Application of C18 ceramide in promoting osteogenic differentiation of adipose-derived stem cells

Abstract

The invention relates to application of C18 ceramide in promoting adipose-derived stem cell osteogenic differentiation and a method for promoting adipose-derived stem cell osteogenic differentiation. The method comprises the following steps: S1, aseptically acquiring adipose tissues of a human body; s2, cutting the adipose tissue into small blocks, mixing the small blocks with collagenase, and incubating at 37 DEG C; s3, after enzymolysis is completed, performing centrifugal treatment and washing, and transferring the cells into a culture bottle containing a culture medium for further culture; s4, when the fusion degree of the cells is 80%, transferring the cells into a culture medium containing C18 ceramide for culture to obtain cultured adipose-derived stem cells; and S5, detecting and cryopreserving. According to the application, C18 ceramide is used for regulating a cell signal channel and membrane fluidity, the osteogenic differentiation capacity of the adipose-derived stem cells is remarkably promoted, the efficiency of differentiation from the adipose-derived stem cells to osteoblasts is remarkably improved through specific bioactive lipid molecule intervention, and a new way is opened up for application of drugs for bone injury and degenerative bone diseases.

Description

Technical Field

[0001] The present application relates to the technical field of biomedicine and regenerative medicine, and in particular to the application of C18 ceramide in promoting osteogenic differentiation of adipose stem cells. Background Art

[0002] Adipose-Derived Stem Cells (ADSCs) are a type of multipotent stem cells derived from adipose tissue that have the potential to differentiate into a variety of cell types, including osteoblasts. Due to their advantages in autologous cell sources, relatively simple acquisition, and good regeneration ability, ADSCs have received extensive attention in bone regeneration and bone repair research.

[0003] The osteogenic differentiation process of adipose-derived stem cells includes multiple stages: stem cells need to be induced to transform into bone precursor cells, and then these precursor cells further differentiate into mature bone cells (osteoblasts). The key steps of osteogenic differentiation include the deposition and mineralization of bone matrix and the expression of bone-related genes, such as bone morphogenetic proteins (BMPs), alkaline phosphatase (ALP) and osteocalcin (OCN). The Wnt / β-catenin signaling pathway plays a vital role in the osteogenesis process.

[0004] However, how to effectively promote the differentiation of ADSCs into osteocytes remains an important challenge in bone tissue engineering. Summary of the invention

[0005] This application proposes the application of C18 ceramide in promoting osteogenic differentiation of adipose stem cells, wherein the chemical structure of ceramide is

[0006] The present application also proposes a method for promoting osteogenic differentiation of adipose stem cells to solve the problem of low osteogenic differentiation ability of adipose stem cells in the prior art.

[0007] The present application proposes a method for promoting osteogenic differentiation of adipose-derived stem cells, comprising the following steps:

[0008] S1: Aseptically obtain human adipose tissue;

[0009] S2: cutting the adipose tissue into small pieces, mixing with collagenase, and incubating at 37° C.;

[0010] S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture;

[0011] S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing C18 ceramide to obtain cultured adipose stem cells;

[0012] S5: Detection and freezing.

[0013] Wherein, the centrifugation condition in step S3 is 300-400g, and the centrifugation time is 5-10 minutes.

[0014] The cell culture in step S3 and step S4 is carried out in an incubator, which is a 37° C. constant temperature incubator, wherein the concentration of the gas containing CO2 is 5%.

[0015] Wherein, the pH value of the culture medium in step S4 is 7.2-7.4, and its pH value is adjusted using Hepes buffer.

[0016] Wherein, the concentration of C18 ceramide in the culture medium in step S4 is between 10-100 uM.

[0017] The cell culture time in step S4 is 14 days, and the culture medium is replaced every 3 days during the culture process.

[0018] Wherein, the culture medium used in step S4 is firstly subjected to ultrasonic treatment after adding C18 ceramide.

[0019] The culture medium used in step S4 is sterilized after adding C18 ceramide, specifically by filtering with a 0.22 um filter.

[0020] The present application also proposes the use of a method for promoting osteogenic differentiation of adipose-derived stem cells in the preparation of a drug for treating bone defects.

[0021] The present application obtains cultured adipose-derived stem cells by transferring the cells to a culture medium containing C18 ceramide when the cells have reached a confluence of 80%. C18 ceramide is used to regulate cell signaling pathways and membrane fluidity, thereby significantly promoting the osteogenic differentiation ability of adipose-derived stem cells. The efficiency of adipose-derived stem cell differentiation into osteoblasts is significantly improved through the intervention of specific bioactive lipid molecules, thus opening up a new path for the application of drugs for bone injury and degenerative bone disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0023] Figure 1 This is a flow cytometric graph of adipose stem cell markers detected by flow cytometry in an embodiment of the present application;

[0024] Figure 2 This is a comparison chart of the proliferation of adipose stem cells treated with C18 ceramide in the examples of this application;

[0025] Figure 3 This is the expression diagram of genes related to osteogenesis of adipose stem cells promoted by C18 ceramide treatment in the examples of this application;

[0026] Figure 4 This is the expression diagram of genes related to osteogenesis of adipose stem cells promoted by C18 ceramide treatment in the examples of this application;

[0027] Figure 5 This is an alkaline phosphatase staining image of the osteogenic differentiation of adipose stem cells after C18 ceramide treatment in the example of the present application;

[0028] Figure 6 This is a comparison chart of the formation of mineralized nodules after C18 ceramide treatment promoted osteogenic differentiation of adipose stem cells in the examples of the present application. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0030] The present application provides an application of C18 ceramide in promoting osteogenic differentiation of adipose stem cells, wherein the chemical structure of ceramide is

[0031] The present invention provides a method for promoting osteogenic differentiation of adipose-derived stem cells, comprising the following steps:

[0032] S1: Aseptically obtain human adipose tissue;

[0033] In this process, the extraction and culture of adipose-derived stem cells (ADSCs) require precise operations to ensure high-quality cells. First, ADSCs are extracted from human adipose tissue. Adipose tissue samples are usually obtained through liposuction or adipose tissue biopsy. The acquisition process needs to be carried out in a sterile environment to reduce the risk of infection.

[0034] S2: cutting the adipose tissue into small pieces, mixing with collagenase, and incubating at 37° C.;

[0035] After the adipose tissue is extracted, it needs to be treated with enzymatic hydrolysis. The enzymatic hydrolysis reagent used in this embodiment is collagenase, which can effectively decompose the extracellular matrix, thereby releasing adipose stem cells. The specific steps of the enzymatic hydrolysis treatment include cutting the adipose tissue into small pieces and then mixing them with the collagenase solution. The enzymatic hydrolysis time and temperature need to be optimized according to the specific experimental conditions to ensure the maximum recovery rate and activity of the cells. In this embodiment, the incubation is carried out at 37°C.

[0036] S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture;

[0037] The centrifugation conditions are 300-400g and the centrifugation time is 5-10 minutes, which effectively removes undissociated tissue components and floating cells. The separated cells need to undergo appropriate washing steps to remove residual enzymes and culture medium components.

[0038] S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing C18 ceramide to obtain cultured adipose stem cells;

[0039] During the culture process, it is crucial to choose a suitable culture medium. The culture medium used in this example is DMEM (Dulbecco's Modified Eagle Medium), which contains nutrients and growth factors required for cell growth. The formula of the culture medium may need to be adjusted according to the specific needs of the cells during the specific experiment, and is not limited here. The culture conditions are set in an incubator at 37°C with a 5% CO2 gas environment. This design helps to maintain the pH value of the cell culture medium within an appropriate range. During cell growth, check the status of the cells regularly to ensure that the cells reach 80% confluence before subsequent processing. The cells reaching 80% confluence means that their growth and proliferation are in good condition and are suitable for further experimental operations.

[0040] When the cells are cultured to 80% confluence, prepare the culture medium and add C18 ceramide. The concentration of C18 ceramide is between 10-100 μM, which is selected according to the experimental requirements. When C18 ceramide is added to the culture medium, it must be ensured to be evenly dissolved to ensure its uniform effect on the cells.

[0041] In order to improve the solubility of C18 ceramide, ultrasonic treatment is used in the examples of this application. In addition, the pH value of the culture medium is adjusted to 7.2-7.4, and the pH value is crucial for the growth and function of cells. Hepes buffer is used in the examples of this application to adjust the pH value to ensure its stability.

[0042] After adding ceramide C18, the culture medium needs to be sterilized to avoid bacterial and fungal contamination. In the present embodiment, a 0.22 μm filter is used for filtration to ensure the sterility of the culture medium.

[0043] The treated adipose stem cells are cultured in a culture medium containing C18 ceramide, wherein the temperature in the incubator is 37°C, the gas environment contains 5% CO2, and the culture time is 14 days. During the culture of adipose stem cells, the culture medium needs to be replaced regularly. In the embodiment of the present application, the culture medium needs to be replaced every 3 days to remove waste and excess secretions produced by cell metabolism and provide fresh nutrients. The replacement of the culture medium not only helps to maintain the cleanliness of the cell growth environment, but also promotes the health of the cells.

[0044] S5: Detection and freezing.

[0045] The cells are subjected to biological efficacy testing, cell differentiation ability testing, etc., and the qualified adipose stem cells are added to a freezing tube, and freezing solution is added thereto. After being mixed and shaken evenly, they are frozen in a refrigerator and transferred to liquid nitrogen for long-term storage. The detection process and the freezing process can be carried out simultaneously to improve processing efficiency.

[0046] Specific examples and comparative examples:

[0047] Sample preparation process

[0048] Adipose-derived stem cells were cultured in an incubator at 37°C and 5% CO2 until the cells reached 80% confluence (usually 2-3 days). The cells were washed with PBS buffer to remove the culture medium, and protease was added and digested at 37°C for 5 minutes to peel off the cell monolayer, and the culture medium was added to terminate the digestion reaction, and the cells were gently blown to disperse.

[0049] The digested cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the cells were resuspended in flow cytometry buffer (PBS containing 1% BSA). Cells were counted using a cell counting plate and the cell concentration was adjusted to 1×10 6 cells / ml.

[0050] Prepare flow cytometry buffer (PBS containing 1% BSA in the present embodiment), which can reduce nonspecific binding, add an appropriate amount of blocking agent to the cell suspension. In the present embodiment, 5-10% human serum is used. The blocking agent can reduce the nonspecific binding of antibodies to non-target cells. Incubate at 4°C in the dark for 15-20 minutes.

[0051] Prepare the diluent of fluorescent labeled antibody and dilute the antibody to the recommended concentration according to the antibody instructions. Add the diluted antibody to the cell suspension, mix gently, and incubate at 4°C in the dark for 30 minutes to avoid high temperature and strong light to reduce the degradation of fluorescent signal. No antibody was added to the blank control group.

[0052] Centrifuge the cell suspension at 1000 rpm for 5 minutes and remove the supernatant. Add flow cytometry buffer, gently resuspend the cells, centrifuge again, and repeat the washing step 2-3 times to ensure that unbound antibodies are removed.

[0053] Set up the flow cytometer, run the flow cytometer and acquire data. Analyze the intensity of the fluorescent signal using the flow cytometer software to check the expression of each marker.

[0054] like Figure 1 As shown, Figure 1 This is a flow cytometric graph of adipose stem cell markers detected by flow cytometry in the embodiment of the present application. The test results show that adipose stem cells are strongly positive for CD73 (96.99%), CD105 (80.55%), and CD90 (96.74%), but do not express CD45 and CD-HLA, indicating that the obtained cells are adipose stem cells.

[0055] Adipose stem cell proliferation rate test

[0056] Adipose-derived stem cells were seeded into a 96-well plate, with 100uL of cell suspension in each well and a cell density of 1×10 5 cells / well. Incubate in an incubator at 37°C and 5% CO2 for 24 hours to allow cells to attach. The control group (Ctrl group) did not receive any treatment, while the experimental group (C18) was treated with 50uL C18 ceramide for 24h, 48h, and 72h, respectively. Remove the 96-well plate from the incubator and add 10uL CCK-8 reagent to each well. Return the 96-well plate to the incubator and continue incubating for 2 hours.

[0057] The optical density (OD) value of each well was measured at a wavelength of 450 nm using an ELISA reader. The average value of 6 replicate wells was taken for each group.

[0058] like Figure 2 As shown, Figure 2 This is a comparison chart of the proliferation of adipose stem cells treated with C18 ceramide in the examples of the present application. The OD value of each sample is calculated to evaluate the metabolic activity and proliferation of the cells. An increase in the OD value usually reflects an increase in cell proliferation or metabolic activity. The results show that after adding C18 ceramide, the proliferation of adipose stem cells was significantly promoted at 24h, 48h, and 72h.

[0059] Real-time fluorescence quantitative PCR test

[0060] The RNA of the experimental group and the control group was extracted, and the RNA concentration was tested. The RNA extraction steps are as follows: take out the sample to be tested, let it stand for a while to melt, add chloroform to shake evenly and then let it stand, centrifuge to obtain RNA, use a pipette to gently shake to fully dissolve the RNA, and then immediately measure its concentration and perform a reverse transcription reaction. The specific processing time, centrifugation processing parameters, etc. can be selected according to the experimental conditions, and will not be elaborated here.

[0061] Figure 3 and Figure 4 : is the expression diagram of C18 ceramide treatment promoting osteogenesis-related genes of adipose stem cells 7 days and 14 days after the embodiment of the present application, as shown in Figure 3 and Figure 4 As shown, the results of QPCR experiments showed that after the addition of C18 ceramide, 7 days after osteogenic induction differentiation, the osteogenic genes RUNX2, ALP, OCN, and COL1A1 in the experimental group (marked in the figure: Osteogenic+C18) were upregulated by 2.4, 2.1, 1.9, and 2.5 times, respectively, compared with the control group (marked in the figure: Osteogenic); after 14 days of osteogenic induction differentiation, the osteogenic genes RUNX2, ALP, OCN, and COL1A1 in the experimental group were upregulated by 1.8, 2.9, 3.3, and 3.2 times, respectively, compared with the control group. The above results indicate that C18 ceramide can significantly promote the osteogenic differentiation of adipose stem cells.

[0062] Furthermore, in the present embodiment, adipose-derived stem cells are inoculated in a culture dish containing an osteogenic induction medium, and the osteogenic induction medium is usually replaced when the cells reach 80%-90% fusion. The medium is replaced every 2-3 days for 14 days to ensure that the cells are fully induced to differentiate into osteocytes. C18 ceramide is added to the osteogenic induction solution, and the effect of C18 ceramide on the osteogenesis of adipose-derived stem cells is detected.

[0063] After induction, gently rinse the cells 1-2 times with PBS buffer to remove the residual culture medium. Add 1 mL of 4% paraformaldehyde or cold propanol fixative to each well and fix for 10-30 minutes. Aspirate the fixative and rinse the cells twice again with PBS buffer to remove the residual fixative. Place the cells in an incubation solution containing sodium β-glycerophosphate, sodium barbital, CaCl2, and MgSO4, incubate at 37°C for 4-6 hours, then soak in 2% cobalt nitrate solution for 3-5 minutes, then treat with 1% ammonium sulfide solution for 2 minutes, rinse with tap water, and seal after natural drying.

[0064] Figure 5 is an alkaline phosphatase staining diagram after C18 ceramide treatment promotes osteogenic differentiation of adipose stem cells in the example of the present application. Figure 5As shown, after staining, the culture plate was placed under a microscope for observation, and blue ALP-positive particles were visible in the cytoplasm of osteoblasts, indicating that the cells successfully differentiated into osteoblasts. Through ALP staining, the experimental group (marked in the figure: Osteogenic+C18) was compared with the control group (marked in the figure: Osteogenic), and it can be seen that after the addition of ceramide C18, the blue ALP-positive particles of adipose stem cells increased, indicating that C18 ceramide promotes the osteogenic differentiation of adipose stem cells.

[0065] Furthermore, the adipose stem cells were subjected to osteogenic induction culture (the osteogenic induction culture conditions were the same as the above conditions), and after the induction, the cells were fixed with a fixative, the fixative was removed and the adipose stem cells were rinsed twice with PBS buffer, the pH value of the PBS buffer was adjusted to 4.2 with 0.1% NH4OH, the PBS buffer was aspirated and sufficient Alizarin Red S staining solution was added to cover the adipose stem cell monolayer, and the culture was carried out at room temperature in a dark environment for 45 minutes, the Alizarin Red S solution outside the cell stem cell monolayer was aspirated, and the culture plate was washed four times with distilled water.

[0066] Figure 6 : is a comparison diagram of the formation of mineralized nodules after C18 ceramide treatment promotes osteogenic differentiation of adipose stem cells in the examples of the present application. Figure 6 As shown, after staining, the culture plate was placed under a microscope for observation, and red or orange-red mineralized nodules were visible in the cytoplasm of osteoblasts, indicating that the cells successfully differentiated into osteoblasts. Through Alizarin red staining, after adding ceramide C18, the red mineralized nodules increased in the experimental group (marked in the figure: Osteogenic+C18) compared with the control group (marked in the figure: Osteogenic), and the experiment showed that ceramide C18 promoted the osteogenic differentiation of adipose stem cells.

[0067] The present application also provides an embodiment of using the method for promoting osteogenic differentiation of adipose-derived stem cells in the preparation of a drug for treating bone defects.

[0068] Analysis shows that the osteogenic differentiation process of adipose stem cells includes multiple stages: stem cells need to be induced to transform into bone precursor cells, and then these precursor cells will further differentiate into mature bone cells (osteoblasts). The key steps of osteogenic differentiation include the deposition and mineralization of bone matrix and the expression of bone-related genes, such as bone morphogenetic proteins (BMPs), alkaline phosphatase (ALP) and osteocalcin (OCN). The Wnt / β-catenin signaling pathway plays a vital role in the process of osteogenesis. C18 ceramide can activate the Wnt signaling pathway and promote the expression of bone-related genes by enhancing the nuclear translocation of β-catenin. This process improves the osteogenic ability of bone precursor cells. C18 ceramide may be able to promote the mineralization of bone matrix and enhance the mineralization process by regulating the expression of mineralization-related proteins in cells, such as osteocalcin. C18 ceramide can change the fluidity of cell membranes, affect signal transduction receptors on cell membranes, and help enhance the response of cells to external osteogenic signals, thereby promoting osteogenic differentiation.

[0069] The present application obtains cultured adipose-derived stem cells by transferring the cells to a culture medium containing C18 ceramide when the cells have reached a confluence of 80%. C18 ceramide is used to regulate cell signaling pathways and membrane fluidity, thereby significantly promoting the osteogenic differentiation ability of adipose-derived stem cells. The efficiency of adipose-derived stem cell differentiation into osteoblasts is significantly improved through the intervention of specific bioactive lipid molecules, thus opening up a new path for the application of drugs for bone injury and degenerative bone disease.

[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0071] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0072] Finally, it should be noted that, in this application, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article including the elements.

[0073] Specific examples are used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core idea of ​​this application. At the same time, for those skilled in the art, according to the idea of ​​this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on this application.

Claims

1. Application of C18 ceramide in promoting osteogenic differentiation of adipose-derived stem cells, including: The chemical structure of ceramide is 2. A method for promoting osteogenic differentiation of adipose-derived stem cells, characterized in that: The following steps are involved: S1: Aseptically obtain human adipose tissue; S2: cutting the adipose tissue into small pieces, mixing with collagenase, and incubating at 37° C.; S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture; S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing C18 ceramide to obtain cultured adipose stem cells; S5: Detection and freezing.

3. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The centrifugation condition in step S3 is 300-400g, and the centrifugation time is 5-10 minutes.

4. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The cell culture in step S3 and step S4 is carried out in an incubator, which is a 37° C. constant temperature incubator with a gas concentration of 5% containing CO2.

5. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The pH value of the culture medium in step S4 is 7.2-7.4, and the pH value is adjusted using Hepes buffer.

6. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The concentration of C18 ceramide in the culture medium in step S4 is between 10-100 uM.

7. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The cell culture time in step S4 is 14 days, and the culture medium is replaced every 3 days during the culture process.

8. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The culture medium used in step S4 is firstly subjected to ultrasonic treatment after adding C18 ceramide.

9. The method for promoting osteogenic differentiation of adipose-derived stem cells according to claim 8, characterized in that: The culture medium used in step S4 is sterilized after adding C18 ceramide, specifically by filtering with a 0.22 um filter.

10. Use of the method according to any one of claims 2 to 9 in preparing a drug for treating bone defects.

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