CN113186154A - Preparation method of chondrocyte exosome capable of regulating osteoarthritis - Google Patents
Preparation method of chondrocyte exosome capable of regulating osteoarthritis Download PDFInfo
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Abstract
The invention discloses a preparation method of a chondrocyte exosome capable of regulating osteoarthritis. The cartilage cell exosome capable of regulating osteoarthritis realizes bidirectional regulation of osteoarthritis through the different types of exosomes, clears the action mechanism of the cartilage cell exosome on osteoarthritis, and provides a new mode for exosome tissue repair.
Description
Technical Field
The invention belongs to the field of biomaterial tissue repair, and particularly relates to a chondrocyte exosome capable of regulating osteoarthritis and a preparation method thereof.
Background
Exosomes are a class of extracellular vesicles secreted by cells, with a particle size of about 60-150nm, and usually carry multiple bioactive molecules that play an important role in cell signaling. Especially mesenchymal stem cell exosomes, have been widely studied in the field of tissue repair, and the action and mechanism of bioactive molecules thereof are relatively clear. However, other cell types, particularly chondrocyte exosomes, have little research and application.
In the development of osteoarthritis, inflammatory damage to osteochondral tissue plays an important role. Under inflammatory conditions, chondrocytes secrete matrix degradation-related proteins such as ADAMTS-5 and MMP13, so that osteochondral tissues are destroyed, and the progress of osteoarthritis is further aggravated.
Research on exosomes and treatment of osteoarthritis are currently focused on stem cell exosomes and drugs, PRP and other treatments. Patent application No. CN201810473041.0 discloses a preparation method of exosome and stem cell proliferation reagent containing exosome, but the preparation method is only limited to mesenchymal stem cell exosome, and the research on the potential of other cell exosomes is limited. Patent application No. CN201810963500.3 discloses an experimental method for treating osteoarthritis by autologous adipose-derived stem cells and PRP, but the penetration force is limited, and the hundred-nanometer tissue penetration effect of exosomes cannot be achieved. At the same time, patents such as these do not address the two-way regulation studies of osteoarthritis with different subtypes of the same material.
Therefore, there is a need for a chondrocyte exosome capable of regulating osteoarthritis, which can effectively regulate osteoarthritis inflammation through strong penetration and contained bioactive molecules, so that the occurrence and development mechanisms of osteoarthritis can be better explored, and osteoarthritis can be effectively treated.
Disclosure of Invention
In view of the above, the present invention provides a chondrocyte exosome capable of regulating osteoarthritis and a preparation method thereof. Various exosome preparation methods are adopted to extract different types of chondrocyte exosomes, and omics analysis is carried out on the exosomes, and the immunoregulation function and mechanism of the exosomes on osteoarthritis are researched.
In order to achieve the purpose, the invention provides the following technical scheme: different types of chondrocytes are respectively cultured, exosomes of different types of chondrocytes are extracted by adopting a plurality of exosome preparation methods, and omics analysis and osteoarthritis immune regulation and control exploration are carried out.
A preparation method of chondrocyte exosomes capable of regulating osteoarthritis comprises the following steps:
the method comprises the following steps: stimulating the different types of chondrocytes for 0-3 days at 5-20ng/mL IL-1 beta when the fusion degree of the chondrocytes reaches 50-60%;
step two: washing and culturing in serum-free medium for 48 hours;
step three: collecting culture medium, and treating by one or more of differential centrifugation, density gradient centrifugation, ultrafiltration, precipitation, immunity separation, and sieving;
step four: obtaining D0 chondrocyte exosomes for repairing and regulating osteoarthritis and D3 chondrocyte exosomes for repairing and regulating osteoarthritis; the D0 chondrocyte exosome for repairing and regulating osteoarthritis is a chondrocyte exosome which is not stimulated by IL-1 beta, and the D3 chondrocyte exosome for repairing and regulating osteoarthritis is a chondrocyte exosome which is stimulated by IL-1 beta for 3 days.
Preferably, the differential centrifugation and ultrafiltration method is adopted to prepare the D0 and D3 chondrocyte exosomes, and the method specifically comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 50%, treating the chondrocytes for 3 days by using 10ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D0 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting culture medium, gradient centrifuging, centrifuging at 300 × g for 15min, and centrifuging at 2500 × g for 15 min;
step three: taking the supernatant, and filtering the supernatant by using a 0.22 mu m filter to remove residual cells and debris;
step four: transferring the solution with an ultracentrifugation filter unit; the solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to 15-25%;
step five: washing the ultrafiltration solution with phosphate buffer and centrifuging repeatedly for 3 times;
step six: ultracentrifuging the washed ultrafiltration liquid containing the exosome for 1 hour; the pellet was resuspended in PBS and centrifuged at 4,000 × g to concentrate the volume to 15-25%.
Preferably, D0 and D3 chondrocyte exosomes under the action of 5ng/mL IL-1 beta are prepared by a density gradient method; the method specifically comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 50%, treating the chondrocytes for 3 days by using 5ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D0 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting the culture medium, centrifuging with Percoll density gradient centrifugate 1000 Xg for 20 min;
step three: sucking the liquid of the exosome layer on the upper interface, adding PBS (2 times the volume of the liquid) and uniformly mixing;
step four: filtering with 0.22 μm filter to remove residual cells and debris, transferring the solution with an ultracentrifuge filter device; the solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to 15-25%; step five: washing with phosphate buffer solution, and centrifuging repeatedly for 3 times;
step six: the pellet was resuspended in 200. mu.L PBS.
Preferably, the differential centrifugation method for preparing D0 and D3 chondrocyte exosomes under the action of 20ng/mL IL-1 beta comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 60%, treating the chondrocytes for 2 days by using 20ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D2 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting the culture medium, performing gradient centrifugation, centrifuging at 300 Xg for 10 min, and collecting the supernatant;
step three: centrifuging at 2000 Xg for 10 min, and collecting supernatant;
step four: centrifuging at 10,000 Xg for 30 min, and collecting supernatant;
step five: 100,000 Xg, at 4 ℃ for 90 minutes, removing the supernatant, left precipitate PBS heavy suspension, again 100,000 Xg centrifugation for 90 minutes;
step six: the pellet was resuspended in 200. mu.L PBS.
The invention has the beneficial effects that:
a chondrocyte exosome capable of regulating osteoarthritis and a preparation method thereof. Various exosome preparation methods are adopted to extract different types of chondrocyte exosomes, omics analysis is carried out on the exosomes, the immunoregulation function and mechanism of the exosomes on osteoarthritis are explored, and the osteoarthritis can be effectively treated by adopting the original chondrocyte exosomes.
Compared with the existing exosomes and the preparation method thereof, the invention has the remarkable progress that:
1) generally, stem cells are used for preparing exosomes, chondrocytes are used as exosome sources, and functions of the exosomes are obviously different from functions of other exosomes.
2) The invention carries out omics analysis on the prepared chondrocyte exosome, researches the immunoregulation function and mechanism of the chondrocyte exosome on osteoarthritis, realizes the bidirectional regulation of osteoarthritis and has clear scientific basis.
Therefore, the chondrocyte exosome capable of regulating osteoarthritis realizes bidirectional regulation of osteoarthritis through different types of exosomes, clears the action mechanism of the chondrocyte exosome on osteoarthritis, and provides a new mode for exosome tissue repair.
Drawings
FIG. 1 is a schematic diagram of the preparation and analysis of original chondrocyte exosomes and degenerated chondrocyte exosomes.
FIG. 2 cellular sources of primary and degenerative chondrocyte exosomes validating immunofluorescence maps.
FIG. 3 particle size analysis of primary and degenerative chondrocyte exosomes.
FIG. 4 Electron microscopy characterization of primary and degenerative chondrocyte exosomes.
FIG. 5 validation of WB proteins of primary and degenerative chondrocyte exosomes.
FIG. 6 Wehn diagram of protein profile of original chondrocyte exosomes and degenerated chondrocyte exosomes
FIG. 7 protein profiles of primary and degenerative chondrocyte exosomes constitute the principal component control.
FIG. 8 immunofluorescence plots of the regulatory effect of primitive chondrocyte exosomes on chondrocyte inflammation.
FIG. 9 immunofluorescence of the mitochondrial rescue mechanism of primary chondrocyte exosomes against chondrocyte inflammation.
FIG. 10 immunofluorescence plots of macrophage regulatory mechanisms of primary chondrocyte exosomes on chondrocyte inflammation.
FIG. 11 is a graph of the in vivo repair effect of primary chondrocyte exosomes on chondrocyte inflammation.
Detailed Description
The chondrocyte exosomes capable of regulating osteoarthritis and the preparation method thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
The preferable example of the chondrocyte exosome capable of regulating osteoarthritis is that D0 and D3 chondrocytes under the action of 10ng/mL IL-1 beta are used, two chondrocyte exosomes are extracted by adopting a differential centrifugation and ultrafiltration method, and omic analysis and osteoarthritis immune regulation and control research are carried out. The specific procedure is as shown in example 1.
The invention can also use the preparation schemes of other IL-1 beta concentrations, other types of chondrocytes and exosomes thereof and other exosomes to obtain the same technical effect.
Example 1 differential centrifugation + Ultrafiltration method for preparing D0 and D3 chondrocyte exosomes under action of 10ng/mL IL-1 beta
1. When the chondrocyte confluence reached 50%, chondrocytes were treated or not with 10ng/mL IL-1 β to give D0 chondrocytes (primary chondrocytes) and D3 chondrocytes (degenerated chondrocytes with IL-1 β acting for 3 days), respectively, which were washed and cultured in serum-free medium for another 48 hours.
2. The medium was collected, centrifuged at 300 Xg for 15min and 2500 Xg for 15 min.
3. The supernatant was taken and filtered using a 0.22 μm filter to remove residual cells and debris.
4. The solution was transferred using an ultracentrifuge filter unit. The solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to about 200. mu.L.
5. The ultrafiltration solution was washed with phosphate buffer and centrifuged 3 times repeatedly.
6. The washed ultrafiltration liquid containing exosomes was ultracentrifuged for 1 hour. The pellet was resuspended in PBS and centrifuged at 4,000 × g to concentrate the volume to about 200 μ L.
Example 2 Density gradient method for preparing D0 and D3 chondrocyte exosomes under effect of 5ng/mL IL-1 beta
1. When the chondrocyte confluence reached 50%, chondrocytes were treated or not treated with 5ng/mL IL-1 β to give D0 chondrocytes (primary chondrocytes) and D3 chondrocytes (degenerated chondrocytes with IL-1 β acting for 3 days), respectively, which were washed and cultured in serum-free medium for another 48 hours.
2. The medium was collected and centrifuged using a Percoll density gradient centrifuge at 1000 Xg for 20 min.
3. And sucking the liquid of the exosome layer on the upper interface, adding 2 times of volume of PBS, and uniformly mixing.
4. Filtration was performed using a 0.22 μm filter to remove residual cells and debris, and the solution was transferred using an ultracentrifuge filter device. The solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to about 200. mu.L.
5. Washed with phosphate buffer and centrifuged 3 times repeatedly.
6. The pellet was resuspended in 200. mu.L PBS,
example 3 differential centrifugation preparation of D0 and D3 chondrocyte exosomes under the action of 20ng/mL IL-1 beta
1. When the chondrocyte confluence reached 50%, chondrocytes were treated or not with 20ng/mL IL-1 β to give D0 chondrocytes (primary chondrocytes) and D3 chondrocytes (degenerated chondrocytes with IL-1 β acting for 3 days), respectively, which were washed and cultured in serum-free medium for another 48 hours.
2. The medium was collected, centrifuged at 300 Xg for 10 minutes with a gradient, and the supernatant was collected.
3. Centrifuge at 2000 Xg for 10 min and take the supernatant.
4. Centrifuge at 10,000 Xg for 30 minutes and collect the supernatant.
5. 100,000 Xg, at 4 ℃ for 90 minutes, removing the supernatant, the remaining precipitation PBS heavy suspension, again 100,000 Xg centrifugation for 90 minutes.
6. The pellet was resuspended in 200. mu.L PBS
Example 1 preparation and analysis of Primary chondrocyte exosomes and degenerative chondrocyte exosomes
As shown in FIG. 1, chondrocytes not treated with IL-1 β were considered to be primitive chondrocytes, and chondrocytes treated with IL-1 β for 3 days were considered to be degenerated chondrocytes and designated D0 chondrocytes and D3 chondrocytes, respectively. Exosomes, named D0 chondrocyte exosome and D3 chondrocyte exosome, were extracted separately for proteome analysis.
Example 1 verification of the cell Source of Primary chondrocyte exosomes and degenerative chondrocyte exosomes immunofluorescence mapping
1. Respectively labeling the original chondrocytes and the degenerated chondrocytes with the antibody proteins of the repair-related protein Collagen II and the degeneration-related protein MMP13 and tracing by using a fluorescent secondary antibody
2. Under a fluorescence microscope, the expression of the original chondrocyte Collagen II is higher than that of the degenerated chondrocyte, and the expression of the degenerated chondrocyte MMP13 is higher than that of the original chondrocyte, as shown in figure 2.
Example 1 particle size analysis of Primary and degenerative chondrocyte exosomes
1. The chondrocyte exosomes D0 and D3 were diluted to appropriate concentrations, respectively, and the particle size was measured using a malvern laser particle sizer.
2. It can be seen that D0 chondrocyte exosomes are similar to D3 chondrocyte exosome subpopulations, all centered between 80-100 nm. As shown in fig. 3.
Example 1 Electron microscopy characterization of Primary and degenerative chondrocyte exosomes
1. Carrying out ultrafiltration and concentration on D0 and D3 chondrocyte exosomes respectively, and then carrying out sample preparation by a cryoelectron microscope.
2. D0 and D3 were observed to be in the form of a classic bowl under a cryo-electron microscope, with a particle size level of about 100nm, as shown in FIG. 4.
Example 1 verification of WB proteins of Primary and degenerative chondrocyte exosomes
1. The intercalary D0, D3 chondrocyte exosomes were lysed using lysine Buffer and subjected to WB western blot experiments.
2. Bands were cut according to the molecular weight of the corresponding protein, and primary antibody and secondary antibody were incubated, respectively.
3. After chemiluminescence exposure, an exosome protein Marker containing TSG101, CD9, CD63 and the like can be seen, as shown in FIG. 5.
In example 1, the protein spectra of primary and degenerative chondrocyte exosomes constitute a wien diagram and a principal component control.
1. And (3) carrying out proteomics detection on D0 and D3 chondrocyte exosomes respectively, annotating corresponding proteins in the result, and carrying out main component analysis.
2. The Wien diagram shows that the components of D0 and D3 chondrocyte exosomes are greatly different, wherein the components of the D3 chondrocyte exosomes are obviously lower than those of the D0 chondrocyte exosomes in the groups of mitochondrial proteins and immune-related proteins, and the D3 chondrocyte exosomes are suggested to have weak capacity of regulating mitochondrial activity and immunoreaction activity. As shown in fig. 6 and 7.
Example 1 immunofluorescence mapping of the modulating effects of primary chondrocyte exosomes on chondrocyte inflammation.
1. Marking original chondrocytes and 3 groups of degenerative chondrocytes and D0 exosomes of degenerative chondrocytes with repair-related proteins Collagen II, Aggrecan and degeneration-related proteins ADAMTS-5 and MMP13 antibody proteins and using fluorescent secondary antibody to trace
2. Under the action of D0 exosome, Collagen II and Aggrecan of degenerated chondrocytes are obviously up-regulated, and ADAMTS-5 and MMP13 are obviously down-regulated. As shown in fig. 8.
Example 1 immunofluorescence of the mitochondrial rescue mechanism of primary chondrocyte exosomes on chondrocyte inflammation.
1. Marking original chondrocytes, degenerated chondrocytes, and a group 3 of degenerated chondrocyte + D0 exosomes by a mitochondrial probe MitoTracker Green and a mitochondrial oxidation probe MitoSOX red respectively.
2. Under the action of D0 exosome, the number and quality of mitochondria of degenerated chondrocytes are obviously increased, and the level of mitochondrial oxidative damage is obviously decreased. As shown in fig. 9.
EXAMPLE 1 immunofluorescence mapping of macrophage regulatory mechanisms of Primary chondrocyte exosomes on chondrocyte inflammation
1. The experimental animals are divided into a pseudo-operation group, a cartilage defect and normal saline treatment group and a cartilage defect and D0 exosome treatment group, and are respectively marked with healing promoting M2 macrophage Marker CD163 and proinflammatory M1 macrophage Marker iNOS protein antibodies at 4 weeks, 6 weeks and 8 weeks after operation, and are marked by fluorescent secondary antibodies.
2. Under a fluorescence microscope, the defect site healing promoting M2 macrophages are remarkably increased and the proinflammatory M1 macrophages are remarkably reduced after D0 exosome treatment, as shown in figure 10.
Example 1 in vivo repair Effect of Primary chondrocyte exosomes on chondrocyte inflammation
1. The experimental animals were divided into a sham operation group, a cartilage defect + saline treatment group, and a cartilage defect + D0 exosome treatment group, and Safranin-O staining and Collagen II staining were performed 4 weeks, 6 weeks, and 8 weeks after the operation, respectively.
2. Under the microscope, the Safranin-O staining and Collagen II staining abundance after D0 exosome treatment are obviously superior to those of a normal saline group, and the significant difference exists after quantitative statistics. As shown in fig. 11.
The results of the cell source verification immunofluorescence, particle size analysis, electron microscopy characterization, WB protein verification, wain diagram composed of protein mass spectrum and principal component comparison, immunofluorescence of the original chondrocyte exosome for regulating and controlling chondrocyte inflammation, immunofluorescence of a mitochondrial rescue mechanism, and immunofluorescence of a macrophage regulation and control mechanism are similar to those of the chondrocyte exosome for regulating and controlling osteoarthritis in example 1, which shows that the preparation of the chondrocyte exosome for regulating and controlling osteoarthritis with similar effects can be realized through the preparation schemes of other IL-1 beta concentrations, other types of chondrocytes and exosomes thereof and other exosomes.
According to the embodiment, the cartilage cell exosome capable of regulating osteoarthritis and the preparation method thereof provided by the invention realize bidirectional regulation of osteoarthritis through different types of exosomes, clear the action mechanism of the cartilage cell exosome on osteoarthritis, and provide a new mode for repairing exosome tissues.
Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A preparation method of chondrocyte exosomes capable of regulating osteoarthritis is characterized by comprising the following steps:
the method comprises the following steps: stimulating the different types of chondrocytes for 0-3 days at 5-20ng/mL IL-1 beta when the fusion degree of the chondrocytes reaches 50-60%;
step two: washing and culturing in serum-free medium for 48 hours;
step three: collecting culture medium, and treating by one or more of differential centrifugation, density gradient centrifugation, ultrafiltration, precipitation, immunity separation, and sieving;
step four: obtaining D0 chondrocyte exosomes for repairing and regulating osteoarthritis and D3 chondrocyte exosomes for repairing and regulating osteoarthritis; the D0 chondrocyte exosome for repairing and regulating osteoarthritis is a chondrocyte exosome which is not stimulated by IL-1 beta, and the D3 chondrocyte exosome for repairing and regulating osteoarthritis is a chondrocyte exosome which is stimulated by IL-1 beta for 3 days.
2. The method for preparing chondrocyte exosomes capable of regulating osteoarthritis according to claim 1, wherein: the differential centrifugation and ultrafiltration method is adopted to prepare D0 and D3 chondrocyte exosomes, and the method specifically comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 50%, treating the chondrocytes for 3 days by using 10ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D0 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting culture medium, gradient centrifuging, centrifuging at 300 × g for 15min, and centrifuging at 2500 × g for 15 min;
step three: taking the supernatant, and filtering the supernatant by using a 0.22 mu m filter to remove residual cells and debris;
step four: transferring the solution with an ultracentrifugation filter unit; the solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to 15-25%;
step five: washing the ultrafiltration solution with phosphate buffer and centrifuging repeatedly for 3 times;
step six: ultracentrifuging the washed ultrafiltration liquid containing the exosome for 1 hour; the pellet was resuspended in PBS and centrifuged at 4,000 × g to concentrate the volume to 15-25%.
3. The method for preparing chondrocyte exosomes capable of regulating osteoarthritis according to claim 1
The method is characterized in that: density gradient method for preparing D0 and D3 chondrocytes under action of 5ng/mL IL-1 beta
A exosome; the method specifically comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 50%, treating the chondrocytes for 3 days by using 5ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D0 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting the culture medium, centrifuging with Percoll density gradient centrifugate 1000 Xg for 20 min;
step three: sucking the liquid of the exosome layer on the upper interface, adding PBS (2 times the volume of the liquid) and uniformly mixing;
step four: filtering with 0.22 μm filter to remove residual cells and debris, transferring the solution with an ultracentrifuge filter device; the solution was centrifuged at 4,000 Xg until the volume in the upper compartment was concentrated to 15-25%; step five: washing with phosphate buffer solution, and centrifuging repeatedly for 3 times;
step six: the pellet was resuspended in 200. mu.L PBS.
4. The method for preparing chondrocyte exosomes capable of regulating osteoarthritis according to claim 1, wherein: the differential centrifugation method for preparing D0 and D3 chondrocyte exosomes under the action of 20ng/mL IL-1 beta specifically comprises the following steps:
the method comprises the following steps: when the fusion degree of the chondrocytes reaches 60%, treating the chondrocytes for 2 days by using 20ng/mL of IL-1 beta or not, respectively obtaining D3 chondrocytes and D2 chondrocytes, washing and culturing the chondrocytes in a serum-free culture medium for 48 hours;
step two: collecting the culture medium, performing gradient centrifugation, centrifuging at 300 Xg for 10 min, and collecting the supernatant;
step three: centrifuging at 2000 Xg for 10 min, and collecting supernatant;
step four: centrifuging at 10,000 Xg for 30 min, and collecting supernatant;
step five: 100,000 Xg, at 4 ℃ for 90 minutes, removing the supernatant, left precipitate PBS heavy suspension, again 100,000 Xg centrifugation for 90 minutes;
step six: the pellet was resuspended in 200. mu.L PBS.
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