Method for preparing macromolecular vesicles containing apoptotic newborn organisms and application of macromolecular vesicles

Abstract

The invention belongs to the field of biological medicine, and relates to a method for preparing apoptotic newborn biological macromolecular vesicles and application of the apoptotic newborn biological macromolecular vesicles. According to the preparation method of the vesicle, cell apoptosis is induced through external force to obtain the induced vesicle, and the vesicle contains the induced vesicle of newborn protein and / or newborn RNA. The inventor finds that biomacromolecules (including protein and RNA) strongly related to apoptosis are newly synthesized in cell apoptosis through research, and also finds that the induced vesicles generated by cell apoptosis still have the function of synthesizing the biomacromolecules (including protein and RNA) after being separated from cells; and the mechanism for synthesizing the new protein in the cell apoptosis process is possibly related to the induced vesicles.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for preparing vesicles containing apoptotic newborn biomacromolecules and application thereof. Background Art

[0002] Extracellular vesicles are nanoscale bilayer lipid molecules that are widely present in the human body and have functional activity. They can be divided into exosomes, microvesicles, and apoptotic vesicles (ApoVs) released by cells under physiological and pathological conditions, and they play an important role in a variety of physiological and pathological processes. Extracellular vesicles can transport proteins, cytokines, lipids, nucleic acids, etc. between cells and mediate a variety of biological reactions.

[0003] Apoptosis is a physiological, programmed, and active form of cell death. Every day, hundreds of millions of cells in the body undergo apoptosis and produce a large number of ApoVs, which have a wide range of effects on the body's homeostasis. In 2018, a study first discovered that circulating ApoVs maintain the homeostasis of stem cells in the body, and exogenous infusion of stem cell-derived ApoVs can be used to treat osteoporosis, suggesting that stem cell-derived ApoVs can be used as a new biological treatment. In recent years, studies have pointed out that stem cell-derived ApoVs can treat a variety of diseases, such as autoimmune diseases such as systemic lupus erythematosus, osteoporosis, sepsis, diabetes, multiple myeloma, coagulation disorders, etc., but the fundamental mechanism by which cell death products have the function of maintaining body homeostasis and treating diseases has not been fully elucidated. The study of apoptosis and ApoVs is of great significance for understanding the damage repair and homeostasis of tissues and organs, the significance of death and its metabolites, and the balance between life and death, and is of strategic significance for guiding the clinical application of ApoVs. Summary of the invention

[0004] In some embodiments, the present invention provides a new inducible vesicle rich in apoptotic neoplastic macromolecules. Such inducible vesicles have a stronger function of promoting osteogenic differentiation of stem cells and may have a better disease treatment effect.

[0005] In some embodiments, the present invention provides a method for preparing inducible vesicles, which produces inducible vesicles by inducing cell apoptosis through external force to obtain inducible vesicles containing nascent macromolecules including proteins and / or RNA.

[0006] In some embodiments, the present invention provides a method for preparing induced vesicles, inducing cell apoptosis by external force to produce induced vesicles, and collecting the produced induced vesicles 1 to 7 hours after applying the external force to obtain induced vesicles containing nascent macromolecules including proteins and / or RNA.

[0007] In some embodiments, the present invention provides a method for preparing induced vesicles, inducing cell apoptosis through external force to produce induced vesicles, and collecting the produced induced vesicles 4 to 7 hours after applying the external force to obtain induced vesicles containing nascent macromolecules including proteins and / or RNA.

[0008] In some embodiments, the inducible vesicles are capable of synthesizing nascent proteins and / or nascent RNAs.

[0009] In some embodiments, the induced vesicles are obtained 4.1 to 6.9 hours after the external force is applied to the cells.

[0010] In some embodiments, the induced vesicles are obtained 4.1 to 6.8 hours after the external force is applied to the cells.

[0011] In some embodiments, the induced vesicles are obtained 4.1 to 6.7 hours after the external force is applied to the cells.

[0012] In some embodiments, the induced vesicles are obtained 4.1 to 6.5 hours after the external force is applied to the cells.

[0013] In some embodiments, the induced vesicles are obtained 4.1 to 6.3 hours after the external force is applied to the cells.

[0014] In some embodiments, the induced vesicles are obtained within 6 hours of applying the external force to the cells.

[0015] In some embodiments, the nascent protein or nascent RNA is an apoptosis-related nascent protein and / or nascent RNA.

[0016] The traditional understanding of apoptosis is the process in which the body follows a set program to eliminate useless cells under certain physiological or pathological conditions. It is characterized by the degradation of cell components caused by the cascade activation of the caspase protein family. The final result is the elimination of apoptotic cells with the participation of immune cells. It is a "passive" biological process. The traditional understanding of apoptotic bodies (or apoptotic vesicles) is a vesicle structure that encapsulates broken chromatin fragments and organelles produced during apoptosis. Its surface characteristic marks can be recognized and engulfed by immune cells, and assist immune cells in eliminating apoptotic cells. The inventors have found that apoptosis is not just a "passive" process characterized by degradation and aimed at elimination. In the process of apoptosis, there is a new synthesis of biomacromolecules that depends on the caspase program, and such new substances can be encapsulated in apoptotic vesicles and delivered into host cells to play a therapeutic role. That is, apoptosis is an important part of the yin-yang balance and life-death balance of life, and it is an "active" biological process.

[0017] In some embodiments, the inventors have found that there is a new synthesis of biomacromolecules (including proteins and RNA) strongly related to apoptosis during cell apoptosis. This process mainly occurs in the late stage of cell apoptosis, during which caspase3 is activated and its downstream corresponding substrates (such as PARP) are cleaved. The inventors also found that the inducible vesicles produced by cell apoptosis still have the function of synthesizing biomacromolecules (including proteins and RNA) after leaving the cells, and the mechanism of synthesizing new proteins during cell apoptosis may be related to the inducible vesicles.

[0018] In some embodiments, the preparation method includes the following steps: (1) culturing cells; (2) in a culture medium, contacting the cells in step (1) with an external force to induce the release of the induced vesicles; (3) collecting the culture supernatant of step (3) and extracting the induced vesicles.

[0019] In some embodiments, the inducible vesicles are extracted from the culture medium by a method selected from the group consisting of polymer precipitation, immunoseparation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and a combination thereof.

[0020] In some embodiments, the step of separating the induced vesicles by the ultracentrifugation method includes: (a) subjecting the collected culture supernatant to a first centrifugation to obtain the supernatant; (b) subjecting the supernatant collected in step (a) to a second centrifugation to obtain the supernatant; (c) subjecting the supernatant received in step (b) to a third centrifugation to obtain the precipitate; (d) subjecting the precipitate received in step (c) to a fourth centrifugation to obtain the precipitate.

[0021] In some embodiments, the first centrifugation is performed at 500-1500 g for 5-30 minutes.

[0022] In some embodiments, the first centrifugation is performed at 500-1000 g for 5-20 minutes.

[0023] In some embodiments, the first centrifugation is performed at 500-900 g for 5-15 minutes.

[0024] In some embodiments, the second centrifugation is performed at 1000-3000 g for 5-30 minutes.

[0025] In some embodiments, the second centrifugation is performed at 1500-2500 g for 5-20 minutes.

[0026] In some embodiments, the second centrifugation is performed at 1500-2200 g for 5-15 minutes.

[0027] In some embodiments, the third centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0028] In some embodiments, the third centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0029] In some embodiments, the third centrifugation is performed at 12,000-20,000 g for 20-40 minutes.

[0030] In some embodiments, the fourth centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0031] In some embodiments, the fourth centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0032] In some embodiments, the fourth centrifugation is performed at 12,000-20,000 g for 20-40 minutes.

[0033] In some embodiments, the external force comprises addition of staurosporine, ultraviolet irradiation, starvation, or heat stress.

[0034] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 1 to 900 nM.

[0035] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 10 to 800 nM.

[0036] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 100-800 nM.

[0037] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 200-700 nM.

[0038] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 300-600 nM.

[0039] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 400-600 nM.

[0040] In some embodiments, the cell comprises a somatic cell or a stem cell.

[0041] In some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells.

[0042] In some embodiments, the stem cells comprise mesenchymal stem cells.

[0043] In some embodiments, the stem cells include mesenchymal stem cells or induced pluripotent stem cells.

[0044] In some embodiments, the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, adipose, placenta, umbilical cord, periosteum, or tendon.

[0045] In some embodiments, the present invention provides an inducible vesicle obtained by the preparation method.

[0046] In some embodiments, the inducible vesicles are collected 4 to 7 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0047] In some embodiments, the inducible vesicles are collected 4.1 to 6.9 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0048] In some embodiments, the inducible vesicles are collected 4.1 to 6.8 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0049] In some embodiments, the inducible vesicles are collected 4.1 to 6.7 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0050] In some embodiments, the inducible vesicles are collected 4.1 to 6.5 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0051] In some embodiments, the inducible vesicles are collected 4.1 to 6.3 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0052] In some embodiments, the inducible vesicles are collected 6 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles.

[0053] In some embodiments, the protein or RNA is a nascent protein and or nascent RNA.

[0054] In some embodiments, the nascent protein or nascent RNA is an apoptosis-associated nascent protein or nascent RNA.

[0055] In some embodiments, the present invention provides the preparation method or the use of the inducible vesicles in synthesizing protein or RNA or promoting osteogenesis.

[0056] In some embodiments, the protein or RNA is a nascent protein or a nascent RNA.

[0057] In some embodiments, the present invention provides a method for obtaining protein or RNA, wherein induced vesicles are produced by inducing cell apoptosis through external force, the produced induced vesicles are collected 4-7 hours after the application of the external force, and the protein or the RNA is extracted from the collected induced vesicles. The induced vesicles are induced vesicles containing nascent proteins and / or RNA.

[0058] In some embodiments, the inducible vesicles are capable of synthesizing nascent proteins and / or RNA.

[0059] In some embodiments, the induced vesicles are obtained 4.1 to 6.9 hours after the external force is applied to the cells.

[0060] In some embodiments, the induced vesicles are obtained 4.1 to 6.8 hours after the external force is applied to the cells.

[0061] In some embodiments, the induced vesicles are obtained 4.1 to 6.7 hours after the external force is applied to the cells.

[0062] In some embodiments, the induced vesicles are obtained 4.1 to 6.5 hours after the external force is applied to the cells.

[0063] In some embodiments, the induced vesicles are obtained 4.1 to 6.3 hours after the external force is applied to the cells.

[0064] In some embodiments, the induced vesicles are obtained within 6 hours of applying the external force to the cells.

[0065] In some embodiments, the nascent protein or nascent RNA is an apoptosis-associated nascent protein or apoptosis-associated nascent RNA.

[0066] In some embodiments, the step of obtaining the induced vesicles includes: (1) culturing cells; (2) contacting the cells in step (1) with an external force in a culture medium to induce the release of the induced vesicles; (3) collecting the culture supernatant of step (2) to extract the induced vesicles.

[0067] In some embodiments, the vesicle-inducing vesicles are extracted from the culture medium by a method selected from the group consisting of polymer precipitation, immunoseparation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and a combination thereof.

[0068] In some embodiments, the step of separating the induced vesicles by the ultracentrifugation method includes: (a) subjecting the collected culture supernatant to a first centrifugation to obtain the supernatant; (b) subjecting the supernatant collected in step (a) to a second centrifugation to obtain the supernatant; (c) subjecting the supernatant received in step (b) to a third centrifugation to obtain the precipitate; (d) subjecting the precipitate received in step (c) to a fourth centrifugation to obtain the precipitate.

[0069] In some embodiments, the first centrifugation is performed at 500-1500 g for 5-30 minutes.

[0070] In some embodiments, the first centrifugation is performed at 500-1000 g for 5-20 minutes.

[0071] In some embodiments, the first centrifugation is performed at 500-900 g for 5-15 minutes.

[0072] In some embodiments, the second centrifugation is performed at 1000-3000 g for 5-30 minutes.

[0073] In some embodiments, the second centrifugation is performed at 1500-2500 g for 5-20 minutes.

[0074] In some embodiments, the second centrifugation is performed at 1500-2200 g for 5-15 minutes.

[0075] In some embodiments, the third centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0076] In some embodiments, the third centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0077] In some embodiments, the third centrifugation is performed at 12,000-20,000 g for 20-40 minutes.

[0078] In some embodiments, the fourth centrifugation is performed at 10,000-30,000 g for 15-60 minutes.

[0079] In some embodiments, the fourth centrifugation is performed at 12000-25000 g for 20-60 minutes.

[0080] In some embodiments, the fourth centrifugation is performed at 12,000-20,000 g for 20-40 minutes.

[0081] In some embodiments, the external force comprises addition of staurosporine, ultraviolet irradiation, starvation, or heat stress.

[0082] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 1 to 900 nM.

[0083] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 10 to 800 nM.

[0084] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 100-800 nM.

[0085] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 200-700 nM.

[0086] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 300-600 nM.

[0087] In some embodiments, apoptosis is induced by adding staurosporine at a concentration of 400-600 nM.

[0088] In some embodiments, the cell comprises a somatic cell or a stem cell.

[0089] In some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells.

[0090] In some embodiments, the stem cells comprise mesenchymal stem cells.

[0091] In some embodiments, the stem cells include mesenchymal stem cells or induced pluripotent stem cells.

[0092] In some embodiments, the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, adipose, placenta, umbilical cord, periosteum, or tendon. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The results show the amount of ApoV produced by hUCMSCs induced by STS at different induction times.

[0094] Figure 2 The positive rate of apoptosis indicators (AnnexinV / PI) of hUCMSCs induced by STS at different induction times is shown.

[0095] Figure 3 The results show the production of new proteins at different stages of STS-induced hUCMSCs apoptosis, where the bar graph corresponds to the fluorescence image of OPP.

[0096] Figure 4It showed that the newly generated proteins produced by STS-induced hUCMSCs apoptosis were transferred to ApoV.

[0097] Figure 5 It showed that the new proteins produced by STS-induced hUCMSCs apoptosis were transferred to host cells through ApoV.

[0098] Figure 6 Showing the fluorescence staining of nascent RNA during STS-induced hUCMSCs apoptosis (A, 5'-EU labeling for 2h) and mass spectrometry detection of nascent RNA (B, 4SU labeling for 2h).

[0099] Figure 7 The image shows that the newly generated RNA during STS-induced hUCMSCs apoptosis is transferred to ApoV (5'-EU marks 4-6h of late apoptosis). The right image is an enlarged image of the left image under different fields of view.

[0100] Figure 8 The results showed that STS induction time at different times induced the osteogenic function of ApoV produced by hUCMSCs.

[0101] Fig. 9 The results show the relationship between the newly generated proteins and Caspase activity in the late stage of STS-induced hUCMSCs apoptosis.

[0102] Fig.10 It showed that RNA nascent process during STS-induced hUCMSCs apoptosis was related to Caspase activity.

[0103] Fig.11 The results showed that ApoV produced by hUCMSCs induced by STS contained ribosome-associated proteins.

[0104] Fig.12 Fluorescence staining showed that ApoV produced by hUCMSCs induced by STS can continue to synthesize new proteins. The right picture is a partial enlargement of the left picture.

[0105] Fig.13 Ribo-seq analysis showed that ApoV produced by hUCMSCs induced by STS could continue to synthesize new proteins.

[0106] Fig.14 It showed that the newly generated proteins in the late stage of STS-induced hUCMSCs apoptosis were associated with vesicle-like structures containing ribosome components.

[0107] Fig.15 It showed that the newly generated proteins in the late stage of STS-induced hUCMSCs apoptosis were related to the production of ApoV.

[0108] Fig.16This shows that ApoV produced by hUCMSCs can continue to synthesize new RNA. The right picture is an enlarged picture of the left picture under different fields of view. DETAILED DESCRIPTION

[0109] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0110] In the text of this article, "AB", "ApoV", "apoptotic vesicles" or "EVs" are the same as "IEVs". IEVs is the abbreviation of induced vesicles, which can be called induced vesicles or induced extracellular vesicles (IEVs). Induced extracellular vesicles refer to a type of subcellular products produced when precursor cells (such as stem cells) are intervened or induced to undergo apoptosis during normal survival. Usually, this type of subcellular product has a membrane structure, expresses apoptotic markers, and partially contains genetic material DNA. The inventors have found that induced extracellular vesicles are a type of substance that is distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.). In some embodiments, the cells in normal survival are, for example, non-apoptotic cells, non-senescent cells, non-aging and proliferation-stagnant cells, non-freezing and resuscitation cells, non-malignant and abnormally proliferating cells, or non-damaged cells. In some embodiments, the cells in normal survival are taken from cells when the cell contact fusion is 80-100% during the cell culture process. In some embodiments, the cells at normal survival are taken from logarithmic phase cells. In some embodiments, the cells at normal survival are taken from primary culture and subculture cells of human or mouse tissue sources. In some embodiments, the cells at normal survival are taken from established cell lines or cell strains. In some embodiments, the precursor cells are taken from early stage cells.

[0111] As used herein, "NasRNA" or "Nas-RNA" refers to "nascent RNA".

[0112] Herein, "NasProtein", "NasProtein" or "NascentProtein" refers to "nascent protein" or "nascent protein".

[0113] In some embodiments, the present invention has found that during apoptosis, it is possible to translate and synthesize proteins, that is, it is possible to produce nascent proteins. In some embodiments, the inducible vesicles produced by apoptotic cells can also continue to translate and synthesize proteins to produce nascent proteins. In some embodiments, during the use of STS to induce hUCMSCs apoptosis, hUCMSCs can translate proteins when they are in the late stage of apoptosis, and the inducible vesicles produced by apoptotic cells can also continue to translate and synthesize proteins therein. In some embodiments, these nascent proteins can be detected by metabolic labeling with amino acid analogs (such as HPG) or aminoacyl tRNA analogs (such as OPP), and the synthesis of nascent proteins is directly related to the initiation of the apoptosis process and the activation of caspase. In some embodiments, such nascent proteins have a significant promoting effect on the functions of ApoV in promoting osteogenesis of stem cells.

[0114] In some embodiments, the present invention has found that during apoptosis, cells can transcribe RNA, that is, they can produce nascent RNA. In some embodiments, the inducible vesicles produced by apoptotic cells can also continue to transcribe and synthesize RNA therein, producing nascent RNA. In some embodiments, during the use of STS to induce hUCMSCs apoptosis, hUCMSCs can transcribe RNA when they are in the late stage of apoptosis, and the inducible vesicles produced by apoptotic cells can also continue to transcribe and synthesize RNA therein. In some embodiments, these nascent RNAs can be detected by metabolic labeling with nucleotide analogs (such as 5'-EU or 4SU), and the synthesis of nascent RNA is directly related to the initiation of apoptosis and the activation of caspase. In some embodiments, RNA, i.e., nascent RNA, can be transcribed and synthesized after the cell initiates apoptosis and activates caspase. In some embodiments, such nascent RNA has a significant promoting effect on the functions of ApoV promoting stem cell osteogenesis.

[0115] In this article, the inventors found that after adding STS to cells to induce apoptosis, more than 80% of the cells entered the apoptotic state in the sixth hour, and when the induction was greater than 6 hours, the cells entered the secondary necrosis state from the apoptotic state, so the induction time of STS-induced cell apoptosis should not be too long to avoid apoptosis and secondary necrosis. At the same time, according to the time span of 6 hours of STS-induced cell apoptosis, the 0-2h of STS-induced induction was named early apoptosis, 2-4h was named mid-term apoptosis, and 4-6h was named late apoptosis.

[0116] Example 1 Method for preparing vesicles

[0117] 1. Human umbilical cord mesenchymal stem cells (hUCMSCs) were obtained from full-term cesarean sections with the informed consent of the donors. The specific extraction process is as follows: flush the umbilical cord, remove the blood vessels, cut the umbilical cord tissue into small pieces, and attach it to the bottom of the culture dish. The umbilical cord tissue cut into small pieces was cultured at 37°C, 5% CO2, and the culture medium was changed every 3 days until the clone formation of hUCMSCs was observed. Flow cytometry was used to detect stem cell surface markers such as CD90, CD44, sca-1, CD45, and CD34. hUCMSCs were then subcultured and cultured in α-MEM containing 15% fetal bovine serum (FBS), 0.1mM vitamin C, 1% glutamine, 100U / mL penicillin, and 100μg / mL streptomycin. Apoptosis was induced using hUCMSCs at passage P6. When hUCMSCs were cultured to 80% confluence in a 10 cm culture dish, apoptosis was induced and apoptotic vesicles were collected.

[0118] 2. Induce apoptosis: Remove the original culture medium of the cells, use 5 ml of PBS buffer filtered through a 0.1 μm filter membrane to gently wash the cells once. Add 6 mL of α-MEM medium (without FBS) containing 500 nM staurosporine (STS) to a 10 cm culture dish and induce apoptosis at 37°C. The duration of apoptosis induction is 0 to 6 hours.

[0119] 3. Collect apoptotic vesicles containing newly synthesized biomacromolecules during apoptosis: Collect vesicles at the 2nd, 4th and 6th hour after the addition of STS to induce apoptosis. At the three time points of 2h, 4h and 6h induced by STS, collect all cells and supernatant by blowing. After centrifugation at 800g, 4℃, for 10min, take the supernatant and transfer it to a new centrifuge tube; after centrifugation at 2000g, 4℃, for 10min, continue to take the supernatant and transfer it to the Ep tube; after centrifugation at 16000g, 4℃, for 30min, carefully remove the supernatant. Resuspend the precipitate with 1mL of PBS buffer filtered with a 0.1μm filter membrane and wash once; centrifuge the resuspended PBS again, 16000g, 4℃, 30min, and carefully discard the supernatant. The resulting precipitate is the apoptotic vesicle.

[0120] 4. Measurement of the number of vesicles produced at different induction times

[0121] Vesicles were collected at the 2nd, 4th and 6th hour after the addition of STS to induce apoptosis (the time was calculated from the start of the addition of STS), and Zetaview was used to detect the total number of apoptotic vesicles produced at different induction times. At the same time, the total number of induced hUCMSCs was counted to calculate the number of vesicles produced by each hUCMSCs.

[0122] The results are as follows Figure 1 As shown, Figure 1 The results showed that the production of apoptotic vesicles of hUCMSCs was different at different time stages of apoptosis induction by adding STS. As the induction time increased, the number of apoptotic vesicles produced by each apoptotic cell gradually increased.

[0123] Table 1

[0124] Vesicle collection time result 2h after STS addition (early apoptosis) Low vesicle yield 4h after STS addition (mid-apoptosis) Low vesicle yield 6h after STS addition (late stage of apoptosis) High vesicle yield

[0125] 5. Flow cytometry analysis of cell apoptosis

[0126] According to the method of steps 1-3 above in this example, hUCMSCs in normal state, hUCMSCs added with STS at 2 / 4 / 6 hours after the start of apoptosis induction, and hUCMSCs added with STS for more than 6 hours (8 hours) were collected respectively, and two apoptosis indicator flow cytometric antibodies APC-AnnexinV and PE-PI were used for labeling, and the apoptosis process of cells was analyzed by flow cytometry.

[0127] Figure 2 It shows that at the 6th hour after the addition of STS to induce apoptosis, more than 80% of the cells enter the apoptotic state, and when the induction is greater than 6 hours (8 hours), the cells enter the secondary necrosis state from the apoptotic state, so the induction time of STS-induced hUCMSCs apoptosis is set to 6 hours to avoid apoptosis and secondary necrosis. At the same time, according to the time span of 6 hours of STS-induced hUCMSCs apoptosis, the 0-2h of STS-induced apoptosis is named early apoptosis, 2-4h is named mid-term apoptosis, and 4-6h is named late apoptosis.

[0128] Example 2 STS induces hUCMSCs apoptosis and produces new proteins

[0129] The preparation method of the vesicles is as shown in Example 1, and the hUCMSCs are pretreated with a methionine-free medium for 1 hour before inducing apoptosis, and 25uM homopropargylglycine (HPG) is added to the culture dish at 0h, 2h and 4h after adding STS, or 30uM O-propargyl-puromycin (OPP) is added to the culture dish at 0h, 2h and 4h after adding STS to induce apoptosis using a normal medium, and the nascent protein translation activity is labeled for 2h, and fluorescent molecules are conjugated to HPG or OPP through a click reaction, and apoptotic cells are observed under a confocal fluorescence microscope, as shown in FIG. Figure 3 As shown in the figure, proteins are newly generated in apoptotic cells at each stage of apoptosis. Figure 3 In the data, the living cell group refers to the hUCMSCs group without the addition of STS to induce apoptosis; the early apoptosis group refers to the group with apoptosis induction time of 2 h (the time is calculated from the addition of STS); the mid-apoptosis group refers to the group with apoptosis induction time of 4 h (the time is calculated from the addition of STS); the late apoptosis group refers to the group with apoptosis induction time of 6 h (the time is calculated from the addition of STS).

[0130] Figure 4 In order to induce hUCMSCs apoptosis by STS, the newly formed proteins were labeled for 2 hours starting from the addition of STS 4 hours ago. ApoV was extracted at 6 hours after STS-induced apoptosis. Under an ultra-high resolution microscope, it was observed that the newly formed proteins in the late stage of apoptosis could be encapsulated and delivered to ApoV.

[0131] Figure 5 For the general Figure 4 The corresponding ApoV (i.e. Figure 4 The extracted ApoV involved was labeled with PKH-26 membrane dye for 30 minutes and then co-cultured with hUCMSCs at a ratio of 1:2 (i.e., 1 dish of hUCMSCSs-derived ApoV treated 2 dishes of hUCMSCs) for 24 hours. It was observed that the newly generated proteins in the late stage of hUCMSCs apoptosis could be transmitted to the host cells through ApoV.

[0132] Example 3: Nascent RNA in apoptotic cells after STS induced apoptosis of hUCMSCs

[0133] The preparation method of the vesicles is as shown in Example 1. At the 0th, 2nd and 4th hour of inducing hUCMSCs apoptosis (i.e., the timing starts when STS is added to hUCMSCs), 500 μM 5'-EU (a nucleotide substitute for synthetic RNA) is added to the culture dish to label the RNA transcription activity for 2 hours. After the induced apoptotic cells are fixed, fluorescent molecules are conjugated to 5'-EU by a click reaction, and the apoptotic cells are stained. The apoptotic cells are observed under a confocal fluorescence microscope, as shown in FIG. Figure 6 As shown in (A), hUCMSCs induced by STS showed RNA nascent at all stages of apoptosis.

[0134] At 0h, 2h and 4h after inducing apoptosis of hUCMSCs, 500 μM 4SU (nucleotide substitute for synthetic RNA) was added to the culture dish to mark the RNA transcription activity for 2h. Figure 6 As shown in (B), mass spectrometry analysis showed that hUCMSCs induced by STS had RNA nascent at each stage of apoptosis. Figure 6 The horizontal axis normal cells -2h in (B) refers to the RNA transcription activity of normal cells labeled with 500 μM 4SU for 2 hours in the group without STS induction (the timing starts from the time when STS in other groups is added to hUCMSCs).

[0135] like Figure 7 As shown, by extracting the apoptotic vesicles produced by hUCMSCs at 6 hours after STS-induced apoptosis and performing ultra-high resolution imaging detection, it was found that the newly synthesized RNA of hUCMSCs during the induction of apoptosis would be delivered to the apoptotic vesicles.

[0136] Example 4 Pro-osteogenic function of apoptotic vesicles produced by STS-induced apoptosis of hUCMSCs

[0137] The preparation method of vesicles is the same as that of Example 1. The apoptotic vesicles derived from hUCMSCs produced at different induction times were added to the osteogenic induction culture supernatant of hUCMSCs. After inducing hUCMSCs for 14 days, alizarin red staining was used and the bottom of the well plate was scanned and photographed. Figure 8 As shown in the figure, the data showed that the osteogenic function of the apoptotic vesicles of hUCMSCs was different under different STS induction time treatments. As the induction time increased, the osteogenic function of the apoptotic vesicles produced was better, especially the apoptotic vesicles of hUCMSCs induced by STS for 6h had the best osteogenic function.

[0138] Among them, CTRL refers to the group without added apoptotic vesicles.

[0139] Example 5 Relationship between the newly generated proteins and caspase activity in the late stage of STS-induced MSCs apoptosis

[0140] like Fig. 9 As shown, when STS was used to induce hUCMSCs apoptosis, Z-VAD (Pan Caspase inhibitor, 40 μM) was added 1 h before adding STS or hUCMSCs were transfected with Caspase3 siRNA for 48 h and then apoptosis was induced by STS (the method of STS-induced hUCMSCs apoptosis and vesicle production was the same as in Example 1), the protein nascent state in the late stage of apoptosis (STS-induced hUCMSCs apoptosis for 6 hours) was inhibited.

[0141] in addition, Fig. 9 It was also shown that STS induced apoptosis of adipose-derived mesenchymal stem cells derived from Caspase3 knockout mice (homozygous Caspase3- / - and heterozygous Caspase3+ / -). It was observed that in the late stage of apoptosis (4-6 hours after the addition of STS), there was a certain positive correlation between protein degeneration and Caspase3 activity.

[0142] Example 6 RNA nascent process during STS-induced hUCMSCs apoptosis is related to Caspase activity

[0143] like Fig.10 As shown, when hUCMSCs apoptosis was induced by STS, the simultaneous addition of Z-VAD (PanCaspase inhibitor, 40 μM) or Z-DEVD (Caspase3 inhibitor, 100 μM) could inhibit RNA nascent during apoptosis.

[0144] hUCMSCs were transfected with Caspase3 siRNA for 48 hours, and then apoptosis was induced by STS. The results showed that RNA nascent process during apoptosis was also inhibited.

[0145] Therefore, RNA neogenesis during STS-induced hUCMSCs apoptosis is dependent on the Caspase molecular pathway.

[0146] Example 7 ApoV protein synthesis

[0147] The preparation method of vesicles is the same as that in Example 1. Fig.11 As shown in the figure, ApoV produced by hUCMSCs at 6h after STS-induced apoptosis was extracted, and both immunofluorescence and Western Blot showed that ApoV produced ribosomal proteins such as RpS3 or RpL4. Among them, Western Blot results showed that exosomes (exo) did not contain ribosomal protein RpL4.

[0148] The steps for obtaining exosomes are as follows: hUCMSCs (cells are the same as in Example 1) are cultured in a serum-free culture medium, and the supernatant is collected after 48 hours. After centrifugation at 800g, 4°C for 10 minutes, the supernatant is transferred to a new centrifuge tube; after centrifugation at 2000g, 4°C for 10 minutes, the supernatant is continued to be transferred to a new centrifuge tube; after centrifugation at 16000g, 4°C for 30 minutes, the supernatant is transferred to a new centrifuge tube; after centrifugation at 120000g, 4°C for 2 hours, the supernatant is carefully removed; the precipitate is resuspended and washed once with 1mL of PBS buffer filtered with a 0.1μm filter membrane; the resuspended PBS is centrifuged again, 120000g, 4°C, 2 hours, and the supernatant is carefully discarded. The resulting precipitate is the exosome.

[0149] like Fig.12As shown, the ApoV produced by hUCMSCs at the 6th hour of STS-induced apoptosis was cultured in vitro using a cell culture medium (such as the hUCMSCs culture medium shown in Example 1) at 37°C, 5% CO2 for 2 hours, and amino acid analogs were co-incubated at the same time. The nascent protein was then labeled by click reaction staining (the same as the detection method of OPP nascent protein in Example 2), and the apoptotic vesicles were found to still have the function of synthesizing nascent protein through detection by an ultra-high resolution imaging system.

[0150] like Fig.13 As shown, the apoptotic vesicles produced by hUCMSCs at 6 hours after STS-induced apoptosis were extracted, and ApoV translational analysis was completed using RNA-seq technology combined with Ribo-seq technology, further confirming that the apoptotic vesicles still have the function of synthesizing new proteins.

[0151] like Fig.14 As shown, the newly generated proteins co-localized with vesicle-like structures containing ribosomal proteins in apoptotic cells within 4-6 h of STS-induced apoptosis;

[0152] like Fig.15 As shown, using ROCKI inhibitor Y-27632 (10uM) to block the apoptotic process and produce apoptotic vesicles 30 minutes before adding STS to induce apoptosis can significantly reduce the synthesis of new proteins in the late stage of apoptosis. It is speculated that the protein synthesis in the late stage of apoptosis may be related to the apoptotic vesicles containing ribosomes.

[0153] Example 8 ApoV RNA nascence

[0154] like Fig.16 As shown, by extracting the apoptotic vesicles produced by hUCMSCs at the 6th hour of STS-induced apoptosis, the cell culture medium (the culture medium of hUCMSCs in Example 1) was used in vitro, and the culture was continued for 2 hours at 37°C and 5% CO2. 5'-EU nucleotide analogs were also given for co-incubation. Then, NasRNA was labeled by click reaction staining, and detected by ultra-high resolution imaging system, it was found that the apoptotic vesicles still had the function of continuing to transcribe and synthesize new RNA.

Claims

1. A method for preparing an inducible vesicle, characterized in that: Inducible vesicles are generated by inducing cell apoptosis through external force, and the generated induced vesicles are collected 4-7 hours after the application of the external force to obtain induced vesicles containing nascent proteins and / or nascent RNA.

2. The preparation method according to claim 1, characterized in that The inducible vesicles are capable of synthesizing nascent proteins and / or nascent RNA; Preferably, the inducible vesicles are obtained 4.1 to 6.9 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.8 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.7 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.5 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.3 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained within 6 hours of applying the external force to the cells; Preferably, the nascent protein or nascent RNA is an apoptosis-related nascent protein or apoptosis-related nascent RNA.

3. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Cultivating cells; (2) contacting the cells in step (1) with an external force in a culture medium to induce the release of the inducible vesicles; (3) collecting the culture supernatant of step (2) and extracting the induced vesicles; Preferably, the inducible vesicles are extracted from the culture medium by a method selected from the group consisting of polymer precipitation, immunoseparation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration and a combination thereof.

4. The preparation method according to claim 1, characterized in that: The step of separating the inducible vesicles by the ultracentrifugation method comprises: (a) centrifuging the collected culture supernatant for the first time and taking the supernatant; (b) centrifuging the supernatant collected in step (a) for a second time and taking the supernatant; (c) centrifuging the supernatant obtained in step (b) for a third time to obtain a precipitate; (d) centrifuging the precipitate obtained in step (c) for a fourth time to obtain a precipitate; Preferably, the first centrifugation is performed at 500-1500 g for 5-30 minutes; Preferably, the first centrifugation is performed at 500-1000 g for 5-20 minutes; Preferably, the first centrifugation is performed at 500-900 g for 5-15 minutes; Preferably, the second centrifugation is performed at 1000-3000 g for 5-30 minutes; Preferably, the second centrifugation is performed at 1500-2500 g for 5-20 minutes; Preferably, the second centrifugation is performed at 1500-2200 g for 5-15 minutes; Preferably, the third centrifugation is performed at 10000-30000 g for 15-60 minutes; Preferably, the third centrifugation is performed at 12000-25000 g for 20-60 minutes; Preferably, the third centrifugation is performed at 12000-20000 g for 20-40 minutes; Preferably, the fourth centrifugation is performed at 10000-30000 g for 15-60 minutes; Preferably, the fourth centrifugation is performed at 12000-25000 g for 20-60 minutes; Preferably, the fourth centrifugation is performed at 12000-20000 g for 20-40 minutes.

5. The preparation method according to claim 1, characterized in that: The external force includes adding staurosporine, ultraviolet irradiation, starvation, or heat stress; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 1 to 900 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 10 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 100 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 200 to 700 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 300-600 nM; Preferably, cell apoptosis is induced by adding staurosporine, and the concentration of staurosporine is 400-600 nM.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The cells include somatic cells or stem cells; Preferably, the stem cells include totipotent stem cells or pluripotent stem cells; Preferably, the stem cells include mesenchymal stem cells; Preferably, the stem cells include mesenchymal stem cells or induced pluripotent stem cells; Preferably, the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or tendon.

7. The inducible vesicle obtained by the preparation method according to any one of claims 1 to 6.

8. The inducible vesicle according to claim 7, characterized in that collecting the inducible vesicles 4 to 7 hours after applying the external force, and extracting the target protein or RNA from the inducible vesicles; Preferably, the inducible vesicles are collected 4.1 to 6.9 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the inducible vesicles are collected 4.1 to 6.8 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the inducible vesicles are collected 4.1 to 6.7 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the inducible vesicles are collected 4.1 to 6.5 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the inducible vesicles are collected 4.1 to 6.3 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the inducible vesicles are collected 6 hours after the external force is applied, and the target protein or RNA is extracted from the inducible vesicles; Preferably, the protein or RNA is a nascent protein and / or nascent RNA; Preferably, the nascent protein or nascent RNA is apoptosis-related nascent protein or nascent RNA.

9. Use of the preparation method according to any one of claims 1 to 6 or the inducible vesicle according to any one of claims 7 to 8 in synthesizing protein or RNA or promoting osteogenesis; Preferably, the protein or RNA is a nascent protein or a nascent RNA.

10. A method for obtaining protein or RNA, characterized in that: Inducing cell apoptosis by external force to generate induced vesicles, collecting the generated induced vesicles 4 to 7 hours after the external force is applied, and extracting the protein or the RNA from the collected induced vesicles, wherein the induced vesicles are induced vesicles containing nascent proteins and / or nascent RNA; Preferably, the inducible vesicles are obtained 4.1 to 6.9 hours after the external force is applied to the cells; Preferably, the inducible vesicles are capable of synthesizing nascent proteins and / or RNA; Preferably, the inducible vesicles are obtained 4.1 to 6.8 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.7 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.5 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained 4.1 to 6.3 hours after the external force is applied to the cells; Preferably, the inducible vesicles are obtained within 6 hours of applying the external force to the cells; Preferably, the nascent protein or nascent RNA is an apoptosis-related nascent protein or apoptosis-related nascent RNA; Preferably, the step of obtaining the inducible vesicles comprises: (1) culturing cells; (2) in a culture medium, exposing the cells in step (1) to an external force to induce the release of the induced vesicles; (3) collecting the culture supernatant of step (2) to extract the induced vesicles; Preferably, the vesicle-inducing vesicles are extracted from the culture medium by a method selected from the group consisting of polymer precipitation, immunoseparation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, or a combination thereof; Preferably, the step of extracting the inducible vesicles by the ultracentrifugation method comprises: (a) centrifuging the collected culture supernatant for the first time and taking the supernatant; (b) centrifuging the supernatant collected in step (a) for a second time and taking the supernatant; (c) centrifuging the supernatant obtained in step (b) for a third time to obtain a precipitate; (d) centrifuging the precipitate obtained in step (c) for a fourth time to obtain a precipitate; Preferably, the first centrifugation is performed at 500-1500 g for 5-30 minutes; Preferably, the first centrifugation is performed at 500-1000 g for 5-20 minutes; Preferably, the first centrifugation is performed at 500-900 g for 5-15 minutes; Preferably, the second centrifugation is performed at 1000-3000 g for 5-30 minutes; Preferably, the second centrifugation is performed at 1500-2500 g for 5-20 minutes; Preferably, the second centrifugation is performed at 1500-2200 g for 5-15 minutes; Preferably, the third centrifugation is performed at 10000-30000 g for 15-60 minutes; Preferably, the third centrifugation is performed at 12000-25000 g for 20-60 minutes; Preferably, the third centrifugation is performed at 12000-20000 g for 20-40 minutes; Preferably, the fourth centrifugation is performed at 10000-30000 g for 15-60 minutes; Preferably, the fourth centrifugation is performed at 12000-25000 g for 20-60 minutes; Preferably, the fourth centrifugation is performed at 12000-20000 g for 20-40 minutes; Preferably, the external force comprises the addition of staurosporine, ultraviolet irradiation, starvation, or heat stress; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 1 to 900 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 10 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 100 to 800 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 200 to 700 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 300-600 nM; Preferably, apoptosis is induced by adding staurosporine, wherein the concentration of staurosporine is 400-600 nM; Preferably, the cells include somatic cells or stem cells; Preferably, the stem cells include totipotent stem cells or pluripotent stem cells; Preferably, the stem cells include mesenchymal stem cells; Preferably, the stem cells include mesenchymal stem cells or induced pluripotent stem cells; Preferably, the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or tendon.

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