturing and screening for four days.

[0044] Day 9 (8:30)

[0045] 6) Replace the stem cell culture medium and culture for 15 days, replacing fresh culture medium every two days.

[0046] Day 24 (8:30)

[0047] 7) Subculturing and expanding mesenchymal stem cells to obtain immortalized mesenchymal stem cell lines.

[0048] An immortalized mesenchymal stem cell line prepared according to the aforementioned construction method.

[0049] A method for constructing immortalized stem cell-derived apoptotic extracellular vesicles comprises the following steps: inducing the aforementioned immortalized mesenchymal stem cell line by IFN-γ and TNF-α to derive immortalized stem cell-derived apoptotic extracellular vesicles.

[0050] Furthermore, the method for constructing the immortalized stem cell-derived apoptotic extracellular vesicles is specifically as follows:

[0051] 1. The immortalized mesenchymal stem cell line successfully constructed above was cultured in an incubator at 37° C., in an environment of 5% CO 2 and 95% air, in a complete MSCs culture medium (MUXMD-90,011) provided by Fumei Biotechnology Co., Ltd.

[0052] 2. Under the above conditions, the cells grew adherently; using trypsin (Beyotime)

[0053] Digest the cells and passage them every 2-3 days.

[0054] 3. IMSCs were treated with IFN-γ 20 ng / mL (PeproTech) + TNF-α

[0055] 20 ng / mL (PeproTech) pre-treated for 24 h.

[0056] 4. After 24 hours, the cells were washed twice with PBS, and the culture medium was replaced with a basal culture medium without cell additives, which contained 500 nM STS. After 16 hours of apoptosis induction, the cell morphology was observed and recorded under an inverted microscope (TE2000-SNikon). At the same time, the apoptotic cells were stained with the Annexin V-FITC / PI (KeyGEN BioTECH) cell apoptosis detection kit according to the instructions, and the apoptosis of hUC-MSCs was detected by flow cytometry.

[0057] 5. After induction of apoptosis of I+T-IMSCs, the supernatant was collected and centrifuged at 800 g for 10 min;

[0058] 6. Take the cell supernatant after centrifugation and centrifuge at 2000g for 10 minutes;

[0059] 7. Take the cell supernatant after centrifugation and centrifuge at 16000g for 30 minutes. The resulting precipitate is the desired I+T-IMSCsapoEVs;

[0060] 8. Finally, the precipitate was resuspended in PBS and quantified using a BCA protein assay kit (Biyuntian), and finally stored in a -80°C refrigerator for subsequent experiments.

[0061] In certain embodiments, the immortalized stem cell-derived apoptotic extracellular vesicles can enhance the expression of immunomodulatory molecules in IMSCs.

[0062] In certain embodiments, the immortalized stem cell-derived apoptotic extracellular vesicles can effectively induce the transformation of M1 macrophages into M2 macrophages.

[0063] An immortalized stem cell-derived apoptotic extracellular vesicle prepared by the aforementioned construction method.

[0064] A use of the aforementioned immortalized mesenchymal stem cell line or the aforementioned immortalized stem cell-derived apoptotic extracellular vesicles in the preparation of drugs for preventing and treating inflammation / immunomodulation / tissue regeneration.

[0065] About the drug:

[0066] Preferably, the drug also includes a pharmaceutically acceptable carrier, such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0067] The drug of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0068] The administration route of the drug of the present invention is not limited as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, local skin or systemic transdermal administration.

[0069] The drug of the present invention can also be used in combination with other drugs for treating inflammation / immunomodulation / tissue regeneration. The combined use of multiple drugs can greatly increase the success rate of treatment.

[0070] Compared with the prior art, the present invention has at least the following advantages:

[0071] The present invention successfully established an infinite and stable human umbilical cord mesenchymal stem cell (IMSC) line by transfecting SV40LT and hTERT, which has a high proliferation capacity and can provide a large number of mesenchymal stem cells. At the same time, it was verified and found that the synergistic effect of TNF-α and IFN-γ can enhance the expression of IMSCs immunoregulatory molecules and enhance its immunoregulatory activity; I+T-IMSCs-apoEVs are rich in a variety of functional proteins involved in a variety of physiological processes and signaling pathways, and can effectively induce the transformation of M1 macrophages into M2 macrophages. At the same time, the present invention also proves that I+T-IMSCs-apoEVs injection shows significant therapeutic effects in CIA mice, which can effectively inhibit inflammation, promote tissue repair, and protect joints from damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0073] Figure 1 The figure is a diagram showing the construction, proliferation and cell morphology and size testing of the immortalized human umbilical cord mesenchymal stem cells of the present invention, wherein Figure 1 A The proliferation and passage rate of UC-MSCs cells transduced with SV40LT or hTERT alone, co-transduced with SV40LT and hTERT, and untransduced; The cells were subcultured for about 50 days, and when the cells reached 90% confluence in the culture flask, they were subcultured, and then the number of culture days and the number of passages were plotted; Figure 1 B Real-time label-free dynamic cell analysis technology was used to record the proliferation curves of hUC-MSCs and IMSCs; Figure 1 C Flow cytometry was used to detect the expression level of Ki-67 in cells; Figure 1 D shows that primary UC-MSCs grow adherently, and the cell morphology is observed and recorded under an inverted microscope every day. Primary UCMSCs cannot continue cell division indefinitely, and after 10 generations of culture, they show obvious signs of aging, accompanied by a decrease in proliferation rate; Figure 1 E is a follow-up observation for about 50 days, during which the cell morphology was observed and recorded under an inverted microscope every day.

[0074] Figure 2 : is a phenotypic analysis diagram of immortalized human umbilical cord mesenchymal stem cells in the present invention; wherein Figure 2 A. Figure 2 B is the phenotypic identification of different generations of IMSC cell lines using flow cytometry; Figure 2 C is a diagram showing the evaluation of IMSCs differentiation ability using cell staining; Figure 2D is a graph showing the differences in the expression levels of senescence-related proteins pRb, P21, and P16 in IMSCs and primary umbilical cord stem cells treated with H2O2 detected by WB; Figure 2 E is the chromosome karyotype analysis of IMSCs;

[0075] Figure 3 The sequence diagram and immunomodulatory ability diagram of mesenchymal stem cells (IMSCs) pretreated with IFN-γ and TNF-α in the present invention; wherein Figure 3 A is the differential expression analysis between two comparison combinations using DESeq2 software (1.20.0). The number of differentially expressed genes is shown in a volcano plot, where up-regulated genes are shown in red and down-regulated genes are shown in blue. Figure 3 B: GO enrichment analysis of differentially expressed genes using clusterProfiler (3.8.1) software; Figure 3 C Heat map analysis showed that combined pretreatment of IFN-γ+TNF-α affected the expression of key genes in IMSCs; Figure 3 D In order to verify the RNA sequencing results, this application used RT-qPCR technology to detect the mRNA expression levels of key genes IDO1 and PD-L1; Figure 3 E The difference in IDO1 protein expression levels was tested by immunofluorescence.

[0076] Figure 4 : is a characterization diagram of I+T-IMSCs-apoEVs in the present invention; wherein the cell morphology was observed and recorded under an inverted microscope after apoptosis of I+T-IMSCs was induced ( Figure 4 A), and at the same time, the apoptotic cells were stained with Annexin V-FITC / PI cell apoptosis detection kit according to the instructions, and the apoptosis of I+T-IMSCs was detected by flow cytometry and immunofluorescence ( Figure 4 B). This application uses an optimized gradient centrifugation protocol to separate apoEVs from apoptotic IMSCs ( Figure 4 C) Figure 4 As shown in DI, the morphology, size distribution, exposure of phosphatidylserine (PtdSer), and contents of the freshly isolated apoEVs were observed by transmission electron microscopy (TEM), scanning electron microscopy, dynamic light scattering (DLS), nanoflow cytometry, and immunofluorescence analysis;

[0077] Figure 5 The biodistribution and safety evaluation of I+T-IMSCs-apoEVs in the present invention; wherein Figure 5 A is a diagram showing the distribution of I+T-IMSCs-apoEVs in vivo using an in vitro imaging system (IVIS); Figure 5B is the pathological result of I+T-IMSCs-apoEVs in human tissues;

[0078] Figure 6 This is a diagram showing the situation in which M1 macrophages pretreated with IFN-γ+LPS in the present invention are treated with I+T-IMSCs-apoEV, IMSCs-apoEV or neutrophil apoEV respectively; wherein Figure 6 A: RAW264.7 mouse macrophages were stimulated with IFN-γ+LPS for 24 h, then replaced with fresh complete medium and added with I+T-IMSCs-apoEVs and IMSCs-apoEVs, respectively. The same volume of PBS and unstimulated macrophages were used as control groups. After 24 h, cells were collected and RT-qPCR was used to detect the differences in the expression levels of TNF-α, Arg-1, IL-10, and iNOS mRNA in each group (n=3). Figure 6 B: RAW264.7 mouse macrophages were stimulated with IFN-γ+LPS for 24h, then replaced with fresh complete medium and added with I+T-IMSCs-apoEVs and neutrophil-derived apoptotic extracellular vesicles. After 24h, the expression levels of TNF-α, Arg-1, IL-6, and iNOS mRNA in each group were detected by RT-qPCR (n=3).

[0079] Figure 7 The therapeutic effect of I+T-IMSCs-apoEVs on CIA mice in the present invention is shown in FIG. Figure 7 A is a schematic diagram of apoEVs used to treat CIA mice; Figure 7 B: The mouse joints were photographed on the 50th day for comparative analysis; Figure 7 C: Mice were grossly evaluated and clinically scored for arthritis every other day, and each limb was scored and summarized and plotted into a statistical graph; Figure 7 D is a diagram showing the enrichment of apoEVs in the joints of CIA mice; Figure 7 E The mice were euthanized, and then the mouse joints were extracted and fluorescent images were captured using IVIS Spectrum. Figure 7 F is the biodistribution of apoEVs in CIA mice.

[0080] Figure 8 The I+T-IMSCs-apoEVs in the present invention has therapeutic benefits on arthritis in CIA mice; wherein Figure 8 A: The mice were humanely sacrificed on day 50, and the joints were quickly separated and radiologically evaluated by vivaCT 40 small animal CT. Figure 8 B and Figure 8C: The joints were quickly separated and fixed overnight with 4% paraformaldehyde, decalcified and embedded in paraffin, and the sections were stained with H&E and Safranin O&Fast Green. All sections were imaged using the NanoZoomer S360 digital slide scanner; Figure 8 D is a diagram for grading the severity of articular cartilage damage from 0 (no damage) to 4 (severe damage);

[0081] Fig. 9 This is a diagram showing the therapeutic effect of I+T-IMSCs-apoEVs on arthritis mice by promoting macrophage polarization to M2 phenotype; wherein Fig. 9 A: The tissue was quickly separated and fixed overnight with 4% paraformaldehyde, decalcified and embedded in paraffin. The paraffin-embedded tissue sections were heat fixed, dewaxed and hydrated, and incubated with antibody iNOS. The sample images were captured using the NanoZoomerS360 digital slide scanner, and the relative IOD of the stained sectioned joint tissue was measured using ImageJ and plotted into a statistical graph (n represents the specific number of joints; Fig. 9 B: The tissue was quickly separated and fixed overnight with 4% paraformaldehyde, decalcified and embedded in paraffin. The paraffin-embedded tissue sections were heat fixed, dewaxed and hydrated, and incubated with antibody CD206. The sample images were captured using the NanoZoomer S360 digital slice scanner, and the relative IOD of the stained sectioned joint tissue was measured using ImageJ and plotted as a statistical graph. DETAILED DESCRIPTION

[0082] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0083] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0084] In the following examples, all data are expressed as mean ± standard deviation. Statistical analysis was performed using Prism 8.0 2 software (GraphPad software, USA). The independent sample t test was used for comparison between the two groups, and the one-way analysis of variance and Newman-Keuls multiple comparison test were used for comparison between multiple groups. Each determination was performed in duplicate and repeated in at least 3 independent experiments. P < 0.05 was considered statistically significant. "*" indicates p < 0.05; "**" indicates p < 0.01; "***" indicates p < 0.001.

[0085] In the following examples, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the umbilical cord tissue of volunteers is used for primary culture to successfully obtain hUC-MSCs and proliferate in vitro. Specifically: In the following examples, human umbilical cord mesenchymal stem cells are purchased from Fumei Stem Cell Biotechnology Co., Ltd. (Chongqing, China), and the obtained hUC-MSCs are cultured in a 37°C incubator in an environment of 5% CO2 and 95% air in a complete MSCs medium (MUXMD-90,011) provided by Fumei Biotechnology Co., Ltd. to obtain hUC-MSCs.

[0086] Example 1

[0087] This example provides a method for constructing immortalized mesenchymal stem cells, which specifically includes: plasmid extraction according to the instructions (Tiangen Biotechnology), wherein the lentiviral vector is packaged by this laboratory, and the specific steps are:

[0088] 1. Reagents:

[0089] 1) HEK 293T cells (purchased from ATCC);

[0090] 2) Transfection reagent Lipofectamine TM LTX Reagent with PLUS TM Reagent (purchased from Invitrogen);

[0091] 3) Opti-MEM serum-free and dual-antibody-free medium, DMEM medium, and fetal bovine serum (all purchased from gbico);

[0092] 2. Lentivirus packaging steps

[0093] Day 0 (8:30) Plate HEK 293T cells

[0094] 293T cells were inoculated into a six-well plate at 830,000 / well.

[0095] Day 1 (8:30) Transfection

[0096] Culture in the incubator for about 24 hours, then transfection can be performed. Specific experimental steps:

[0097] 1) Take two 1.5 ml aliquots and centrifuge them, and add 150 μL Opti-MEM to each aliquot;

[0098] 2) Take one of the tubes and add psPAX2: pMD2.G: vector plasmid with target gene (hTERT gene or SV40LT gene) at a mass ratio of (1.5μg: 0.5μg: 2μg), then add 4μL PLUS TM Reagent (included in the transfection reagent), mix well and mark as tube A;

[0099] 3) Take one of the tubes and add 12 μL of transfection reagent, mix well and mark it as tube B;

[0100] 4) Add tube B to tube A, mix well and let stand at room temperature for 5 minutes;

[0101] 5) Slowly add the rested transfection reagent (A+B) into the HEK 293T cells.

[0102] Day 1(17:00)

[0103] Fluid exchange

[0104] After transfection for more than 6 h, replace the HEK 293T supernatant containing the transfection reagent with 2 mL of DMEM complete medium (containing 10% fetal bovine serum);

[0105] Day 3(8:30)

[0106] Collecting lentivirus

[0107] 48 hours after transfection, the virus was collected in a 15 mL centrifuge tube and centrifuged at 2000 g for 5 minutes to remove cell debris and impurities to obtain the lentivirus (hTERT lentivirus or SV40LT lentivirus), which was then aliquoted and stored at -80°C for later use.

[0108] 3. Lentivirus titer determination

[0109] 4. Lentiviral infection of MSCs

[0110] Day 0(8:30)

[0111] 1) Human umbilical cord mesenchymal stem cells (hUC-MSCs) were seeded into 12-well plates at 5 cells per well, with 1 ml of stem cell culture medium per well.

[0112] Day 0(15:00)

[0113] 2) After MSCs adhered to the wall for 6 hours, SV40LT lentivirus was added at an MOI of 10, and 40 μl of lentivirus enhanced infection solution P (Hitrans GP) (Genechem) was immediately added.

[0114] Day 0 (21:00);

[0115] 3) After 6 hours, hTERT lentivirus was added at an MOI of 10.

[0116] Day 1(8:30)

[0117] 4) Replace with fresh culture medium and continue subculturing for four days.

[0118] Day 5(8:30)

[0119] 5) Replace the culture medium with fresh one and add 50 μg / ml neomycin (calculated in activity units), and continue culturing and screening for four days.

[0120] Day 9 (8:30)

[0121] 6) Replace the stem cell culture medium and culture for 15 days, replacing fresh culture medium every two days.

[0122] Day 24 (8:30)

[0123] 7) Human umbilical cord mesenchymal stem cells are passaged and expanded to obtain immortalized human umbilical cord mesenchymal stem cell lines.

[0124] Performance Testing:

[0125] 1) Proliferation ability test

[0126] The present application tests the construction, proliferation and cell morphology and size of the immortalized cell line obtained, as shown in the following examples: Figure 1 As shown, Figure 1 A The proliferation and passage rate of UC-MSCs cells transduced with SV40LT or hTERT alone, co-transduced with SV40LT and hTERT, and untransduced; The cells were subcultured for about 50 days, and when the cells reached 90% confluence in the culture flask, they were subcultured, and then the number of culture days and the number of passages were plotted; Figure 1 B Real-time label-free dynamic cell analysis technology was used to record the proliferation curves of hUC-MSCs and IMSCs; Figure 1 C Flow cytometry was used to detect the expression level of Ki-67 in cells. Figure 1 A, 1B, and 1C show that the proliferation potential of the untransfected parental hUC-MSCs cells significantly decreases with the increase in the number of passages, and reaches proliferation stagnation at P12 ( Figure 1B); hTERT and SV40LT transfection significantly enhanced the proliferation capacity of hUC-MSCs. Compared with hUC-MSCs transfected with SV40LT or hTERT alone, hUC-MSCs transfected with both hTERT and SV40LT did not show a reduced proliferation rate in long-term culture and maintained their proliferation capacity. hTERT and SV40LT transfected IMSCs cells also did not show any signs of proliferation arrest at high generations, and the results of the proliferation experiment confirmed that the proliferation capacity of IMSCs at the 30th generation was significantly stronger than that of the original cells at the 8th generation.

[0127] 2) Morphological change test

[0128] The present application explored the morphological changes during the expansion of IMSCs. Figure 1 D and Figure 1 E, where Figure 1 D shows that primary UC-MSCs grow adherently, and the cell morphology is observed and recorded every day under an inverted microscope. Primary UCMSCs cannot continue cell division indefinitely, and after 10 generations of culture, they show obvious signs of aging, accompanied by a decrease in proliferation rate. Figure 1 E is a follow-up observation of about 50 days, and the cell morphology was observed and recorded every day under an inverted microscope. As can be seen from the figure, untransfected MSCs and IMSCs attached to the surface of the plastic culture medium. As the cells approached fusion, they showed a spindle-shaped fiber-like morphology, but as the number of generations in culture continued to increase, IMSCs maintained their initial morphology, although in some areas the cells overgrew and still formed a very dense cell monolayer, however, the morphology of hUC-MSCs showed significant changes, with increased cell volume and different sizes.

[0129] 3) Characteristic performance test

[0130] At the same time, phenotypic analysis was performed on the IMSC cell lines of different generations constructed in this application. Figure 2 A and 2B, where Figure 2 A and Figure 2 B is the phenotype identification of different generations of IMSC cell lines by flow cytometry; as can be seen from the figure, these cells (> 95% of cells) express CD90, CD105, CD73, CD44 and CD29, but do not express (< 2% of cells) CD45 and CD34. These results clearly show that long-term cultured IMSCs do not change the expression of specific markers after immune immortality.

[0131] 4) Aging performance test

[0132] The present application tests the aging performance of the prepared immortalized cell line, and the results are as follows Figure 2C, to verify that IMSCs can be induced to differentiate into adipogenic, osteogenic, and chondrogenic cell lineages after immortalization, for each differentiation, IMSCs were cultured in conditioned medium and their differentiation ability was evaluated by combining cell staining ( Figure 2 C); As can be seen from the figure, IMSCs can maintain the three-way differentiation potential of mesoderm after immune immortality and can be induced to differentiate into adipocytes, osteoblasts and chondrocytes. Cell senescence will impair the proliferation and differentiation ability of MSCs in vivo during long-term culture, thus greatly limiting the use of MSCs. Senescence-related protein detection results (such as Figure 2 As shown in D, Figure 2 D is the difference in the expression levels of senescence-related proteins pRb, P21, and P16 in IMSCs and primary umbilical cord stem cells treated with H2O2 detected by WB. It shows that IMSCs do not show obvious signs of senescence in long-term culture, that is, the experiment shows that the migration ability of IMSCs is significantly stronger than that of original MSCs. Figure 2 As shown in E, Figure 2 E is the representative data of the chromosome karyotype analysis experiment of IMSCs; as can be seen from the figure, IMSCs still maintain a normal karyotype after long-term passaging.

[0133] In summary, the present application has successfully established a cell line that can be infinitely expanded and will not age. These cells not only retain the characteristics of the original mesenchymal stem cells, but also have a high proliferation capacity, thereby enabling the mass production of mesenchymal stem cells.

[0134] Example 2

[0135] This example provides a method for preparing apoptotic extracellular vesicles (I+T-IMSCs-apoEVs).

[0136] 1. Example 1 Successfully constructed immortalized human umbilical cord mesenchymal stem cells were cultured in an incubator at 37° C., in an environment of 5% CO 2 and 95% air, in a complete MSCs culture medium (MUXMD-90,011) provided by Fumei Biotechnology Co., Ltd.

[0137] 2. Under these conditions, cells grew adherently. Cells were digested with trypsin (Beyotime) and passaged every 2-3 days.

[0138] 3. IMSCs were pretreated with IFN-γ 20 ng / mL (PeproTech) + TNF-α 20 ng / mL (PeproTech) for 24 h.

[0139] 4. After 24 hours, the cells were washed twice with PBS, and the culture medium was replaced with a basal culture medium without cell additives, which contained 500 nM STS. After 16 hours of apoptosis induction, the cell morphology was observed and recorded under an inverted microscope (TE2000-SNikon). At the same time, the apoptotic cells were stained with the Annexin V-FITC / PI (KeyGEN BioTECH) apoptosis detection kit according to the instructions, and the apoptosis of hUC-MSCs was detected by flow cytometry.

[0140] 5. After induction of apoptosis of I+T-IMSCs, the supernatant was collected and centrifuged at 800 g for 10 min;

[0141] 6. Take the cell supernatant after centrifugation and centrifuge at 2000g for 10 minutes;

[0142] 7. Take the cell supernatant after centrifugation and centrifuge at 16000g for 30 minutes. The resulting precipitate is the desired I+T-IMSCsapoEVs;

[0143] 8. Finally, the precipitate was resuspended in PBS and quantified using a BCA protein assay kit (Biyuntian), and finally stored in a -80°C refrigerator for subsequent experiments.

[0144] 9. The morphological information of apoEVs was obtained by transmission electron microscopy (JEM-2100, JEOL Ltd.) and scanning electron microscopy (SU8100, Hitachi). Dynamic light scattering (DLS) analysis was performed using Zetasizer Nano ZSE (Malvern) to measure the particle size distribution of apoEVs. Annexin V-FITC / PI apoptosis detection kit (KeyGEN BioTECH) was used to stain apoEVs with Annexin V, and the expression of Annexin V in apoEVs was detected by fluorescence microscopy.

[0145] Performance test results:

[0146] 1) Mesenchymal stem cells (IMSCs) pretreated with IFN-γ and TNF-α showed stronger immunoregulatory ability.

[0147] In order to understand the effect of IFN-γ combined with TNF-α stimulation on the immunoregulatory function of IMSCs, the present invention performed RNA sequencing analysis on IMSCs pretreated with IFN-γ 20 ng / mL + TNF-α 20 ng / mL and untreated IMSCs. The results are as follows Figure 3 As shown, Figure 3A is the differential expression analysis between two comparison combinations using DESeq2 software (1.20.0). The number of differentially expressed genes is shown in a volcano plot, where up-regulated genes are shown in red and down-regulated genes are shown in blue. Figure 3 B: GO enrichment analysis of differentially expressed genes using clusterProfiler (3.8.1) software; Figure 3 C Heat map analysis showed that combined pretreatment of IFN-γ+TNF-α affected the expression of key genes in IMSCs; Figure 3 D In order to verify the RNA sequencing results, this application used RT-qPCR technology to detect the mRNA expression levels of key genes IDO1 and PD-L1; Figure 3 E The difference in IDO1 protein expression levels was tested by immunofluorescence. IMSC_I_T, IFN-γ+TNF-αenabled IMSCs; **p<0.01, ****p<0.0001. As can be seen from the figure, a total of 177 genes (100 of which were upregulated and 77 were downregulated) were differentially expressed between the two groups ( Figure 3 A). It can be seen that pretreatment with IFN-γ combined with TNF-α extensively reprogrammed the genes of IMSCs. After GO enrichment analysis, it was found that the differentially expressed genes were involved in many key regulatory pathways, such as interferon γ response, inflammatory response regulation, cytokine production regulation, immune system process regulation, NF-κB pathway, and macrophage polarization, etc. ( Figure 3 B). It is worth noting that heat map analysis showed that combined pretreatment with IFN+TNF-α upregulated a series of immunosuppressive molecular profiles of IMSCs, such as ICAM1, CD83, IDO1, PD-L1, and VCAM1, which further enhanced the immunosuppressive potential of IMSCs ( Figure 3 C). RT-qPCR was used to detect the mRNA expression levels of key genes. The results are shown in Figure 3 As shown in D, it was found that after IFN-γ+TNF-α pretreatment, the expression levels of IDO and PD-L1 in the immortalized cell line IMSCs group were significantly higher than those in the original MSCs group, while the up-regulation level of IDO in the I+T IMSCs group was significantly higher than that in the original MSCs group after pretreatment. Consistent with the RT-qPCR results, immunofluorescence results showed that after IFN-γ+TNF-α combined treatment, the IDO protein level was significantly increased, and the IDO protein level in I+T IMSCs was significantly higher than that in the original cells treated with the same treatment ( Figure 3 E). Our results showed that the synergistic effect of TNF-α and IFN-γ could enhance the expression of immunomodulatory molecules and strengthen the immunomodulatory activity of IMSCs, which had a stronger immunomodulatory capacity than that of primary MSCs under the same treatment.

[0148] 2) Characterization of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs)

[0149] After inducing apoptosis of I+T-IMSCs, the cell morphology was observed and recorded under an inverted microscope ( Figure 4 A), and at the same time, the apoptotic cells were stained with Annexin V-FITC / PI cell apoptosis detection kit according to the instructions, and the apoptosis of I+T-IMSCs was detected by flow cytometry and immunofluorescence ( Figure 4 B). This application uses an optimized gradient centrifugation protocol to separate apoEVs from apoptotic IMSCs ( Figure 4 C) Figure 4 As shown in DI, the morphology, size distribution, exposure of phosphatidylserine (PtdSer) and contents of the newly isolated apoEVs were observed by transmission electron microscopy (TEM), scanning electron microscopy, dynamic light scattering (DLS), nano-flow cytometry, and immunofluorescence analysis. The results showed that the apoptotic vesicles used in the present application were spherical, with dispersed particle sizes ranging from 100 to 1000 nanometers, expressed higher PS (shown by Annexin V binding, 91.78%), and were rich in mitochondria.

[0150] 3) Biodistribution test of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs)

[0151] This application studied the distribution of I+T-IMSCs-apoEVs in mice. I+T-IMSCs-apoEVs were labeled with DiR, injected in vivo via tail vein injection, and analyzed using an in vitro imaging system (IVIS). The results are shown in Figure 5 As shown in A, the figure shows that the fluorescent signal is mainly distributed in the liver and can be observed 2 hours after injection, and the signal remains at a high level for 1-3 days.

[0152] 4) Safety evaluation test of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs)

[0153] This application also studies the safety of I+T-IMSCs-apoEVs in vivo. The results of routine blood tests (percentages of red blood cells, white blood cells, lymphocytes, and neutrophils, etc.) and serum biochemical indicators (serum alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine, etc.) are shown in Tables 1 and 2:

[0154] Table 1 Biochemical index detection

[0155]

[0156] DBIL, direct bilirubin; TBIL, total bilirubin; IBIL, indirect bilirubin; AST, aspartate aminotransferase;

[0157] ALT, alanine transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyltranspeptidase; UA, uric acid.

[0158] Table 2 Routine blood test

[0159]

[0160] WBC, white blood cell; NEUTRO, neutrophil; LYM, lymphocyte; MONO, monocyte; RBC, red blood cell; PLT, platelets; Hb, hemoglobin.

[0161] As can be seen from Tables 1 and 2, there were no significant differences. These results indicate that I+T-IMSCs-apoEVs is safe in vivo. (The differences between the I+T-IMSCs-apoEVs group and the PBS group are shown in Tables 1 and 2)).

[0162] The test results of multiple cytokines are shown in Table 3:

[0163] Table 3 Multi-cytokine detection

[0164]

[0165] From the data in Table 3, it can be seen that I+T-IMSCs-apoEVs do not cause an increase in the levels of inflammatory factors (such as TNF-α, IL-1β, IFN-γ, etc.) in the blood serum of mice, indicating that I+T-IMSCs-apoEVs do not cause a significant immune response. At 7d, 4W and 8W after injection of I+T-IMSCs-apoEVs, in the six tissues analyzed (lung, liver, kidney, heart, spleen and colon), pathological results showed that I+T-IMSCs-apoEVs had no adverse effects on the organs of mice. These results show that the I+T-IMSCs-apoEVs we developed are safe for in vivo application ( Figure 5 B).

[0166] Example 4: Apoptotic extracellular vesicles (I+T-IMSCs-apoEVs) have an enhanced ability to promote the transformation of macrophages from M1 phenotype to M2 phenotype

[0167] The present application tests the enhanced ability of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs) to promote the transformation of macrophages from M1 phenotype to M2 phenotype. Specifically, ApoEVs have multiple biological functions. Compared with other EVs, ApoEVs have unique biological functions, such as the ability to actively target macrophages at inflammatory sites and regulate macrophage phenotypes, and the higher surface area / volume ratio of ApoEVs promotes its specific surface interaction with target cells. Therefore, the present application comparatively evaluated the effect of I+T-IMSCs-apoEV on macrophage polarization, and treated M1 macrophages pretreated with IFN-γ+LPS with I+T-IMSCs-apoEV, IMSCs-apoEV or neutrophil apoEV respectively; the results are as follows: Figure 6 As shown, Figure 6 A: RAW264.7 mouse macrophages were stimulated with IFN-γ+LPS for 24 h, then replaced with fresh complete medium and added with I+T-IMSCs-apoEVs and IMSCs-apoEVs, respectively. The same volume of PBS and unstimulated macrophages were used as control groups. After 24 h, cells were collected and RT-qPCR was used to detect the differences in the expression levels of TNF-α, Arg-1, IL-10, and iNOS mRNA in each group (n=3). Figure 6 B shows that RAW264.7 mouse macrophages were stimulated with IFN-γ+LPS for 24h, and then replaced with fresh complete medium and I+T-IMSCs-apoEVs and neutrophil-derived apoptotic extracellular vesicles were added respectively. After 24 hours, the expression levels of mRNA of TNF-α, Arg-1, IL-6, and iNOS in each group were detected by RT-qPCR (n=3). As can be seen from the figure, as expected, compared with the control group, the mRNA expression of genes related to the M1 phenotype (including TNF-α and iNOS) in the I+T-IMSCs-apoEV, IMSCs-apoEV, and neutrophil apoEV groups was significantly downregulated, while the expression of M2 macrophage markers Arg-1 and IL-10 was significantly upregulated. It is worth noting that the effect of I+T-IMSCs-apoEV was the most significant. Studies have shown that I+T-IMSCs-apoEVs can more effectively induce the transformation of M1 macrophages to M2 macrophages, thereby inhibiting persistent inflammation and producing a favorable anti-inflammatory effect, opening up a new avenue for the treatment of inflammation-related diseases ( Figure 6 ).

[0168] Example 5: Application of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs) in arthritis

[0169] This example provides the application of apoptotic extracellular vesicles (I+T-IMSCs-apoEVs) in arthritis, and the specific application method is as follows:

[0170] IFN-γ+TNF-α-enabled immortalized stem cell-derived apoptotic extracellular vesicles for the treatment of DBA mouse arthritis model:

[0171] 1) DBA / 1 mice were first immunized on day 0 by subcutaneous injection (at the base of the tail) with 100 μl of emulsion (containing 100 μg of type II collagen (Chondrex. Inc.) and Complete Freund's Adjuvant (Chondrex. Inc.)) per mouse.

[0172] 2) On day 21, booster immunization was performed, with each mouse injected with 100 μl of emulsion (containing 100 μg type II collagen and incomplete Froude's adjuvant).

[0173] 3) To evaluate the therapeutic effect, treatment was started at the onset of the disease, when the arthritis score reached 3 points or more (i.e., about 27 days after the initial immunization). For experimental mice with established CIA, 2.5 μg / g of I+T-IMSCs-apoEVs, IMSCs-apoEVs, neutrophil-derived apoEVs, or an equal amount of PBS were injected into the mice by intravenous injection. The drugs were administered once every 2 days for a total of 3 times.

[0174] 4) Clinical features were monitored by two unbiased investigators who were blind to the experimental groups, who performed gross assessments of the mice every other day and performed clinical arthritis scores. Clinical arthritis was assessed using the following system: 0, no swelling; 1, mild swelling and erythema; 2, marked edema; 3, joint stiffness. Scores were collected for each limb and recorded.

[0175] 5) Mice were humanely sacrificed on day 50, and limbs and joints were rapidly separated for radiological evaluation and histological assessment (H&E, Safranin O&FastGreen, and histochemistry) by vivaCT 40 small animal CT (SCANO MEDICAL, Switzerland).

[0176] Performance test results:

[0177] 1) The therapeutic effect of I+T-IMSCs-apoEVs was studied in a mouse CIA model;

[0178] The therapeutic effect of I+T-IMSCs-apoEVs was studied in the mouse CIA model. Figure 7 and Figure 8 As shown, Figure 7 A is a schematic diagram of apoEVs used to treat CIA mice; Figure 7 B: The mouse joints were photographed on the 50th day for comparative analysis; Figure 7 C: Mice were grossly evaluated and clinically scored for arthritis every other day. Each limb was scored and summarized and plotted into a statistical graph; Figure 7 D In order to detect whether apoEVs can be enriched in the joints of CIA mice, the present application used the lipid-soluble membrane dye Dil to label ApoEVs, and then injected them into the mice through the tail vein; after different time intervals, the mice were anesthetized and the fluorescent images of the mice were captured using IVIS Spectrum; Figure 7 E The mice were euthanized, and then the mouse joints were extracted and fluorescent images were captured using IVIS Spectrum. Figure 7 To detect the biodistribution of apoEVs in CIA mice, the present application labeled ApoVs and then injected them into mice through the tail vein. After different time intervals, the mice were euthanized, and then the main organs were extracted and fluorescent images of the organs were taken using IVIS Spectrum.

[0179] As can be seen from the figure, the targeting ability of I+T-IMSCs-apoEVs in mice was verified by an in vitro imaging system to determine whether they would be targeted and enriched in inflamed joints. By injecting DiR-labeled I+T-IMSCs-apoEVs into the tail vein of mice to detect their distribution in the body, it was found that I+T-IMSCs-apoEVs could be efficiently enriched in swollen joints in a time-dependent manner, reaching a peak at 10 hours, and continued to be enriched in the joints within 24 hours without being metabolically cleared. In summary, the I+T-IMSCs-apoEVs in this application have the ability to target and enrich in inflamed joints ( Figure 7 D, Figure 7 E). We also found that apoptotic vesicles did not remain in the mouse organs for a long time and did not cause damage to the organs ( Figure 7 F).

[0180] At the same time, this application also compares and analyzes the degree of inflammatory edema in the paws of mice treated with I+T-IMSCs-apoEVs, IMSCs-apoEVs, neutrophil-derived apoEVs or PBS.

[0181] The results are as follows Figure 7 B. Figure 7As shown in Figure C, the paws of mice treated with PBS were significantly swollen, and the ankle joints, foot pads and toes were obviously red, thickened and even deformed. After treatment with apoEVs, the edema of the paws of all three groups of mice was alleviated, and the improvement in the I+T-IMSCs-apoEVs group was the most significant.

[0182] 2) I+T-IMSCs-apoEVs have therapeutic benefits on arthritis in CIA mice

[0183] Figure 8 I+T-IMSCs-apoEVs have therapeutic benefits on arthritis in CIA mice, including Figure 8 A: Mice were humanely sacrificed on day 50, and the joints were quickly isolated and radiologically evaluated by vivaCT 40 small animal CT. Figure 8 B and Figure 8 C: The joints were quickly separated and fixed overnight with 4% paraformaldehyde, decalcified and embedded in paraffin, and the sections were stained with H&E and Safranin O&Fast Green. All sections were imaged using the NanoZoomer S360 digital slide scanner; Figure 8 D is the severity of articular cartilage damage, which is graded from 0 (no damage) to 4 (severe damage). We calculated the histological scores of arthritis, and two researchers calculated the histological scores of these specimens and plotted them into statistical graphs (*p < 0.05, ****p < 0.0001). Specifically, using imaging technology, microCT scans were performed on each group of mice to observe the degree of bone erosion. The I+T-IMSCs-apoEVs group showed a significant improvement in protecting joints from damage ( Figure 8 A). Throughout the course of the disease, mice treated with apoEVs had low clinical scores for joint inflammation, and the effect of I+T-IMSCs-apoEVs treatment was significantly better than that of IMSCs-apoEVs and neutrophil-derived apoEVs. Histopathological evaluation showed that pathological damage in the joint tissues of CIA mice treated with apoptotic vesicles, especially synovial proliferation and cartilage erosion, was significantly reduced compared with the PBS group (and histological scores showed that the improvement effect of the I+T-IMSCs-apoEVs group was significantly better than that of the IMSCs-apoEVs group and neutrophil-derived apoEVs group (( Figure 8 B. Figure 8 C. Figure 8 D)). In summary, the powerful therapeutic effect of I+T-IMSCs-apoEVs in this application can protect joints and delay joint destruction.

[0184] 3) Immunomodulatory ability of I+T-IMSCs-apoEVs on macrophage infiltration

[0185] Furthermore, the present application further evaluated the immunomodulatory ability of I+T-IMSCs-apoEVs on macrophage infiltration by immunohistochemical analysis, and the results were as follows Fig. 9 As shown, Fig. 9 A: The tissue was quickly separated and fixed overnight with 4% paraformaldehyde, decalcified and embedded in paraffin. The paraffin-embedded tissue sections were heat fixed, dewaxed and hydrated, and incubated with antibody iNOS. The sample images were captured using the NanoZoomer S360 digital slide scanner, and the relative IOD of the stained sectioned joint tissue was measured using ImageJ and plotted into a statistical graph (n represents the specific number of joints; Fig. 9 B is the rapid separation of tissue and fixation with 4% paraformaldehyde overnight, decalcification and embedding in paraffin, the paraffin-embedded tissue sections were heat fixed, dewaxed and hydrated, and incubated with antibody CD206; the sample images were captured using the NanoZoomer S360 digital slice scanner, and the relative IOD of the stained sliced ​​joint tissue was measured using ImageJ and plotted into a statistical graph. As can be seen from the figure, the figure shows that the expected results are consistent with the I+T-IMSCs-apoEVs injection, which significantly upregulated the expression level of CD206, a marker of M2 macrophages, in the joint tissue, and downregulated the expression level of iNOS, a marker of M1 macrophages, compared with PBS and the other two treatment groups. In summary, I+T-IMSCs-apoEVs effectively inhibited inflammation and promoted tissue repair. In addition, I+T-IMSCs-apoEVs can also protect joints from damage by converting macrophages into an anti-inflammatory phenotype. Finally, no adverse reactions or deaths occurred in mice treated with I+T-IMSCs-apoEVs before the end of the experiment. In summary, these research results collectively indicate that I+T-IMSCs-apoEVs have excellent therapeutic effects on RA without obvious side effects.

[0186] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for constructing an immortalized mesenchymal stem cell line, characterized in that: The method involves infecting mesenchymal stem cells with a lentivirus carrying the hTERT gene and the SV40LT gene to obtain an immortalized mesenchymal stem cell line.

2. The construction method according to claim 1, characterized in that: The immortalized mesenchymal stem cells are immortalized human umbilical cord mesenchymal stem cells.

3. The construction method according to claim 2, characterized in that: The immortalization genes are hTERT gene and SV40LT gene.

4. An immortalized mesenchymal stem cell line obtained by the construction method according to any one of claims 1 to 3.

5. A method for constructing immortalized stem cell-derived apoptotic extracellular vesicles, characterized in that: The method comprises the following steps: inducing the immortalized mesenchymal stem cell line in claim 4 by IFN-γ and TNF-α to derive immortalized stem cell-derived apoptotic extracellular vesicles.

6. The method for constructing immortalized stem cell-derived apoptotic extracellular vesicles according to claim 6, characterized in that: The immortalized stem cell-derived apoptotic extracellular vesicles can enhance the expression of IMSCs immunoregulatory molecules.

7. The method for constructing immortalized stem cell-derived apoptotic extracellular vesicles according to claim 6, characterized in that: The immortalized stem cell-derived apoptotic extracellular vesicles can effectively induce the transformation of M1 macrophages into M2 macrophages.

8. An immortalized stem cell-derived apoptotic extracellular vesicle obtained according to the construction method of any one of claims 5 to 7.

9. Use of the immortalized mesenchymal stem cell line according to claim 4 or the immortalized stem cell-derived apoptotic extracellular vesicles according to claim 8 in the preparation of a drug for preventing and treating inflammation / immunomodulation / tissue regeneration.

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