WO2023232099A1 - 一种获取囊泡的方法 - Google Patents
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- WO2023232099A1 WO2023232099A1 PCT/CN2023/097692 CN2023097692W WO2023232099A1 WO 2023232099 A1 WO2023232099 A1 WO 2023232099A1 CN 2023097692 W CN2023097692 W CN 2023097692W WO 2023232099 A1 WO2023232099 A1 WO 2023232099A1
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Definitions
- the present disclosure belongs to the field of biomedicine and relates to a method of obtaining vesicles.
- Apoptosis produces a large number of apoptotic extracellular vesicles (ApoEVs), which contain a variety of cellular components, including microRNA, mRNA, DNA, proteins and lipids. ApoEVs can not only promote intercellular communication through the transfer of cytokines, but also function as a carrier of small molecule drugs, which has feasible clinical application prospects.
- ApoEVs apoptotic extracellular vesicles
- the present disclosure provides a method for producing vesicles, comprising the following steps: (1) applying negative pressure to a cell to induce the cell to produce vesicles; (2) collecting the vesicles; (3) in the The remaining cells in step (2) are continued to be cultured; (4) steps (1) and (2) are performed again.
- step (3) after completing step (4), return to step (3) to repeat the cycle, and the cycle is one round, two rounds or more.
- step (3) the mixture is placed in a carbon dioxide incubator to continue culturing.
- the negative pressure has a value of about -0.1Mpa to -0.01Mpa. In some embodiments, the negative pressure has a value of about -0.1Mpa to -0.02Mpa. In some embodiments, the negative pressure has a value of about -0.1Mpa to -0.06Mpa. In some embodiments, the negative pressure has a value of about -0.08Mpa to -0.01Mpa. In some embodiments, the negative pressure has a value of about -0.07Mpa to -0.01Mpa. In some embodiments, the negative pressure has a value of about -0.06Mpa to -0.02Mpa. In some embodiments, the negative pressure has a value of about -0.04Mpa to -0.02Mpa.
- the time for inducing cells to produce vesicles is about 6h-50h. In some embodiments, in step (1), the time for inducing cells to produce vesicles is about 6h-40h. In some embodiments, in step (1), the time for inducing cells to produce vesicles is about 6h-24h. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is about 20°C to 50°C. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is about 25°C to 40°C. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is about 25°C to 37°C.
- the vesicles are inducible vesicles.
- the vesicles are vesicles produced by applying negative pressure to the cells to induce the cells when the cells are in normal survival.
- the cells include stem cells, somatic cells, or tumor cells.
- the stem cells include totipotent stem cells or pluripotent stem cells.
- the stem cells include mesenchymal stem cells or induced pluripotent stem cells.
- the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or tendon.
- the somatic cells include Jurkat, red blood cells, or PBMC.
- the present disclosure provides a vesicle-generating device comprising a negative pressure regulating component and a temperature regulating component.
- the device includes a box, which is provided with a pressure sensor, a temperature sensor, a heater, a vacuum pump, and a controller; the controller is connected to the temperature sensor and the pressure sensor respectively.
- the controller controls the operation of the vacuum pump through a pressure sensor; the controller controls the operation of the heater through the temperature sensor.
- the device is a cell culture incubator. In some embodiments, the device is a negative pressure cell culture incubator.
- the present disclosure provides a vesicle-producing system, comprising the device, and further comprising a cell culture incubator.
- the present disclosure provides a method of using any of the devices, including the following steps: (1) controlling a heater and a vacuum pump through the controller to make the pressure in the device a negative pressure; ( 2) place the cells in the device and culture them to induce the cells to produce vesicles; (3) collect the vesicles; (4) use the remaining cells in step (3) Continue to culture the cells; (5) Perform the steps of (2) and (3) again.
- step (5) after completing step (5), return to step (4) to repeat the cycle, and the cycle is one round, two rounds or more.
- step (4) the mixture is placed in a carbon dioxide incubator to continue culturing.
- the temperature in the device is about 20°C-50°C, and the value of the negative pressure is about -0.1Mpa ⁇ -0.01Mpa.
- the temperature in the device is about 25°C-40°C. In some embodiments, the temperature in the device is about 25°C-37°C.
- the negative pressure has a value of about -0.1Mpa to -0.02Mpa. In some embodiments, the negative pressure has a value of about -0.1Mpa to -0.06Mpa.
- step (2) the cells are placed in the device and cultured for about 6h-50h. In some embodiments, in step (2), the cells are placed in the device and cultured for about 6h-40h. In some embodiments, in step (2), the cells are placed in the device and cultured for about 6h-24h.
- the vesicles are inducible vesicles. In some embodiments, the vesicles are vesicles produced by applying negative pressure to the cells to induce the cells when the cells are in normal survival.
- the cells include stem cells, somatic cells, or tumor cells.
- the stem cells include totipotent stem cells or pluripotent stem cells.
- the stem cells include mesenchymal stem cells or induced pluripotent stem cells.
- the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or tendon.
- the somatic cells include Jurkat, red blood cells, or PBMC.
- the present disclosure provides a method of generating vesicles using any of the devices described.
- the method includes the following steps: (1) controlling the heater and the vacuum pump through the controller so that the pressure in the device is negative pressure; (2) placing the cells in the The cells were cultured in the above-mentioned device, the cell culture supernatant was collected, and the vesicles were collected from the cell culture supernatant.
- the method further includes the following steps: (3) after obtaining the vesicles in step (2), placing the cells in a carbon dioxide incubator to continue culturing; (4) repeating the steps Describe steps (1) and (2).
- the method of collecting vesicles includes isolating the vesicles from the cell culture supernatant using ultracentrifugation or differential centrifugation.
- the step of isolating the vesicles by the ultracentrifugation method includes: (a) centrifuging the collected culture supernatant for the first time and taking the supernatant; (b) centrifuging the collected culture supernatant in step (a) Centrifuge the obtained supernatant for the second time and take the supernatant; (c) Centrifuge the supernatant collected in step (b) for the third time and take the precipitate; (d) Centrifuge the precipitate collected in step (c) Perform the fourth centrifugation and collect the precipitate.
- the first centrifugation is about 500-1500 g for about 5-30 minutes. In some embodiments, the first centrifugation is about 500-1000 g for about 5-20 minutes. In some embodiments, the first centrifugation is about 500-900 g for about 5-15 minutes. In some embodiments, the second centrifugation is performed at about 1000-3000 g for about 1-30 minutes. In some embodiments, the second centrifugation is performed at about 1500-2500g for about 1-20 minutes. In some embodiments, the second centrifugation is performed at about 1500-2200g for about 1-15 minutes. In some embodiments, the third centrifugation is performed at about 10,000-30,000 g for about 15-60 minutes.
- the third centrifugation is performed at about 12,000-25,000 g for about 20-60 minutes. In some embodiments, the third centrifugation is performed at about 12,000-20,000 g for about 20-40 minutes. In some embodiments, the fourth centrifugation is performed at about 10,000-30,000 g for about 15-60 minutes. In some embodiments, the fourth centrifugation is performed at about 12,000-25,000 g for about 20-60 minutes. In some embodiments, the fourth centrifugation is performed at about 12,000-20,000 g for about 20-40 minutes.
- steps (1), (2), and (3) are repeated two or more times.
- the temperature in the device is about 20°C-50°C, and the value of the negative pressure is about -0.1Mpa ⁇ -0.01Mpa. In some embodiments, the negative pressure has a value of about -0.1Mpa to -0.02Mpa. In some embodiments, the negative pressure has a value of about -0.1Mpa to -0.06Mpa. In some embodiments, the temperature in the device is about 25°C-40°C. In some embodiments, the temperature in the device is about 25°C-37°C. In some embodiments, in step (2), the cells are placed in the device and cultured for about 6h-50h. In some embodiments, in step (2), the cells are placed in the device and cultured for about 6h-40h. In some embodiments, in step (2), the cells are placed in the device and cultured for about 6h-24h.
- the vesicles are inducible vesicles.
- the vesicles are vesicles produced by applying negative pressure to the cells to induce the cells when the cells are in normal survival.
- the cells include stem cells, somatic cells, or tumor cells.
- the stem cells include totipotent stem cells or pluripotent stem cells.
- the stem cells include mesenchymal stem cells or induced pluripotent stem cells.
- the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or tendon.
- the somatic cells include Jurkat, red blood cells, or PBMC.
- the present disclosure provides a vesicle or a vesicle produced by the above method, said vesicle having markers CD63, TSG101, ALIX, syntaxin 4, Annexin V, intergrin ⁇ 5, calnexin or careticulin, cleaved caspase 3. Lamin B1, VDAC2, piezo1 or active- ⁇ -catenin.
- the expression level of CD63, TSG101, ALIX, syntaxin 4, Annexin V, intergrin ⁇ 5, calnexin, careticulin, cleaved caspase 3, Lamin B1, or VDAC2 in the vesicles is lower than that in STS-induced syngeneic cells obtained vesicles.
- the careticulin appears as two cleaved bands in a Western blotting assay.
- the vesicles highly express piezo1 or active- ⁇ -catenin. In some embodiments, the expression level of piezo1 or active- ⁇ -catenin in the vesicles is higher than that in vesicles obtained from STS-induced cells of the same type.
- UMSC-NP-EV may be a new and unique vesicle.
- the inventors have found that cells die through apoptosis under positive pressure, while cells may die through other cell death methods under negative pressure. In some embodiments, it was found that negative pressure induces cells through lysosome-dependent cell death.
- the present disclosure provides a use of the vesicle in fat regulation or osteogenic differentiation; or in the preparation of a fat modulating agent or an osteogenic differentiation agent.
- the adipogenesis is inhibited.
- the vesicles are used in the preparation of products for treating diseases, anti-aging, promoting skin function, and/or cosmetics for non-therapeutic purposes.
- the present disclosure provides a method of treating disease, anti-aging, promoting skin function, and cosmetology, the method comprising administering the vesicles to a patient or subject.
- treating a disease includes promoting wound healing.
- the present disclosure provides a method of treating a wound comprising administering the vesicles to a patient or subject.
- This disclosure also contains the following items:
- Item 1 a method for preparing vesicles, applying mechanical pressure to cells to induce cell death, thereby producing the vesicles.
- the mechanical pressure includes positive pressure or negative pressure.
- the negative pressure is about -0.1 to -0.005Mpa; in some embodiments, the negative pressure is about -0.1 to -0.01Mpa; in some embodiments, the negative pressure is The size is about -0.1 ⁇ -0.02Mpa; in some embodiments, the size of the negative pressure is about -0.1 ⁇ -0.03Mpa; in some embodiments, the size of the negative pressure is about -0.1 ⁇ -0.05Mpa; In some embodiments, the negative pressure is about -0.1 to -0.06Mpa; in some embodiments, the negative pressure is about -0.1 to -0.07Mpa; in some embodiments, the positive pressure is The magnitude of the positive pressure is about 2 to 6 g/cm 2 ; in some embodiments, the magnitude of the positive pressure is about 2 to 5 g/cm 2 ; in some embodiments, the magnitude of the positive pressure is about 2 to 4 g/cm 2
- the cells include stem cells, somatic cells, or tumor cells; in some embodiments, the stem cells include totipotent stem cells or pluripotent stem cells; in some embodiments, the stem cells include mesenchymal stem cells or induced pluripotent stem cells. Competent stem cells; in some embodiments, the source of mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon; in some embodiments, the somatic cells include Jurkat, PBMC or red blood cells.
- the negative pressure is achieved through a negative pressure incubator; in some embodiments, the positive pressure is achieved by placing a glass slide on the cells, and then placing a container containing a heavy object.
- the cells are cultured on the glass slide to apply pressure to the cells; in some embodiments, the weights include steel beads; in some embodiments, the glass slides include quartz glass slides.
- the time for inducing cell death is about 3 to 72 hours; the time for inducing cell death is about 3 to 50 hours; in some embodiments, the time for inducing cell death is about 3 to 50 hours.
- the time for inducing cell death is about 3-48 hours; in some embodiments, the time for inducing cell death is about 3-24 hours; in some embodiments, when the mechanical pressure is negative pressure, the time for inducing cell death The time for inducing cell death is about 3-40 hours; in some embodiments, when the mechanical pressure is negative pressure, the time for inducing cell death is about 3-30 hours; in some embodiments, when the mechanical pressure is negative pressure , the time for inducing cell death is about 3-24 hours; in some embodiments, when the mechanical pressure is negative pressure, the time for inducing cell death is about 5-24 hours.
- the temperature at which the cells are induced to produce vesicles is about 20°C-50°C; in some embodiments, the temperature at which the cells are induced to produce vesicles is about 25°C-40°C; in some embodiments, the temperature at which the cells are induced to produce vesicles The temperature is about 25°C-37°C.
- the vesicles express markers CD63, TSG101, ALIX, syntaxin 4, Annexin V, cleaved caspase 3, Lamin B1, intergrin ⁇ 5, VDAC2, calnexin, careticulin.
- the negative pressure-induced vesicles low-express CD63, TSG101, ALIX, syntaxin 4, Annexin V, intergrin ⁇ 5, calnexin, and careticulin, wherein careticulin appears as two cleaved bands.
- Item 7 The vesicle according to item 6, the diameter of the vesicle is about 0.05-0.4 ⁇ m.
- the diameter of the vesicles is about 0.05-0.38 ⁇ m; in some embodiments, the diameter of the vesicles is about 0.05-0.35 ⁇ m; in some embodiments, the diameter of the vesicles is about 0.05 ⁇ 0.32 ⁇ m; in some embodiments, the diameter of the vesicles is about 0.05-0.3 ⁇ m; in some embodiments, the diameter of the vesicles is about 0.05-0.25 ⁇ m; in some embodiments, the diameter of the vesicles The diameter is about 0.05-0.22 ⁇ m; in some embodiments, the diameter of the vesicles is about 0.55-0.22 ⁇ m.
- Item 9 A composition containing the vesicle according to any one of Items 6 to 8.
- the composition is a differentiation medium; in some embodiments, the composition is an osteogenic differentiation medium.
- Item 10 The use of the vesicles as described in any one of items 6 to 8 in fat regulation or osteogenic differentiation; or in the preparation of fat modulating agents or osteogenic differentiation agents; in some embodiments, the Fat regulation inhibits adipogenesis.
- Figure 1 is a schematic diagram of the negative pressure incubator of Embodiment 1.
- Figure 2 is a flow chart of vesicle collection.
- Figure 3 shows the morphological changes of UMSCs observed under a light microscope after UMSCs were induced in a negative pressure incubator.
- Figure 4 shows the dynamic changes in the vesicle production process of UMSCs observed under a super-resolution microscope after induction of UMSCs in a negative pressure incubator.
- Figure 5 shows the cell morphology observed under an electron microscope after UMSCs were induced in a negative pressure incubator.
- Figure 6 is a negative-stained transmission electron microscope image of ApoV generated after UMSCs were induced in a negative pressure incubator.
- Figure 7 shows the results of analyzing the number, particle size, and potential of vesicles after induction by different pressures in a negative pressure incubator of UMSCs using Zeta View.
- Figure 8 shows the results of analyzing the number, particle size, and potential of vesicles after induction at different temperatures in a negative pressure incubator of UMSCs using Zeta View.
- Figure 9 shows the morphological changes of Jurkat/PBMC observed under a light microscope after being induced in a negative pressure incubator.
- Figure 10 shows the results of analyzing the quantity, particle size, and potential of ApoV produced by Jurkat/PBMC under negative pressure through Zeta View.
- Figure 11 is a diagram showing the morphological changes of RBCs during vesicle production after induction in a negative pressure incubator using ultra-high resolution microscopy.
- Figure 12 shows the results of Zeta View’s analysis of the quantity, particle size, and potential of ApoV produced by RBC under negative pressure.
- Figure 13 shows the morphological changes of UMSCs observed under a light microscope before and after the first negative pressure induction and STS induction.
- Figure 14 shows the morphological changes of UMSCs observed under a light microscope before and after the second negative pressure induction and STS induction.
- Figure 15 shows the morphological changes of UMSCs observed under a light microscope after continuing to culture the cells after the second induction of negative pressure.
- Figure 16 shows the results of Zeta View’s analysis of the quantity, particle size, and potential of ApoV produced by UMSC under negative pressure.
- Figure 17 shows the effect of UMSC vesicles on the stemness of MSCs.
- C Western blot results show that the expression of osteogenic markers Runx2 and ALP is up-regulated in MSCs treated with UMSC vesicles. The expression of adipogenic marker PPAR- ⁇ was reduced.
- ns means that compared with the control group, the difference is not statistically significant, P>0.05.
- Figure 18 shows that UMSC vesicles promote skin wound healing in mice.
- B In vitro culture results of mouse skin tissue blocks show that UMSC vesicles can promote the migration and growth of tissue block cells.
- C CCK8 results show that UMSC vesicles can proliferate SMSCs.
- D,E In vivo animal imaging results show the dynamic changes in fluorescence and tissue and organ distribution after local injection of UMSC vesicles around the wound. *Indicates that compared with the control group, the difference is statistically significant, P ⁇ 0.05. ns means that compared with the control group, the difference is not statistically significant, P>0.05.
- Figure 19 shows a comparison of the characterization of UMSC-NP-EV and UMSC-MF-EV.
- A Transmission electron microscope (TEM) images of UMSC-NP-EV and UMSC-MF-EV, scale bar: 200nm.
- B Images of PKH26 dye-labeled UMSC-NP-EV and UMSC-MF-EV taken by Elyra 7 Lattice SIM, scale bar: 200nm.
- C NTA analysis of the median particle size of UMSC-NP-EV and UMSC-MF-EV.
- D NTA analysis of particle size distribution of UMSC-NP-EV and UMSC-MF-EV.
- E Number of EVs produced by a single UMSC cell.
- Figure 20 shows protein spectrum analysis of UMSC vesicles.
- A,B Volcano plot and cluster heat map show the differential protein distribution between NP-EV group and STS-EV group.
- C-E GO-C enrichment analysis, GO-F and GO-P enrichment analysis of up-regulated proteins in the NP-EV group compared with the STS-EV group.
- Figure 21 shows protein expression of UMSC vesicles.
- Western blotting analysis showed that UMSC vesicles expressed some specific biomarkers and mechanically related functional molecules of apoptotic vesicles.
- Figure 22 shows the morphological changes and the time course of apoptosis rate of UMSCs under the stimulation of positive and negative pressure.
- A Morphological changes in UMSC cell death after mechanical positive pressure and negative pressure treatment.
- B-C Changes in the apoptosis rate of UMSCs under positive and negative pressure.
- D-E After treatment with apoptosis inhibitors, the apoptosis rate of UMSC changes under the action of positive and negative pressure.
- MF mechanical force
- NP negative pressure.
- ns means that compared with the control group, the difference is not statistically significant, P>0.05.
- Figure 23 shows the activation of specific death pathways in UMSC under stimulation of positive and negative pressure.
- A-E Expression of key proteins in UMSC apoptosis after mechanical positive pressure and negative pressure treatment.
- F Activation of cleaved caspase3 in UMSCs under positive and negative pressure. Scale bar: 5 ⁇ m. MF, mechanical force; NP, negative pressure. *Indicates that compared with the control group, the difference is statistically significant, P ⁇ 0.05. ns means that compared with the control group, the difference is not statistically significant, P>0.05.
- Figure 24 is a comparison of UMSC mechanical death and other cell death methods.
- A-G Expression of autophagy, ferroptosis, cell necrosis, pyroptosis and other proteins during UMSC cell death after mechanical positive pressure and negative pressure treatment.
- H-L Expression of key proteins in UMSC death under negative pressure after treatment with autophagy inhibitors. *Indicates that compared with the control group, the difference is statistically significant, P ⁇ 0.05. ns means that compared with the control group, the difference is not statistically significant, P>0.05.
- Figure 25 shows the functional verification of LC3II during the negative pressure death process of UMSC.
- A-G After treatment with lysosomal function inhibitors BafA1 and CQ, the expression of autophagy-related proteins in UMSCs under negative pressure and rapamysin.
- H After treatment with lysosomal function inhibitors BafA1 and CQ, the amount of secreted vesicles in UMSC changed under the action of negative pressure and rapamysin.
- I-J Extracellular vesicle levels in rapamysin-induced autophagy group after siRNA knockdown of LC3II.
- Figure 26 shows the functional verification of negative pressure-induced cell death of UMSCs.
- A Flow cytometry detects changes in cell apoptosis rate after treatment with different inhibitors.
- B Heat map results show changes in cell apoptosis rate after treatment with different inhibitors. The more obvious the green color, the more living cells and the fewer apoptotic cells; the more obvious the red color, the fewer living cells and the more apoptotic cells.
- C Line chart results show changes in cell apoptosis rate after treatment with different inhibitors.
- Figure 27 shows the mechanical positive pressure adding device.
- the terms “set”, “connected” or “connected” should be understood in a broad sense.
- it can be a fixed connection or a fixed connection. It is a detachable connection or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two components.
- the specific meanings of the above terms in this disclosure can be understood on a case-by-case basis.
- ApoV refers to "vesicles", which can also be called IEVs, which are a type of extracellular vesicles (i.e. EVs).
- the umbilical cord mesenchymal stem cells used are human umbilical cord mesenchymal stem cells of the P4-P8 generation; the P4 generation cells are specifically used in the following examples.
- mouse bone marrow mesenchymal stem cells used in the experiments are P3 generation cells.
- the terms “include,” “have,” “contains,” and “include” and other similar forms, as well as their grammatical equivalents, are equivalent in meaning and are open-ended, that is, in these words Any following item or items is not meant to be an exhaustive enumeration of the item or items, nor is it meant to be limited to the listed item or items.
- something “comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain more than components A, B , and C, and may include one or more other components. Accordingly, it is intended and understood that “comprising” and similar forms and its grammatical equivalents include disclosure of embodiments that "consist essentially of” or “consist of.”
- Extract mouse bone marrow mesenchymal stem cells (1) Prepare 3-5 week WT mice and die suddenly due to cervical dislocation; (2) Take the femurs and tibias from both sides of the mice, remove the muscle tissue and place them in iced PBS; (3) Rinse the bones twice with ice-cold PBS, use a 10ml syringe to draw 1% BSA prepared in PBS to rinse the bone marrow, and filter it with a 70 ⁇ m filter; (4) Centrifuge the cell suspension at 1500RPM ⁇ 4°C ⁇ 5min to collect the cell pellet. Take 1 ml of complete culture medium to resuspend the cells; (5) Count the cells, inoculate 1 ⁇ 10 6 cells, and transfer to the cell culture incubator.
- PBMC-NP-EV refers to the vesicles produced by PBMC induced by negative pressure (NP);
- BMSC-NP-EV refers to the vesicles produced by BMSC induced by negative pressure (NP);
- UMSC- MF-EV refers to the vesicles produced by UMSC induced by positive pressure (MF);
- UMSC-STS-EV refers to the vesicles produced by UMSC induced by STS; and so on.
- Staurosporine (STS) induces cell apoptosis: When the cell density reaches 80%, wash twice with PBS, induce cell apoptosis with 500nM staurosporine (STS), and observe changes in cell morphology. Cell apoptosis is determined when typical cell apoptosis morphological changes such as cell shrinkage, disappearance of connections with surrounding cells, plasma membrane bubbles, and nucleoplasmic condensation;
- the negative pressure incubator described in this article adds a temperature module and a negative pressure module, which can adjust the pressure (such as negative pressure) and temperature in the incubator.
- the negative pressure incubator includes a box 1, which is provided with a pressure sensor 2, a temperature sensor 3, a controller 4, a heater 5 and a vacuum pump 6; the controller 4 is respectively connected with The temperature sensor 3 and the pressure sensor 2 are connected; the controller 4 controls the negative pressure generated by the vacuum pump 6 through the pressure sensor 2; the controller 4 communicates The temperature sensor 3 controls the operation of the heater 5.
- the incubator culture parameters as follows: 1Temperature: 25°C-50°C; 2Negative pressure value: -0.1Mpa ⁇ -0.01Mpa; 3Induction time: 6h-50h.
- the gas component in the negative pressure incubator is air.
- Example 2 In vitro negative pressure induces vesicle production from umbilical cord mesenchymal stem cells
- UMSCs umbilical cord mesenchymal stem cells
- Vesicle collection After the cells are induced and cultured, collect UMSCs and culture them in a centrifuge tube. Perform differential centrifugation of the culture medium containing vesicles obtained above (the process is shown in Figure 2): centrifuge at 800g for 10 minutes and discard the cells.
- Precipitate collect the supernatant, centrifuge at 2000g for 5 minutes, discard the cell debris and other precipitates, collect the supernatant, centrifuge at 16000g for 30 minutes, collect the precipitate which is apoptotic vesicle (apoV), then resuspend ApoV in 1ml PBS and centrifuge at 16000g Collect the precipitate after 30 minutes, resuspend apoV in PBS and store it at 4°C for subsequent identification and analysis;
- apoV apoptotic vesicle
- Negative staining transmission electron microscopy image of vesicles (Figure 6); Analysis of the number, particle size, and potential of vesicles after induction by different pressures in UMSCs negative pressure incubator using Zeta View ( Figure 7); Analysis of UMSCs negative pressure incubator using Zeta View The number, particle size, and potential of vesicles after induction at different temperatures were analyzed ( Figure 8).
- Figure 6 is a negative-stained transmission electron microscope image of the vesicles of the experimental group.
- the vesicles are derived from UMSCs induced by negative pressure: after culturing for 20 hours at 37°C in a negative pressure incubator with a negative pressure induction condition of -0.08Mpa. .
- Example 3 In vitro negative pressure induces vesicle production in Jurkat cells and monocytes
- the isolated human peripheral blood mononuclear cells were directly inoculated into 10cm culture dishes and cultured in serum-free 1640 medium (experimental group), 500nM STS-free and PBMC-free.
- the serum was cultured in 1640 medium (control group); the experimental group was placed in a negative pressure incubator ( Figure 1), and the control group was placed in a conventional carbon dioxide incubator.
- Vesicle collection After the cells are induced and cultured, collect the cell culture base in a centrifuge tube, and perform differential centrifugation of the culture medium containing vesicles obtained above (the process is shown in Figure 2): centrifuge at 800g for 10 minutes and discard the cells.
- Precipitate collect the supernatant, centrifuge at 2000g for 5 minutes, discard the cell debris and other precipitates, collect the supernatant, centrifuge at 16000g for 30 minutes, collect the precipitate which is apoptotic vesicle (apoV), then resuspend ApoV in 1ml PBS and centrifuge at 16000g Collect the precipitate after 30 minutes, resuspend apoV in PBS and store it at 4°C for subsequent identification and analysis;
- apoV apoptotic vesicle
- Vesicle collection After the cells are induced and cultured, collect the cell culture base in a centrifuge tube, and perform differential centrifugation of the culture medium containing vesicles obtained above (process shown in Figure 2 (shown): Centrifuge at 800g for 10 minutes to discard the cell pellet, collect the supernatant, centrifuge at 2000g for 5 minutes, discard the cell debris and other precipitates, collect the supernatant, centrifuge at 16000g for 30 minutes and collect the precipitate, which is apoptotic vesicle (apoV). Then resuspend ApoV in 1 ml of PBS and centrifuge at 16000g for 30 minutes to collect the precipitate. Resuspend apoV in PBS and store at 4°C for subsequent identification and analysis.
- apoV apoptotic vesicle
- Umbilical cord mesenchymal stem cells were inoculated into a 10cm culture dish and cultured in a conventional carbon dioxide incubator using MEM ALPHA medium containing 10% FBS until the cells grew to 90-95%. , replaced with serum-free MEM-ALPHA medium (experimental group), and serum-free MEM-ALPHA medium containing 500nM STS (control group); the experimental group was placed in a negative pressure incubator ( Figure 1) -0.06Mpa, 12 hours after induction at 37°C, vesicles were collected; the control group was cultured in a conventional carbon dioxide incubator and vesicles were collected.
- a negative pressure incubator Figure 1 -0.06Mpa
- the method of collecting vesicles is the same as in Example 2.
- MEM ALPHA medium containing 10% FBS to the remaining cells in the culture dish after the first induction and place them in a conventional carbon dioxide incubator for culture. Then it was replaced with serum-free MEM-ALPHA medium (experimental group), and serum-free MEM-ALPHA medium containing 500nM STS (control group); the experimental group was placed in a negative pressure incubator again, and the control group Cultivate again in a conventional carbon dioxide incubator. Repeat the above steps of cell culture, negative pressure induction, and vesicle collection for the first induction until the end of the experiment.
- this device can achieve the purpose of repeatedly collecting vesicles produced by UMSCs by reasonably setting different negative pressure and temperature induction times.
- the specific program design is as follows:
- UMSCs umbilical cord mesenchymal stem cells
- Vesicle collection After the cells are induced and cultured, collect UMSCs and culture them in a centrifuge tube. Perform differential centrifugation of the culture medium containing vesicles obtained above (the process is shown in Figure 2): centrifuge at 800g for 10 minutes and discard the cells.
- Precipitate collect the supernatant, centrifuge at 2000g for 5 minutes, discard the cell debris and other precipitates, collect the supernatant, centrifuge at 16000g for 30 minutes, collect the precipitate which is apoptotic vesicle (apoV), then resuspend ApoV in 1ml PBS and centrifuge at 16000g Collect the precipitate after 30 minutes, resuspend apoV in PBS and store it at 4°C for subsequent identification and analysis;
- apoV apoptotic vesicle
- the vesicles induced by the above were collected separately, and the number, particle size, and potential of the vesicles after induction in the UMSCs negative pressure incubator were analyzed through Zeta View ( Figure 16). It can be seen that the number of vesicles induced by STS is small, and the number of vesicles induced by negative pressure is The induced vesicles have a large yield and can be cultured and induced many times. The potential and particle size of the produced vesicles are basically consistent. The number of vesicles is far greater than that of vesicles induced by STS, and the two are not of the same order of magnitude.
- STS-7h-1 and STS-7h-1 refer to the first and second inductions of STS (induction for 7 hours) respectively; similarly, -0.06Mpa-37°C-12h-1, - 0.06Mpa-37°C-12h-2 and -0.06Mpa-37°C-12h-3 refer to the first, second and third times of negative pressure induction respectively.
- the ordinate apoV Nb in Figure 16 refers to the number of apoV.
- Example 7 In vitro functional identification of vesicles producing UMSC positive and negative stress sources
- the STS concentration is 500nM
- the induction time is 8h
- the MF pressure value is 4g/cm2
- the induction time is 24h
- the NP pressure value is -60Kpa
- the induction time is 20h.
- Example 8 In vivo functional verification of UMSC positive and negative pressure source vesicles
- mice In order to further verify the in vivo function of UMSC vesicles, an equal amount of vesicles was used to locally inject around the skin wound of mice.
- the STS concentration is 500nM
- the induction time is 8h
- the MF pressure value is 4g/cm2
- the induction time is 24h
- the NP pressure value is -60Kpa
- the induction time is 20h.
- UMSC vesicles were isolated by differential centrifugation.
- GO-C enrichment analysis results showed that 1789 proteins with higher expression in UMSC-NP-EV than UMSC-STS-EV were mainly concentrated in mitochondria, ribosomes, mitochondrial inner membrane, mitochondrial matrix, mitochondrial large ribosome subunit, nucleolus, Cytoplasmic large ribosomal subunits, mitochondrial nucleoids, integral components of membranes, glyconucleoprotein complexes, endoplasmic reticulum and other cellular components (Figure 20C).
- GO-F enrichment analysis results show that 1789 proteins are mainly concentrated in ribosome structural components, RNA binding, ribonucleic acid binding, proton transport ATP synthase activity, nucleosome DNA binding, aminoacyl-tRNA ligase activity, nucleosomes Binding, ribonucleoprotein complex binding, SNAP receptor activity and other molecular functions (Figure 20D).
- GO-P enrichment analysis results show that 1789 proteins are mainly concentrated in translation, mitochondrial translation elongation, mitochondrial translation termination, SRP-dependent co-translation proteins, viral transcription, translation initiation, nuclear transcription, mRNA catabolic process, RNA processing, mitochondria Translation, RNA splicing, mRNA processing and other biological processes ( Figure 20E).
- UMSC-NP-EV lowly expresses the common vesicle markers CD63, TSG101, ALIX, and syntaxin 4 and the apoptotic vesicle markers Annexin V, intergrin ⁇ 5, calnexin, and careticulin, but careticulin appears as two cleaved bands; UMSC -NP-EV has low expression of apoptotic vesicle markers cleaved caspase 3, Lamin B1, and VDAC2, and high expression of mechanical-related proteins piezo1 and active- ⁇ -catenin (Figure 21).
- UMSC-MF-EV may be produced in a similar process to UMSC-STS-EV, both of which are produced during apoptosis, while UMSC-NP-EV may be a new and unique vesicle.
- the STS concentration is 500nM
- the induction time is 8h
- the MF pressure value is 4g/cm 2
- the induction time is 24h
- the NP pressure value is -60Kpa
- the induction time is 20h.
- the method for obtaining UMSC vesicles is the same as in Example 8.
- UMSC exhibit caspase-independent cell death under negative pressure.
- UMSC mechanical death proteins were further collected and the expression of key proteins such as autophagy, ferroptosis, cell necrosis, and pyroptosis were detected using western blotting technology.
- the results showed that the autophagy marker protein LC3II was highly expressed during the negative pressure chemical death process of UMSC, but the key protein BECN1 for autophagy initiation, ferroptosis marker proteins GPX4, COX2, cell necrosis marker protein RIP3, and pyroptosis marker protein GSDMD were expressed at low levels (Figure 24A - Figure 24G). It is suggested that LC3II may be involved in the negative pressure chemical death process of UMSC and play a key role.
- rapamysin-induced autophagy and STS-induced apoptosis were used as control groups, and the autophagy initiation process inhibitor 3MA and the lysosomal function inhibitor BafA1 were used to treat Perform negative pressure treatment.
- Western blotting results showed that LC3II was still highly expressed under the action of 3MA and BafA1, while the expression of lysosomal membrane protein LAMP1 decreased, and the expression of LAMP2A increased and was highly cleaved (Figure 24H- Figure 24L). It is suggested that the negative pressure chemical death process of UMSC may be a non-classical autophagy process involving LC3II and may be related to lysosomes.
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Abstract
一种获取囊泡的方法,提供了一种产囊泡的方法,通过负压的作用,使得细胞产生囊泡。细胞经过这种负压产囊泡之后,还可以继续添加培养基正常培养,从而达到一株细胞可以继续多次重复用于产囊泡的目的,可以较好地节约细胞来源,简化操作流程,便于产业化大量产囊泡。
Description
本公开属于生物医药领域,涉及一种获取囊泡的方法。
细胞新生和死亡伴随着机体生命的整个过程,二者的平衡对于维持机体的稳态具有非常重要的意义,然而目前对于细胞死亡的认识仍在不断深入。细胞死亡及其功能失调是各种病理和生理过程的基础,包括细胞稳态,组织重塑和肿瘤发生。1972年,Kerr及其同事发现细胞死亡的一种典型形态变化,提出了“细胞凋亡”的概念:具有核变化包括核染色质浓缩和核碎裂,细胞收缩以及与相邻细胞的附着丧失等明显的形态特征。之后30年里陆续有十余种新型细胞死亡形式被发现命名,如细胞焦亡、细胞坏死、铁死亡、细胞自噬等,提示细胞死亡是多样性的,可能有许多细胞新型死亡形式亟需探索,这对于疾病治疗和对生命的认识都具有非常重要的意义。
细胞凋亡会产生大量凋亡外囊泡(Apototic extracellular vesicles,ApoEVs),其中包含多种细胞成分,包括microRNA,mRNA,DNA,蛋白质和脂质。ApoEVs既可通过细胞因子等转移促进细胞间通讯发挥作用,又可作为小分子药物的载体发挥作用,具有可行的临床应用前景。
发明内容
在一些实施方案中,本公开提供了一种产囊泡的方法,包括以下步骤:(1)将负压施加至细胞诱导细胞产囊泡;(2)收集囊泡;(3)在所述步骤(2)中剩下的细胞进行继续培养;(4)再次实施(1)和(2)的步骤。
在一些实施方案中,完成步骤(4)后,返回步骤(3)进行循环重复,所述的循环为一轮、两轮或以上。在一些实施方案中,步骤(3)中,放置于二氧化碳培养箱中继续培养。
在一些实施方案中,步骤(1)中,所述负压的值约为-0.1Mpa~-0.01Mpa。在一些实施方案中,所述负压的值约为-0.1Mpa~-0.02Mpa。在一些实施方案中,所述负压的值约为-0.1Mpa~-0.06Mpa。在一些实施方案中,所述负压的值约为-0.08Mpa~-0.01Mpa。在一些实施方案中,所述负压的值约为-0.07Mpa~-0.01Mpa。在一些实施方案中,所述负压的值约为-0.06Mpa~-0.02Mpa。在一些实施方案中,所述负压的值约为-0.04Mpa~-0.02Mpa。
在一些实施方案中,步骤(1)中,诱导细胞产囊泡的时间约为6h–50h。在一些实施方案中,步骤(1)中,诱导细胞产囊泡的时间约为6h–40h。在一些实施方案中,步骤(1)中,诱导细胞产囊泡的时间约为6h–24h。在一些实施方案中,所述步骤(1)中,诱导细胞产囊泡的温度约为20℃-50℃。在一些实施方案中,所述步骤(1)中,诱导细胞产囊泡的温度约为25℃-40℃。在一些实施方案中,所述步骤(1)中,诱导细胞产囊泡的温度约为25℃-37℃。
在一些实施方案中,所述的囊泡为诱导性囊泡。
在一些实施方案中,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡。
在一些实施方案中,所述细胞包括干细胞、体细胞或肿瘤细胞。在一些实施方案中,所述干细胞包括全能干细胞或多能干细胞。在一些实施方案中,所述干细胞包括间充质干细胞或诱导性多能干细胞。在一些实施方案中,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱。在一些实施方案中,所述的体细胞包括Jurkat、红细胞或PBMC。
在一些实施方案中,本公开提供了一种产囊泡的装置,包含负压调节组件和温度调节组件。
在一些实施方案中,所述的装置包括箱体,箱体内设置有压力传感器、温度传感器、加热器、真空泵、控制器;所述控制器分别与温度传感器、压力传感器连接。
在一些实施方案中,所述控制器通过压力传感器控制所述真空泵的工作;所述控制器通过所述温度传感器控制所述加热器的工作。
在一些实施方案中,所述装置为细胞培养箱。在一些实施方案中,所述装置为负压细胞培养箱。
以往的实验中,使用STS等诱导后,会发现细胞大量死亡,从而一般只会一株细胞只用STS诱导一次产囊泡后就不再使用。而在一些实施方案中,发明人研究发现,细胞经过这种负压培养箱产囊泡之后,还可以继续添加培养基正常培养,即使细胞进行第二次培养产生的囊泡数量也是远远多于STS第一次诱导产生的囊泡,甚至远远多于STS诱导细胞两次加起来的总共产生的囊泡。从而一株细胞可以继续多次重复用于产囊泡,可以较好地节约细胞来源,简化操作流程,便于产业化大量产囊泡。
一些实施方案中,本公开提供了一种产囊泡的系统,包含所述的装置,还包括细胞培养箱。
在一些实施方案中,本公开提供了任一所述的装置的使用方法,包括以下步骤:(1)通过所述控制器控制加热器和真空泵,使所述装置中的压力为负压;(2)将细胞放置于所述的装置中培养诱导细胞产生囊泡;(3)收集囊泡;(4)将步骤(3)中剩下的细
胞进行继续培养;(5)再次实施如(2)和(3)的步骤。
在一些实施方案中,完成步骤(5)之后,返回步骤(4)进行循环重复,所述的循环为一轮、两轮或以上。
在一些实施方案中,步骤(4)中,放置于二氧化碳培养箱中继续培养。
在一些实施方案中,所述步骤(1)中,所述装置中的温度约为20℃-50℃,所述负压的值约为-0.1Mpa~-0.01Mpa。
在一些实施方案中,所述装置中的温度约为25℃-40℃。在一些实施方案中,所述装置中的温度约为25℃-37℃。
在一些实施方案中,所述负压的值约为-0.1Mpa~-0.02Mpa。在一些实施方案中,所述负压的值约为-0.1Mpa~-0.06Mpa。
在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–50h。在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–40h。在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–24h。
在一些实施方案中,所述的囊泡为诱导性囊泡。在一些实施方案中,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡。在一些实施方案中,所述细胞包括干细胞、体细胞或肿瘤细胞。在一些实施方案中,所述干细胞包括全能干细胞或多能干细胞。在一些实施方案中,所述干细胞包括间充质干细胞或诱导性多能干细胞。在一些实施方案中,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱。在一些实施方案中,所述的体细胞包括Jurkat、红细胞或PBMC。
在一些实施方案中,本公开提供了一种产囊泡的方法,所述方法使用了任一所述的装置。
在一些实施方案中,所述方法包括以下步骤:(1)通过所述控制器控制所述加热器和所述真空泵,使所述装置中的压力为负压;(2)将细胞放置于所述的装置中培养,收集细胞培养上清,从所述细胞培养上清中收集囊泡。
在一些实施方案中,所述方法还包括以下步骤:(3)在所述步骤(2)获得所述囊泡之后,将所述的细胞放置于二氧化碳培养箱中继续培养;(4)重复所述步骤(1)和(2)。
在一些实施方案中,收集囊泡的方法包括选用超速离心或差速离心的方法从细胞培养上清中分离所述囊泡。
一些实施方案中,所述超速离心的方法分离所述囊泡的步骤包括:(a)将收集到的培养上清进行第一次离心,取上清;(b)将步骤(a)中收集到的上清进行第二次离心,取上清;(c)将步骤(b)中收集到的上清进行第三次离心,取沉淀;(d)将步骤(c)中收集到的沉淀进行第四次离心,取沉淀。
一些实施方案中,所述第一次离心约为500-1500g离心约5-30分钟。一些实施方案中,所述第一次离心约为500-1000g离心约5-20分钟。一些实施方案中,所述第一次离心约为500-900g离心约5-15分钟。一些实施方案中,所述第二次离心约为1000-3000g离心约1-30分钟。一些实施方案中,所述第二次离心约为1500-2500g离心约1-20分钟。一些实施方案中,所述第二次离心约为1500-2200g离心约1-15分钟。一些实施方案中,所述第三次离心约为10000-30000g离心约15-60分钟。一些实施方案中,所述第三次离心约为12000-25000g离心约20-60分钟。一些实施方案中,所述第三次离心约为12000-20000g离心约20-40分钟。一些实施方案中,所述第四次离心约为10000-30000g离心约15-60分钟。一些实施方案中,所述第四次离心约为12000-25000g离心约20-60分钟。一些实施方案中,所述第四次离心约为12000-20000g离心约20-40分钟。
在一些实施方案中,重复步骤(1)、(2)和(3)2次以上。
在一些实施方案中,步骤(1)中,所述装置中的温度约为20℃-50℃,所述负压的值约为-0.1Mpa~-0.01Mpa。在一些实施方案中,所述负压的值约为-0.1Mpa~-0.02Mpa。在一些实施方案中,所述负压的值约为-0.1Mpa~-0.06Mpa。在一些实施方案中,所述装置中的温度约为25℃-40℃。在一些实施方案中,所述装置中的温度约为25℃-37℃。在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–50h。在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–40h。在一些实施方案中,步骤(2)中,将细胞放置于所述的装置中培养约6h–24h。
在一些实施方案中,所述的囊泡为诱导性囊泡。
在一些实施方案中,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡。在一些实施方案中,所述细胞包括干细胞、体细胞或肿瘤细胞。在一些实施方案中,所述干细胞包括全能干细胞或多能干细胞。在一些实施方案中,所述干细胞包括间充质干细胞或诱导性多能干细胞。在一些实施方案中,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱。在一些实施方案中,所述的体细胞包括Jurkat、红细胞或PBMC。
在一些实施方案中,本公开提供了一种囊泡或上述方法产的囊泡,所述囊泡具有标志物CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin或careticulin、cleaved caspase 3、Lamin B1、VDAC2、piezo1或active-β-catenin。
在一些实施方案中,所述囊泡低表达标志物CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin、careticulin、cleaved caspase 3、Lamin B1或VDAC2。在一些实施方案中,所述囊泡中的CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin、careticulin、cleaved caspase 3、Lamin B1、或VDAC2的表达量低于STS诱导同种细胞获得的囊泡。在一些实施方案中,所述careticulin在Western blotting检测中显示为被切割的两条条带。在一些实施方案中,所述囊泡高表达piezo1或active-β-catenin。在一些实施方案中,所述囊泡中piezo1或active-β-catenin的表达量高于STS诱导同种细胞获得的囊泡。
在一些标志物表达或者的机制研究中,发明人研究发现,正压力诱导产的囊泡与星形孢菌素诱导产的囊泡产生过程相似,均是细
胞凋亡过程中产生的,而UMSC-NP-EV可能是一种新型独特的囊泡。
在一些实施方案中,发明人研究发现,细胞在正压力作用下通过细胞凋亡走向死亡,而负压力作用下细胞可能通过其他细胞死亡方式走向死亡。在一些实施方案中,研究发现,负压诱导细胞通过溶酶体依赖性细胞死亡。
一些实施方案中,本公开提供了一种所述的囊泡在脂肪调节、或成骨分化方面的用途;或者在制备脂肪调节剂或成骨分化剂方面的用途。一些实施方案中,所述的脂肪调节为抑制成脂。一些实施方案中,所述的囊泡在在制备用于治疗疾病、抗衰老、促进皮肤机能、和/或非治疗目的的美容的产品中的应用。一些实施方案中,本公开提供了一种治疗疾病、抗衰老、促进皮肤机能和美容的方法,所述方法包括给予患者或者受试者所述的囊泡。
一些实施方案中,所述治疗疾病包括促进伤口愈合。一些实施方案中,本公开了提供了一种治疗伤口的方法,所述方法包括给予患者或者受试者所述囊泡。
本公开还包含以下项:
项1,一种囊泡的制备方法,将机械压力施加至细胞诱导细胞死亡,从而产生所述的囊泡。
项2,如项1所述的方法,所述机械压力包括正压力或负压力。一些实施方案中,所述负压力的大小约为-0.1~-0.005Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.01Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.02Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.03Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.05Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.06Mpa;一些实施方案中,所述负压力的大小约为-0.1~-0.07Mpa;一些实施方案中,所述正压力的大小约为2~6g/cm2;一些实施方案中,所述正压力的大小约为2~5g/cm2;一些实施方案中,所述正压力的大小约为2~4g/cm2。
项3,如项1或2所述的方法,所述囊泡是细胞处于正常存活时将机械压力施加至细胞诱导细胞死亡而产生的囊泡。一些实施方案中,所述细胞包括干细胞、体细胞或肿瘤细胞;一些实施方案中,所述干细胞包括全能干细胞或多能干细胞;一些实施方案中,所述干细胞包括间充质干细胞或诱导性多能干细胞;一些实施方案中,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱;一些实施方案中,所述的体细胞包括Jurkat、PBMC或红细胞。
项4,如项2或3所述的方法,所述负压力通过负压培养箱达到;一些实施方案中,所述正压力通过放置玻璃片于细胞上,然后将载有重物的容器置于玻璃片上培养细胞对细胞施加压力达到;一些实施方案中,所述重物包括钢珠;一些实施方案中,所述玻璃片包括石英玻璃片。
项5,如项1-4任一所述的方法,所述诱导细胞死亡的时间约为3~72小时;所述诱导细胞死亡的时间约为3~50小时;一些实施方案中,所述诱导细胞死亡的时间约为3-48小时;一些实施方案中,所述诱导细胞死亡的时间约为3-24小时;一些实施方案中,所述机械压力为负压力时,所述诱导细胞死亡的时间约为3-40小时;一些实施方案中,所述机械压力为负压力时,所述诱导细胞死亡的时间约为3-30小时;一些实施方案中,所述机械压力为负压力时,所述诱导细胞死亡的时间约为3-24小时;一些实施方案中,所述机械压力为负压力时,所述诱导细胞死亡的时间约为5-24小时。一些实施方案中,诱导细胞产囊泡的温度约为20℃-50℃;一些实施方案中,诱导细胞产囊泡的温度约为25℃-40℃;一些实施方案中,诱导细胞产囊泡的温度约为25℃-37℃。
项6,项1-5任一所述的方法获得的囊泡。一些实施方案中,所述囊泡表达标志物CD63、TSG101、ALIX、syntaxin 4、Annexin V、cleaved caspase 3、Lamin B1、intergrin α5、VDAC2、calnexin、careticulin。一些实施方案中,所述负压诱导的囊泡低表达CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin、careticulin,其中careticulin表现为被切割的两条条带。项7,如项6所述的囊泡,所述囊泡的直径约为0.05-0.4μm。一些实施方案中,所述囊泡的直径约为0.05-0.38μm;一些实施方案中,所述囊泡的直径约为0.05~0.35μm;一些实施方案中,所述囊泡的直径约为0.05~0.32μm;一些实施方案中,所述囊泡的直径约为0.05~0.3μm;一些实施方案中,所述囊泡的直径约为0.05~0.25μm;一些实施方案中,所述囊泡的直径约为0.05~0.22μm;一些实施方案中,所述囊泡直径约为0.55~0.22μm。
项9,一种组合物,含有如项6-8任一所述的囊泡。一些实施方案中,所述组合物为分化培养基;一些实施方案中,所述的组合物为成骨分化培养基。
项10,如项6-8任一所述的囊泡在脂肪调节、或成骨分化方面的用途;或者在制备脂肪调节剂或成骨分化剂方面的用途;一些实施方案中,所述的脂肪调节为抑制成脂。
图1为实施例1的负压培养箱的示意图。
图2为囊泡收集的流程图。
图3为负压培养箱诱导UMSCs后光镜下观察UMSCs形态改变图。
图4为负压培养箱诱导UMSCs后超高分辨率显微镜观察UMSCs产囊泡过程的动态变化图。
图5为负压培养箱诱导UMSCs后电镜下观察细胞形态图。
图6为负压培养箱诱导UMSCs后生成的ApoV负染透射电镜图。
图7为通过Zeta View对UMSCs负压培养箱不同压力诱导后囊泡的数量、粒径、电位进行分析的结果。
图8为通过Zeta View对UMSCs负压培养箱不同温度诱导后囊泡的数量、粒径、电位进行分析的结果。
图9为Jurkat/PBMC负压培养箱诱导后光镜下观察Jurkat/PBMC形态改变图。
图10为通过Zeta View对Jurkat/PBMC在负压力下产的ApoV的数量、粒径、电位进行分析的结果。
图11为超高分辨率显微镜观察RBC经负压培养箱诱导后产囊泡过程的形态变化图。
图12为Zeta View对RBC在负压力下产的ApoV的数量、粒径、电位进行分析结果。
图13为第一次负压诱导和STS诱导UMSCs前后,光镜下观察UMSCs形态改变图。
图14为第二次负压诱导和STS诱导前后,光镜下观察UMSCs形态改变图。
图15为负压第二次诱导后继续培养细胞,光镜下观察UMSCs形态改变图。
图16为Zeta View对UMSC在负压力下产的ApoV的数量、粒径、电位进行分析结果。
图17为UMSC囊泡对MSCs干性的影响。(A)茜素红染色实验结果显示,UMSC囊泡处理的MSCs矿化结节形成能力显著增强(n=3)。(B)油红O染色(n=3)显示,经UMSC囊泡处理的MSCs在成脂诱导培养条件下分化成脂肪细胞的能力显著降低。(C)Western blot结果显示UMSC囊泡处理的MSCs中成骨标志物Runx2和ALP表达上调。成脂标志物PPAR-γ表达降低。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图18为UMSC囊泡促进小鼠皮肤伤口愈合。(A)WT小鼠等囊泡量处理后具有代表性的皮肤损伤区域宏观图像(n=3)。(B)小鼠皮肤组织块体外培养结果显示UMSC囊泡可以促进组织块细胞迁徙生长。(C)CCK8结果显示UMSC囊泡可以SMSC增殖。(D,E)活体动物成像结果显示UMSC囊泡局部注射伤口周围后荧光动态变化及组织器官分布。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图19为UMSC-NP-EV与UMSC-MF-EV表征的比较。(A)UMSC-NP-EV与UMSC-MF-EV的透射电镜(TEM)图像,比例尺:200nm。(B)Elyra 7 Lattice SIM拍摄的PKH26染料标记的UMSC-NP-EV与UMSC-MF-EV图像,比例尺:200nm。(C)NTA分析UMSC-NP-EV与UMSC-MF-EV的中位粒径。(D)NTA分析UMSC-NP-EV与UMSC-MF-EV的粒径分布。(E)单个UMSC细胞产生的EV数量。(F)UMSC-NP-EV与UMSC-MF-EV的Zeta电位。(G)BCA检测等蛋白量的UMSC细胞正负压力所产的EV的蛋白量。(H)BCA检测单个UMSC-NP-EV与UMSC-MF-EV的蛋白含量。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图20为UMSC囊泡的蛋白质谱分析。(A,B)火山图和聚类热图显示NP-EV组与STS-EV组的差异蛋白分布。(C-E)NP-EV组较STS-EV组上调蛋白的GO-C富集分析、GO-F和GO-P富集分析。
图21为UMSC囊泡的蛋白表达。Western blotting分析显示,UMSC囊泡表达部分凋亡囊泡的特异性生物标记物和力学相关功能分子。
图22为UMSC在正负压力的刺激下形态变化及凋亡率时程变化。(A)机械正压力、负压力处理后UMSC细胞死亡形态变化。(B-C)正负压力作用下UMSC的凋亡率变化情况。(D-E)凋亡抑制剂处理后,正负压力作用下UMSC的凋亡率变化情况。MF,mechanical force;NP,negative pressure。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图23为UMSC在正负压力的刺激下特异性死亡通路激活情况。(A-E)机械正压力、负压力处理后UMSC凋亡关键蛋白表达情况。(F)正负压力作用下UMSC的cleaved caspase3激活情况。比例尺:5μm。MF,mechanical force;NP,negative pressure。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图24为UMSC力学死亡与其他细胞死亡方式的比较。(A-G)机械正压力、负压力处理后UMSC细胞死亡过程中自噬、铁死亡、细胞坏死、细胞焦亡等蛋白的表达情况。(H-L)自噬抑制剂处理后UMSC负压力学死亡关键蛋白表达情况。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图25为UMSC负压力学死亡过程中LC3II的功能验证。(A-G)溶酶体功能抑制剂BafA1、CQ处理后,UMSC在负压力及rapamysin作用下自噬相关蛋白的表达情况。(H)溶酶体功能抑制剂BafA1、CQ处理后,UMSC在负压力及rapamysin作用下分泌囊泡量的变化。(I-J)siRNA敲低LC3II后,rapamysin诱导自噬组胞外囊泡水平的情况。(K)凋亡抑制剂处理细胞后,正负压力诱导细胞生产囊泡的量的变化情况。*代表与对照组相比,差异有统计学意义,P<0.05。ns代表与对照组相比,差异没有统计学意义,P>0.05。
图26为负压力诱导UMSC细胞死亡的功能验证。(A)流式细胞术检测不同抑制剂处理后细胞凋亡率的变化。(B)热图结果显示不同抑制剂处理后细胞凋亡率的变化。绿色颜色越明显,代表活细胞越多,凋亡细胞越少;红色颜色越明显,代表活细胞越少,凋亡细胞越多。(C)折线图结果显示不同抑制剂处理后细胞凋亡率的变化。
图27为机械正压力加力装置。
附图中部件名称对应的标号如下:
箱体1;压力传感器2;温度传感器3;控制器4;加热器5;真空泵6。
箱体1;压力传感器2;温度传感器3;控制器4;加热器5;真空泵6。
以下通过具体的实施例进一步说明本公开的技术方案,具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
在本公开实施例的描述中,还需要说明的是,除非另有明确的规定和限定,术语“设置”、“连接”或“相连”应做广义理解,例如,可以是固定连接,也可以是可拆卸连接,或一体地连接;可以是机械连接,也可以是电连接;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通。对于本领域的普通技术人员而言,可以具体情况理解上述术语在本公开中的具体含义。
本文中,ApoV指的是“囊泡”,也可以称为IEVs,为细胞外囊泡(即EV)的一种。
本文实施例中,所使用的脐带间充质干细胞为P4-P8代的人脐带间充质干细胞;以下实施例具体使用的为P4代细胞。
以下实施例中,实验使用的小鼠骨髓间充质干细胞为P3代细胞。
本文实施例中,各组实验中,如若未指明具体的温度的话,温度为37℃。
除非另有定义,本文所用的所有技术和科学术语具有与本领域(例如,在细胞培养、分子遗传学、核酸化学、杂交技术和生物化学中)普通技术人员通常理解的相同的含义。
本文使用的术语“包括”、“具有”、“含有”和“包含”以及其他类似的形式,以及其语法上的等同形式,其含义是等同的,并且是开放式的,即这些词语中的任何一个后面的一个或多个项并不意味着是对该项或多个项的详尽列举,也不意味着只限于列出的项或多个项。例如,“包括”组分A、B、和C的物可以由组分A、B、和C组成(即,仅含有组分A、B、和C),或者可以不仅含有组分A、B、和C,而且可以包括一或更多个其它组分。因此,意图并理解的是,“包括”及其类似形式及其语法等同形式包含“基本上由……组成”或“由……组成”的实施例的公开。
统计分析:所有实验数据以均数±标准差表示,使用GraphPad Prism 8.0软件分析。采用独立非配对双尾学生t检验分析两组之间的比较,采用单因素方差分析(one-way ANOVA)和Bonferroni处理两组以上的比较分析。检验水准为双侧α=0.05,P<0.05差异有统计学意义。
本研究及相关实验使用的细胞均经医学伦理委员会批准。
提取小鼠骨髓间充质干细胞(1)准备3-5周WT小鼠,脱颈猝死;(2)取小鼠两侧股骨和胫骨,去除肌肉组织后放于冰PBS中;(3)将骨头用冰PBS涮洗2次,使用10ml注射器吸取PBS配制的1%BSA冲洗骨髓,并用70μm的滤网过筛;(4)将细胞悬液离心,1500RPM×4℃×5min,收集细胞沉淀,吸取1ml完全培养基重悬细胞;(5)细胞计数,取1×106细胞接种,转移至细胞培养箱。
皮肤组织块体外培养实验:(1)取7周WT小鼠,脱颈猝死,背部剃毛并消毒;
(2)取小鼠背部全层皮肤组织,用75%酒精清洗2次,PBS清洗2次,将皮肤切成0.5cm3的组织块;(3)把消毒后的皮肤组织块贴在培养皿表面,周围轻轻滴加少许培养基,转移至培养箱2-3h;(4)待皮肤组织块与培养皿贴合牢固,在培养皿中继续加入培养基,使组织块完全浸入培养基。(5)用倒置荧光显微拍照记录周围MSC生长情况,每间隔6h观察一次。
本文中,“PBMC-NP-EV”指的是负压(NP)诱导PBMC产的囊泡;“BMSC-NP-EV”指的是负压(NP)诱导BMSC产的囊泡;“UMSC-MF-EV”指的是正压(MF)诱导UMSC产生的囊泡;“UMSC-STS-EV”指的是STS诱导UMSC产的囊泡;其他的依此类推。
本文实施例中,如无特别说明,则按照以下方式进行:
(1)机械正压力(mechanical force,MF)诱导细胞死亡(图27):将细胞接种到六孔板中,当细胞密度达到90%左右时,将完全培养基吸出,PBS洗涤2次,每孔加入1mLα-MEM,于细胞上方放置一块石英玻璃圆片,玻璃片上继续放置一定数量的不锈钢球使压力达到4g/cm2。观察细胞收缩,失粘附,质膜气泡时即确定细胞死亡;24/48/72h后取出压在细胞上的装置,然后收集含有死亡UMSCs的培养基于离心管中。
(2)星形孢菌素(Staurosporine,STS)诱导细胞凋亡:待细胞密度达到80%时,PBS洗涤2次,500nM星形孢菌素(STS)诱导细胞凋亡,观察细胞形态变化,细胞收缩,与周围细胞连接消失,质膜气泡,核质浓缩等典型的细胞凋亡形态改变时确定细胞发生凋亡;
(3)负压诱导细胞方法同实施例2;
(4)提取囊泡的方式同实施例7。
实施例1负压培养箱
本文所述的负压培养箱相对于传统的二氧化碳细胞培养箱增加了温度模块和负压模块,能够调节培养箱内的压力(如负压)和温度。如图1所示,所述的负压培养箱包括箱体1,箱体1内设置有压力传感器2,温度传感器3,控制器4,加热器5和真空泵6;所述控制器4分别与温度传感器3、压力传感器2连接;所述控制器4通过压力传感器2控制所述真空泵6的产生负压;所述控制器4通
过温度传感器3控制加热器5的工作。
根据培养需要,设置培养箱培养参数为:①温度:25℃-50℃;②负压值:-0.1Mpa~-0.01Mpa;③诱导时间:6h–50h。
其中负压培养箱中的气体成分为空气。
实施例2体外负压力诱导脐带间充质干细胞产囊泡
1、实验方法
(1)将脐带间充质干细胞(umbilical cord mesenchymal stem cells,UMSCs)接种于10cm培养皿里,加入含10%FBS的MEM-ALPHA培养基,放置在常规的二氧化碳培养箱中培养。待细胞长至90-95%时,实验组更换为不含血清的MEM-ALPHA培养基培养,然后置于负压培养箱中(图1);对照组更换为含有500nM星形孢菌素(Staurosporine,STS)、不含血清的MEM-ALPHA培养基,然后置于常规的二氧化碳培养箱中培养。
(2)负压诱导;
(3)囊泡收集:待细胞诱导培养后,收集UMSCs培养基于离心管中,将上述获得的含有囊泡的培养基进行差速离心(流程如图2所示):800g离心10min弃去细胞沉淀,收集上清,2000g离心5min后弃去细胞碎片等沉淀,收集上清,16000g离心30min后收集沉淀即为凋亡囊泡(apoptotic vesicle,apoV),然后用1ml PBS重悬ApoV后16000g离心30min后收集沉淀,PBS重悬apoV后4℃保存,用于后续鉴定分析;
2、实验结果
2.1 MSCs经负压培养箱诱导后细胞形态
将UMSCs置于-0.08Mpa的负压培养箱中37℃培养20h后,光镜下观察UMSCs形态改变(图3);通过细胞染料标记细胞膜、细胞核,然后超高分辨率显微镜观察UMSCs产囊泡过程的动态变化(图4)。电镜下观察细胞形态(图5)。
2.2 UMSCs经负压培养箱诱导后生成的ApoV的鉴定分析
囊泡负染透射电镜图(图6);通过Zeta View对UMSCs负压培养箱不同压力诱导后囊泡的数量、粒径、电位进行分析(图7);通过Zeta View对UMSCs负压培养箱不同温度诱导后囊泡的数量、粒径、电位进行分析(图8)。
其中,图6为实验组的囊泡负染透射电镜图,所述的囊泡来源于经负压诱导后的UMSCs:负压诱导条件为-0.08Mpa的负压培养箱中37℃培养20h后。
实施例3体外负压力诱导Jurkat细胞和单核细胞产囊泡
1、实验方法
(1)将悬浮细胞Jurkat细胞接种于10cm培养皿里,使用含10%FBS的1640培养基在常规的二氧化碳培养箱中培养,待细胞长至90-95%时,更换为不含血清的1640培养基培养(实验组),和含有500nM STS、不含血清的1640培养基(对照组);实验组置于负压培养箱中培养(图1),对照组置于常规的二氧化碳培养箱中培养。
将分离得到的人外周血来源的单核细胞(peripheral blood mononuclear cell,PBMC)直接接种于10cm培养皿里,分别用不含血清的1640培养基培养(实验组),和含有500nM STS、不含血清的1640培养基培养(对照组);实验组置于负压培养箱中(图1),对照组置于常规的二氧化碳培养箱中培养。
(2)负压诱导;
(3)囊泡收集:待细胞诱导培养后,收集细胞培养基于离心管中,将上述获得的含有囊泡的培养基进行差速离心(流程如图2所示):800g离心10min弃去细胞沉淀,收集上清,2000g离心5min后弃去细胞碎片等沉淀,收集上清,16000g离心30min后收集沉淀即为凋亡囊泡(apoptotic vesicle,apoV),然后用1ml PBS重悬ApoV后16000g离心30min后收集沉淀,PBS重悬apoV后4℃保存,用于后续鉴定分析;
2、实验结果
2.1 Jurkat/PBMC经负压培养箱诱导后的形态
光镜下观察Jurkat/PBMC形态改变(图9)。
2.2 Jurkat/PBMC负压培养箱诱导后生成的ApoV的鉴定分析
通过Zeta View对Jurkat/PBMC在负压力下产的ApoV的数量、粒径、电位进行分析(图10)。
实施例4体外负压力诱导红细胞产囊泡
1、实验方法
(1)将人来源的红细胞(red blood cell,RBC)接种于10cm培养皿中,于不含血清的1640培养基中培养(实验组),和在红细胞裂解液(中国CWBIO)中培养4h(对照组);实验组置于负压培养箱中(图1),对照组置于常规的二氧化碳培养箱中培养。
(2)负压诱导;
(3)囊泡收集:待细胞在诱导培养后,收集细胞培养基于离心管中,将上述获得的含有囊泡的培养基进行差速离心(流程如图2
所示):800g离心10min弃去细胞沉淀,收集上清,2000g离心5min后弃去细胞碎片等沉淀,收集上清,16000g离心30min后收集沉淀即为凋亡囊泡(apoptotic vesicle,apoV),然后用1ml PBS重悬ApoV后16000g离心30min后收集沉淀,PBS重悬apoV后4℃保存,用于后续鉴定分析。
2、实验结果
2.1 RBC经负压培养箱诱导后的形态
将RBC置于-0.08Mpa的负压培养箱中37℃培养48h后,通过细胞染料标记细胞膜,然后超高分辨率显微镜观察RBC产囊泡过程的形态变化(图11)。
2.2 RBC负压培养箱诱导后生成的ApoV的鉴定分析
通过Zeta View对RBC在负压力下产的ApoV的数量、粒径、电位进行分析(图12)。
实施例5连续负压诱导UMSCs的细胞形态观察
1、实验方法
(1)第一次诱导
将脐带间充质干细胞(umbilical cord mesenchymal stem cells,UMSCs)接种于10cm培养皿里,使用含10%FBS的MEM ALPHA培养基在常规的二氧化碳培养箱中培养,待细胞长至90-95%时,更换为不含血清的MEM-ALPHA培养基培养(实验组),和含有500nM STS、不含血清的MEM-ALPHA培养基(对照组);实验组置于负压培养箱中(图1)-0.06Mpa,37℃诱导12h后,收集囊泡;对照组置于常规的二氧化碳培养箱中培养,收集囊泡。
收集囊泡的方法同实施例2。
(2)第二次诱导
向第一次诱导后培养皿中剩下的细胞中加入含10%FBS的MEM ALPHA培养基后放置在常规的二氧化碳培养箱中培养。然后更换为不含血清的MEM-ALPHA培养基培养(实验组),和含有500nM STS、不含血清的MEM-ALPHA培养基(对照组);实验组再次置于负压培养箱中,对照组再次置于常规的二氧化碳培养箱中培养。重复以上第一次诱导的细胞培养、负压诱导、囊泡收集步骤,直至实验结束。
2、实验结果
第一次负压诱导12h后光镜下观察UMSCs形态改变(图13),其中图13中的STS组为诱导时间7小时得到的结果图。
第一次诱导后重复培养2d后光镜下观察UMSCs形态改变,可见对照组STS诱导的UMSCs诱导一次后细胞重复培养无法恢复正常形态,细胞数量减少,说明细胞已经死亡;而负压诱导组的细胞重复培养后可以继续长满培养皿,形态恢复正常(图14左下图)。
第二次负压诱导12h和STS诱导7h后,可见STS诱导的UMSCs数量进一步减少,形态无明显变化,进一步证实细胞死亡,之后不再重复培养(图14右上图);而第二次负压诱导后,其变化与第一次诱导基本相同(图14右下图)。再次重复培养4d后光镜下观察UMSCs形态改变,可见负压诱导的细胞重复培养后可以继续长满培养皿,部分细胞体积变大(图15)。
实施例6连续负压诱导UMSCs后生成的ApoV的鉴定分析
1、实验方法
区别于过往利用STS等化学药物诱导干细胞不可逆性死亡,本装置可以通过合理设置不同的负压和温度诱导时间,达到重复性多次收集UMSCs产的囊泡的目的。具体程序设计如下:
(1)将脐带间充质干细胞(umbilical cord mesenchymal stem cells,UMSCs)接种于10cm培养皿里,待细胞长至90-95%时,更换为不含血清的MEM-ALPHA培养基培养(实验组),和含有500nM星型孢菌素(Staurosporine,STS)、不含血清的MEM-ALPHA培养基(对照组),然后实验组置于负压培养箱中(如图1);对照组置于常规的二氧化碳培养箱中培养,收集囊泡。
(2)负压诱导:-0.06Mpa-37℃-12h;
(3)囊泡收集:待细胞诱导培养后,收集UMSCs培养基于离心管中,将上述获得的含有囊泡的培养基进行差速离心(流程如图2所示):800g离心10min弃去细胞沉淀,收集上清,2000g离心5min后弃去细胞碎片等沉淀,收集上清,16000g离心30min后收集沉淀即为凋亡囊泡(apoptotic vesicle,apoV),然后用1ml PBS重悬ApoV后16000g离心30min后收集沉淀,PBS重悬apoV后4℃保存,用于后续鉴定分析;
(4)细胞继续培养:将步骤(3)中被收集培养基后剩下的UMSCs添加含有10%FBS的MEM-ALPHA培养基培养继续48-96h后,待细胞长至90-95%时,更换为不含血清的MEM-ALPHA培养基置于负压培养箱中,重复(2)的诱导程序诱导;
(5)重复(2)、(3)、(4)的步骤。
2、实验结果
分别收集上述诱导产生的囊泡,通过Zeta View对UMSCs负压培养箱诱导后囊泡的数量、粒径、电位进行分析(图16),可见STS诱导产生的囊泡数量较少,而负压诱导的囊泡产量大,又可重复培养诱导多次,产生的囊泡的电位、粒径基本保持一致,产的囊
泡的数量远远多于STS诱导产生的囊泡,两者完全不是一个数量级的。
图16中,STS-7h-1、STS-7h-1分别指的是STS(诱导7小时)诱导第1次和诱导第2次;同理,-0.06Mpa-37℃-12h-1、-0.06Mpa-37℃-12h-2、-0.06Mpa-37℃-12h-3分别指的是负压诱导第1次、第2次和第3次。图16中纵坐标apoV Nb的指的是apoV的数量。
实施例7产UMSC正负压力来源的囊泡的体外功能鉴定
为了进一步鉴定UMSC囊泡的功能,使用囊泡与UMSC共培养,并通过成骨、成脂诱导鉴定其对MSC干性的影响。茜素红结果显示UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著促进UMSC成骨(图17A)。油红O结果显示UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著抑制UMSC成脂(图17B)。Western blotting结果显示UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著提高成骨相关蛋白ALP、RUNX2的表达,更显著抑制成脂相关蛋白PPAR-γ(图17C)。以上结果提示UMSC囊泡可能具有更好的提升MSC干性的潜力,更适合用于临床转化治疗。
本实施例中,STS浓度500nM,诱导时间8h;MF压力值4g/cm2,诱导时间24h;NP压力值-60Kpa,诱导时间20h。
实施例8 UMSC正负压力来源的囊泡的体内功能验证
为了进一步验证UMSC囊泡的体内功能,使用等量的囊泡局部注射到小鼠皮肤伤口周围,方法包括:(1)气麻机麻醉小鼠,背部剃毛并消毒。(2)在小鼠背部皮肤剪去1.8cm×1.8cm的全厚方形皮肤创面。(3)随机将小鼠分为4组:PBS组、STS-EV组、MF-EV组和NP-EV组。(4)手术后第0、3和6天,用100μL PBS加8μL肝素重悬EV,以每只小鼠1.6×108EV局部给药。(5)局部注射EV后第0、8、10、12和14天,拍摄图片并使用Image-Pro Plus软件量化伤口愈合。
结果显示14d后,UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著地促进小鼠皮肤伤口愈合(图18A)。进一步提取小鼠的皮肤组织块,通过与囊泡共培养鉴定其促进皮肤细胞生长的能力。结果显示UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著地促进皮肤组织块周围的细胞生长迁徙(图18B)。CCK8结果进一步证实UMSC-MF-EV与UMSC-NP-EV较UMSC-STS-EV更显著地提高SMSC细胞活性(图18C)。为了进一步分析UMSC囊泡在体内的分布,使用DIR标记的囊泡注射到皮肤伤口周围,通过活体动物成像设备进行观察囊泡的分布规律。结果显示UMSC-MF-EV、UMSC-NP-EV、UMSC-STS-EV三者均可以富集到小鼠伤口区域,1d-3d达到高峰,之后逐渐降低(图18D)。一周后将小鼠出死后分离各组织脏器,发现UMSC-MF-EV、UMSC-NP-EV、UMSC-STS-EV三者均可以富集到心、肝、肺、肾、骨,富集到脾脏组织较少(图18E)。以上结果提示UMSC囊泡可能具有较好的促进组织损伤愈合的能力。
本实施例中,STS浓度500nM,诱导时间8h;MF压力值4g/cm2,诱导时间24h;NP压力值-60Kpa,诱导时间20h。
实施例9 UMSC正负压力来源的囊泡的表征鉴定
为了探究UMSC在正负压力下生成囊泡的特征,通过差速离心法分离获得UMSC囊泡。
透射电镜和超高分辨率显微镜结果显示UMSC-MF-EV与UMSC-NP-EV均表现为单层膜的典型囊泡结构,并含有一定的核物质(图19A,图19B)。NTA分析结果显示UMSC-NP-EV直径略大于UMSC-MF-EV,产量高一倍左右,电位无明显差异(图19C-图19F)。
BCA蛋白定量结果显示相同质量细胞来源的UMSC-NP-EV蛋白含量较UMSC-MF-EV高,但单个UMSC-NP-EV蛋白含量较UMSC-MF-EV低(图19G-图19H)。提示负压力诱导细胞生成的囊泡可能更加高效,可能更适合用于临床转化应用。
为了进一步区分UMSC-NP-EV与UMSC-STS-EV的内含物差异,使用DIA蛋白质谱对二者进行鉴定。火山图和聚类热图结果显示UMSC-NP-EV较UMSC-STS-EV高表达的蛋白有1789个(图20A-20B)。GO-C富集分析结果显示UMSC-NP-EV较UMSC-STS-EV高表达的1789个蛋白主要集中在线粒体、核糖体、线粒体内膜、线粒体基质、线粒体大核糖体亚基、核仁、胞质大核糖体亚基、线粒体类核、膜的整体组件、糖核蛋白复合物、内质网等细胞成分方面(图20C)。
GO-F富集分析结果显示1789个蛋白主要集中在核糖体结构成分、RNA结合、核糖核酸结合、质子转运ATP合酶活性、核小体DNA结合、氨基酷-tRNA连接酶活性、核小体结合、核糖核蛋白复合物结合、SNAP受体活性等分子功能方面(图20D)。GO-P富集分析结果显示1789个蛋白主要集中在翻译、线粒体翻译延伸、线粒体翻译终止、SRP依赖的共翻译蛋白、病毒转录、翻译起始、核转录、mRNA分解代谢过程、RNA加工、线粒体翻译、RNA剪接、mRNA加工等生物过程方面(图20E)。
为了进一步鉴定UMSC-STS-EV、UMSC-MF-EV、UMSC-NP-EV的蛋白表达特征,使用Western blotting技术进行鉴定。
结果显示UMSC-MF-EV与UMSC-STS-EV蛋白表达模式基本相同,高表达囊泡共同标志物CD63、TSG101、ALIX、syntaxin 4和凋亡囊泡标志物Annexin V、cleaved caspase 3、Lamin B1、intergrin α5、VDAC2、calnexin、careticulin(图21)。此外UMSC-MF-EV较UMSC-STS-EV高表达力学相关蛋白piezo1。UMSC-NP-EV低表达囊泡共同标志物CD63、TSG101、ALIX、syntaxin 4和凋亡囊泡标志物Annexin V、intergrin α5、calnexin、careticulin,但careticulin表现为被切割的两条条带;UMSC-NP-EV低表达凋亡囊泡标志物cleaved caspase 3、Lamin B1、VDAC2,高表达力学相关蛋白piezo1和active-β-catenin(图21)。以上结果提示UMSC-MF-EV可能是与UMSC-STS-EV产生过程相似,均是细胞凋亡过程中产生的,而UMSC-NP-EV可能是一种新型独特的囊泡。
本实施例中,STS浓度500nM,诱导时间8h;MF压力值4g/cm2,诱导时间24h;NP压力值-60Kpa,诱导时间20h。
实施例10机制研究
获得UMSC囊泡的方法同实施例8。
1、UMSC在负压力作用下表现为不依赖caspase的细胞死亡方式
为了探究UMSC在正负压力作用下形态变化,首先用CellMask标记细胞膜,hochest标记细胞核后,使用高分辨活细胞成像系统及超高分辨率显微镜拍摄了UMSC在正负压力作用下的死亡形态变化(图22A-22E)。结果显示,机械正压力处理过程中,UMSC发生一系列的典型细胞凋亡形态变化,包括细胞膜和细胞质收缩、细胞膜出泡和核皱缩等;负压力作用下,UMSC表现为细胞膜和细胞质、细胞核收缩,细胞呈“花环状”剧烈出泡死亡(图22A)。
为了进一步比较UMSC在正负压力作用下细胞死亡速率,使用流式细胞术检测不同时间点的细胞凋亡率的变化。结果显示,在力学作用早期(0h-6h),正负压力作用下UMSC凋亡率无明显差异;在力学作用中晚期(6h-24h),负压力作用下UMSC死亡速率高于正压处理(图22B,图22C)。
接下来为了比较UMSC在正负压力作用下是否是通过细胞凋亡途径走向死亡,使用细胞凋亡抑制剂Z-VAD处理后进行流式细胞术检测。结果显示Z-VAD处理后,正压力诱导UMSC死亡的速率减慢,总凋亡率减少;而负压力诱导UMSC死亡的速率无明显改变(图22D,图22E)。提示UMSC在正压力作用下通过细胞凋亡走向死亡,而负压力作用下UMSC可能通过其他细胞死亡方式走向死亡。
为了研究UMSC在正负压力作用下细胞凋亡通路关键蛋白的表达情况,使用Western blotting和细胞免疫荧光进行检测。Western blotting结果显示,机械正压力处理24h后,UMSC中凋亡关键执行蛋白cleaved caspase3、cleaved caspase8、cleaved caspase9高度表达,凋亡作用底物PARP被大量切割。负压力处理24后,UMSC中凋亡关键执行蛋白cleaved caspase3、cleaved caspase8、cleaved caspase9表达较弱,凋亡作用底物PARP被切割(图23A-图23E)。细胞免疫荧光结果显示,正压力作用下,UMSC中cleaved caspase3高度表达;负压力作用下,UMSC中cleaved caspase3基本不活化(图23F)。提示UMSC在正压力作用下表现为经典的细胞死亡方式,而负压力作用下UMSC表区别于经典细胞凋亡方式,可能是其他细胞死亡方式。
2、UMSC负压力学死亡与其他细胞死亡方式的鉴别
为了探究UMSC在负压力作用下的细胞死亡方式,进一步收集UMSC力学死亡蛋白通过western blotting技术检测细胞自噬、铁死亡、细胞坏死、细胞焦亡等关键蛋白表达情况。结果显示UMSC负压力学死亡过程中高表达自噬标志蛋白LC3II,但自噬启动关键蛋白BECN1及铁死亡标志蛋白GPX4、COX2、细胞坏死标志蛋白RIP3、细胞焦亡标志蛋白GSDMD表达较低(图24A-图24G)。提示LC3II可能参与了UMSC负压力学死亡过程,并发挥关键作用。
为了进一步鉴定UMSC负压力学死亡与细胞自噬的区别,使用rapamysin诱导的自噬与STS诱导的细胞凋亡作为对照组,使用自噬启动过程抑制剂3MA以及溶酶体功能抑制剂BafA1处理后进行负压处理。Western blotting结果显示3MA和BafA1作用下LC3II仍高表达,而溶酶体膜蛋白LAMP1表达降低,LAMP2A表达升高并被高度切割(图24H-图24L)。提示UMSC负压力学死亡过程可能是LC3II参与的非经典自噬过程,并且可能与溶酶体有关。
为了进一步验证LC3II参与UMSC负压囊泡形成的功能,使用溶酶体功能抑制剂BafA1和CQ处理细胞后,再给予负压和rapamysin处理,通过差速离心的方式提取胞外囊泡。结果显示BafA1和CQ处理细胞后,rapamysin诱导的自噬组LC3II、P62、Cathepsin B表达量升高,而负压组则表现为LC3II降低,P62升高,Cathepsin B表达恢复至正常细胞水平(图25A-图25G)。
此外,BafA1和CQ处理细胞后,rapamysin诱导的自噬组胞外囊泡水平升高,负压组胞外囊泡量减少(图25H)。
进一步使用siRNA敲低LC3II后,rapamysin诱导的自噬组胞外囊泡水平大量降低,而负压组胞外囊泡量降低1/2左右(图25I-图25J)。提示在细胞自噬过程中溶酶体可能降解了自噬小体,减少了自噬过程中胞外囊泡的分泌,而在细胞负压死亡过程中LC3II与溶酶体参与了负压囊泡的形成。
我们进一步使用凋亡抑制剂处理细胞后,发现正压力诱导细胞生产囊泡的量大量降低,而负压组无明显变化(图25K)。提示凋亡抑制剂可以抑制正压力诱导生成的囊泡。
3、负压力诱导UMSC溶酶体依赖性细胞死亡的验证
为了进一步验证负压力诱导UMSC溶酶体依赖性细胞死亡的机制,使用钙离子拮抗剂BAPTA-AM/EGTA-AM、calpain抑制剂PD150606、组织蛋白酶B抑制剂CA-074、溶酶体丝氨酸蛋白酶和半胱氨酸蛋白酶抑制剂E64D/Leupeptin Hemisulfate处理后进行流式细胞凋亡率检测。结果表明BAPTA-AM/EGTA-AM可有效抑制负压力诱导的UMSC死亡,其次是E64D/Leupeptin Hemisulfate、PD150606,而Z-VAD、CA-074基本没有抑制作用(图26A-图26C)。提示抑制负压力诱导UMSC死亡的早期钙离子内流及calpain的活化、晚期释放的溶酶体蛋白水解酶功能可有效抑制UMSC溶酶体依赖性细胞死亡。
Claims (10)
- 一种产囊泡的方法,其中,包括以下步骤:(1)将负压施加至细胞诱导细胞产囊泡;(2)收集囊泡;(3)将步骤(2)中剩下的细胞进行继续培养;(4)再次实施如(1)和(2)的步骤;优选地,完成步骤(4)后,返回步骤(3)进行循环重复,所述的循环为一轮、两轮或以上;优选地,步骤(3)中,放置于二氧化碳培养箱中继续培养;优选地,步骤(1)中,所述负压的值约为-0.1Mpa~-0.01Mpa;优选地,所述负压的值为约为-0.1Mpa~-0.02Mpa;优选地,所述负压的值约为-0.1Mpa~-0.06Mpa;优选地,所述负压的值约为-0.08Mpa~-0.01Mpa;优选地,所述负压的值约为-0.07Mpa~-0.01Mpa;优选地,所述负压的值约为-0.06Mpa~-0.02Mpa;优选地,所述负压的值约为-0.04Mpa~-0.02Mpa;优选地,步骤(1)中,诱导细胞产囊泡的时间约为6h–50h;优选地,步骤(1)中,诱导细胞产囊泡的时间约为6h–40h;优选地,步骤(1)中,诱导细胞产囊泡的时间约为6h–24h;优选地,所述步骤(1)中,诱导细胞产囊泡的温度约为20℃-50℃;优选地,所述步骤(1)中,诱导细胞产囊泡的温度约为25℃-40℃;优选地,所述步骤(1)中,诱导细胞产囊泡的温度约为25℃-37℃;优选地,所述的囊泡为诱导性囊泡;优选地,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡;优选地,所述细胞包括干细胞、体细胞或肿瘤细胞;优选地,所述干细胞包括全能干细胞或多能干细胞;优选地,所述干细胞包括间充质干细胞或诱导性多能干细胞;优选地,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱;优选地,所述的体细胞包括Jurkat、红细胞或PBMC。
- 一种产囊泡的装置,其中,包含负压调节组件和温度调节组件。
- 如权利要求2所述的装置,其中,包括箱体,箱体内设置有压力传感器、温度传感器、加热器、真空泵、控制器;所述控制器分别与温度传感器、压力传感器连接。优选地,所述控制器通过压力传感器控制所述真空泵的工作;所述控制器通过所述温度传感器控制所述加热器的工作;优选地,所述装置为负压细胞培养箱。
- 一种产囊泡的系统,其中,包含如权利要求3所述的装置,还包括细胞培养箱。
- 权利要求3所述的装置的使用方法,其中,包括以下步骤:(1)通过所述控制器控制加热器和真空泵,使所述装置中的压力为负压;(2)将细胞放置于所述的装置中培养诱导细胞产囊泡;(3)收集囊泡;(4)将步骤(3)中剩下的细胞进行继续培养;(5)再次实施如(2)和(3)的步骤;优选地,完成步骤(5)之后,返回步骤(4)进行循环重复,所述的循环为一轮、两轮或以上;优选地,步骤(4)中,放置于二氧化碳培养箱中继续培养。
- 如权利要求5所述的方法,其中,所述步骤(1)中,所述装置中的温度为20℃-50℃,所述负压的值约为-0.1Mpa~-0.01Mpa;优选地,所述装置中的温度约为25℃-40℃;优选地,所述装置中的温度约为25℃-37℃;优选地,所述负压的值约为-0.1Mpa~-0.02Mpa;优选地,所述负压的值约为-0.1Mpa~-0.06Mpa;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–50h;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–40h;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–24h;所述的囊泡为诱导性囊泡;优选地,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡;优选地,所述细胞包括干细胞、体细胞或肿瘤细胞;优选地,所述干细胞包括全能干细胞或多能干细胞;优选地,所述干细胞包括间充质干细胞或诱导性多能干细胞;优选地,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱;优选地,所述的体细胞包括Jurkat、红细胞或PBMC。
- 一种产囊泡的方法,其中,所述方法使用了权利要求3-5任一所述的装置;优选地,所述方法包括以下步骤:(1)通过所述控制器控制所述加热器和所述真空泵,使所述装置中的压力为负压;(2)将细胞放置于所述的装置中培养,收集细胞培养上清,从所述细胞培养上清中获得囊泡;优选地,所述方法还包括以下步骤:(3)在所述步骤(2)获得所述囊泡之后,将所述的细胞放置于二氧化碳培养箱中继续培养;(4)重复所述步骤(1)和(2);优选地,重复步骤(1)、(2)和(3)两次以上。
- 如权利要求7所述的方法,其中,步骤(1)中,所述装置中的温度约为20℃-50℃,所述负压的值约为-0.1Mpa~-0.01Mpa;优选地,所述负压的值约为-0.1Mpa~-0.02Mpa;优选地,所述负压的值约为-0.1Mpa~-0.06Mpa;优选地,所述装置中的温度约为25℃-40℃;优选地,所述装置中的温度约为25℃-37℃;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–50h;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–40h;优选地,步骤(2)中,将细胞放置于所述的装置中培养约6h–24h。优选地,所述的囊泡为诱导性囊泡;优选地,所述囊泡是细胞处于正常存活时负压施加至细胞诱导细胞而产生的囊泡;优选地,所述细胞包括干细胞、体细胞或肿瘤细胞;优选地,所述干细胞包括全能干细胞或多能干细胞;优选地,所述干细胞包括间充质干细胞或诱导性多能干细胞;优选地,所述间充质干细胞来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或肌腱;优选地,所述的体细胞包括Jurkat、红细胞或PBMC。
- 权利要求1所述的方法或权利要求7-8任一所述的方法产的囊泡,其中,所述careticulin在Western blotting检测中显示为被切割的两条条带;所述囊泡具有标志物CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin、careticulin,cleaved caspase 3、Lamin B1、VDAC2、piezo1或active-β-catenin;优选地,所述囊泡低表达标志物CD63、TSG101、ALIX、syntaxin 4、Annexin V、intergrin α5、calnexin、careticulin,cleaved caspase 3、Lamin B1或VDAC2;优选地,所述囊泡中CD63、TSG101、ALIX、syntaxin 4,Annexin V、intergrin α5、calnexin、careticulin、cleaved caspase 3、Lamin B1或VDAC2的表达量低于STS诱导同种细胞获得的囊泡;优选地,所述囊泡高表达piezo1或active-β-catenin;优选地,所述囊泡中piezo1或active-β-catenin的表达量高于STS诱导同种细胞获得的囊泡。
- 权利要求9所述的囊泡在脂肪调节、或成骨分化方面的用途;或者在制备脂肪调节剂或成骨分化剂方面的用途;优选地,所述的脂肪调节为抑制成脂;优选地,所述的囊泡在在制备用于治疗疾病、抗衰老、促进皮肤机能、和/或非治疗目的的美容的产品中的应用;优选地,所述治疗疾病包括促进伤口愈合。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103333800A (zh) * | 2013-06-09 | 2013-10-02 | 中国人民解放军第四军医大学 | 一种体外细胞动静态正-负压加载实验系统及方法 |
US20140099649A1 (en) * | 2011-06-10 | 2014-04-10 | Hitachi Chemical Research Center, Inc. | Vesicle capturing devices and methods for using same |
KR101827291B1 (ko) * | 2016-12-12 | 2018-03-22 | (주)나비바이오텍 | 외부 환경 영향 배제가 가능한 세포 배양용 인큐베이터 |
CN107893050A (zh) * | 2017-10-17 | 2018-04-10 | 杜水果 | 一种细胞外囊泡及其制备方法和用途 |
KR20180118326A (ko) * | 2017-04-21 | 2018-10-31 | 고려대학교 산학협력단 | 미세 소포체의 분리 장치 및 방법 |
CN214004660U (zh) * | 2020-11-05 | 2021-08-20 | 甘肃中医药大学 | 一种正负压力可调型细胞培养箱 |
WO2023024637A1 (zh) * | 2021-08-26 | 2023-03-02 | 中山大学 | 一种力学性囊泡的制备方法 |
-
2023
- 2023-06-01 CN CN202310638877.2A patent/CN117165519A/zh active Pending
- 2023-06-01 WO PCT/CN2023/097692 patent/WO2023232099A1/zh unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140099649A1 (en) * | 2011-06-10 | 2014-04-10 | Hitachi Chemical Research Center, Inc. | Vesicle capturing devices and methods for using same |
CN103333800A (zh) * | 2013-06-09 | 2013-10-02 | 中国人民解放军第四军医大学 | 一种体外细胞动静态正-负压加载实验系统及方法 |
KR101827291B1 (ko) * | 2016-12-12 | 2018-03-22 | (주)나비바이오텍 | 외부 환경 영향 배제가 가능한 세포 배양용 인큐베이터 |
KR20180118326A (ko) * | 2017-04-21 | 2018-10-31 | 고려대학교 산학협력단 | 미세 소포체의 분리 장치 및 방법 |
CN107893050A (zh) * | 2017-10-17 | 2018-04-10 | 杜水果 | 一种细胞外囊泡及其制备方法和用途 |
CN214004660U (zh) * | 2020-11-05 | 2021-08-20 | 甘肃中医药大学 | 一种正负压力可调型细胞培养箱 |
WO2023024637A1 (zh) * | 2021-08-26 | 2023-03-02 | 中山大学 | 一种力学性囊泡的制备方法 |
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