Adipose stem cell apoptotic bodies and preparation method and application thereof

Abstract

The application provides a kind of adipose-derived stem cell apoptosis corpuscle and its preparation method and application, belong to medical technology field.The preparation method includes the following steps: step (1), by collagenase type I digestion method to obtain adipose-derived stem cell, and in vitro culture to induction period;Step (2), take the adipose-derived stem cell of induction period, STS method induces concentrated apoptosis, add trypsin digestion, room temperature differential centrifugation, the obtained precipitate is the adipose-derived stem cell apoptosis corpuscle.The application first discovers that adipose-derived stem cell apoptosis corpuscle has the effect of inhibiting the expression of CDC20 in proliferative scar fibroblasts to reduce Wnt / β-catenin signal pathway, and has a significant inhibitory effect on the pathological phenotype and horizontal migration of proliferative scar-derived fibroblasts.In addition, the application is simple in operation, low in cost, safe and efficient, and convenient for clinical popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an apoptotic body of adipose stem cells, its preparation method, and its application. Background Technology

[0002] Scars are among the most common and difficult-to-completely resolve conditions in Chinese plastic and reconstructive surgery. Due to the diversity of ethnic groups, regional climates, and lifestyles in my country, scar subtypes are complex and their causes varied. Hypertrophic scars, in particular, are pathological scars caused by excessive proliferation of fibroblasts during wound healing. They not only affect appearance but can also lead to local functional impairment, soft tissue displacement, or joint deformities due to contractures.

[0003] Recent studies have found that cell-free biological derivatives can influence scar development by regulating the fibroblast microenvironment. The mechanisms mainly include anti-fibrosis, immunomodulation and anti-inflammation, vascularization regulation, and promotion of tissue regeneration. These cell-free biological derivatives mainly include conditioned mediums for cells and extracellular vesicles (exosomes, apoptotic bodies). Extracellular vesicles contain various active substances and hold promise for comprehensive targeted therapy of scars, thus gradually becoming a new direction in scar treatment research. Apoptotic bodies, compared to exosomes and other vesicles, have the advantages of higher yield and easier preparation, thus becoming a recent research hotspot.

[0004] The composition of apoptotic vesicles is closely related to their source cells, and there is evidence that vesicles from different sources carry different characteristics of their origin cells. Fat (derived from liposuction, abdominoplasty, etc.) is a readily available research subject in plastic and reconstructive surgery. Adipose-derived stem cells extracted from adipose tissue have been shown to have scar-regulating capabilities. Apoptotic stem cell bodies (ASCs-ApoVs), as a subclass of extracellular vesicles, are derived from adipose-derived stem cells, making them more readily available, easier to prepare, and more biosafety-tolerant. Therefore, they have high research value in the treatment of hypertrophic scars.

[0005] Current scar treatments primarily focus on exosomes and other non-apoptotic vesicles derived from traditional stem cells (SCs). However, these types of vesicles are all physiologically secreted from living stem cells, resulting in problems such as low yield, long production cycles, high preparation costs, and unstable product performance.

[0006] Therefore, there is an urgent clinical need for simpler, more efficient, and safer strategies for treating hypertrophic scars. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an apoptotic body of adipose stem cells, its preparation method, and its application.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing apoptotic bodies of adipose-derived stem cells, the specific steps of which are as follows: Step (1): Adipose stem cells are obtained by digestion with type I collagenase and cultured in vitro to the induction phase, which is the 2nd to 4th generation of adipose stem cells cultured in vitro.

[0009] Step (2): Take the 2nd to 4th generation adipose stem cells cultured in vitro, induce apoptosis using the STS method, digest with trypsin, collect the precipitate by differential centrifugation at room temperature, and the precipitate is the apoptotic body of the adipose stem cells.

[0010] In one embodiment of the present invention, the specific operation of the type I collagenase digestion method is as follows: Take adipose tissue and add an equal volume of 1 mg / ml type I collagenase solution. After digestion in a water bath at 37°C for 2 hours, take the non-oil layer liquid, filter it through a 70 μm filter to remove the incompletely digested tissue, and then centrifuge the filtered collagenase digestion solution at 1000 rpm. The resulting cell precipitate is adipose stem cells.

[0011] In one embodiment of the present invention, the STS induction operation is as follows: 2nd to 4th generation adipose stem cells cultured in vitro are taken, given 5 μM STS, and induced in a 37°C incubator for 12 h to induce concentrated apoptosis of adipose stem cells.

[0012] In one embodiment of the present invention, the trypsin digestion operation is as follows: take the STS-induced adipose stem cells, add 0.25% trypsin, digest at 37°C for 2-3 minutes, and then add complete culture medium to end the digestion.

[0013] In one embodiment of the present invention, the room temperature differential centrifugation operation includes the following steps: S1, take the trypsin digest solution from step (2), centrifuge at 300 rpm for 10-20 min, and collect the supernatant; S2, take the supernatant collected in S1, centrifuge at 3000 rpm for 10-20 min, collect the precipitate, the precipitate obtained is the apoptotic body of adipose stem cells; S3: Take the precipitate collected in S2, resuspend it in PBS, centrifuge at 3000 rpm for 10-20 min, and collect the precipitate. Repeat S3 twice. The resulting precipitate is the purified apoptotic body of adipose stem cells.

[0014] A second aspect of the present invention provides an apoptotic body of adipose stem cells, which is prepared by the above-described preparation method.

[0015] The third aspect of this invention provides the use of the above-mentioned adipose stem cell apoptosis bodies in the preparation of products for treating hypertrophic scars.

[0016] A fourth aspect of the present invention provides a medicament for treating hypertrophic scars, comprising the aforementioned apoptotic bodies of adipose stem cells.

[0017] In one embodiment of the present invention, the drug is a drug that inhibits the expression of CDC20 in proliferative scar fibroblasts.

[0018] In one embodiment of the present invention, the drug is used to inhibit the activity of the Wnt / β-catenin signaling pathway.

[0019] In one embodiment of the present invention, the drug is used to inhibit the migration ability of fibroblasts derived from hypertrophic scars, and / or, the drug is used to inhibit the expression of α-SMA, COL1A1, COL3A1, Vimentin, and TGF-β1 genes.

[0020] Furthermore, the drug is a drug that inhibits the lateral migration ability of fibroblasts originating from hypertrophic scars.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for preparing adipose-derived stem cell apoptotic bodies (ASCs-ApoVs) provided by this invention is simple to operate, has a short preparation cycle, and a high product yield, enabling rapid transformation from adipose tissue to therapeutic products. Specifically, after inducing concentrated apoptosis of adipose-derived stem cells via STS, vesicles are extracted using room temperature differential centrifugation. Compared with traditional ultracentrifugation or cryogenic centrifugation, this method eliminates the need for expensive ultracentrifuges and cryogenic environmental control, significantly reducing equipment requirements and preparation costs.

[0022] The ASCs-ApoVs prepared by the method of this invention exhibit excellent stability. Experiments have shown that after repeated freeze-thaw cycles (3 cycles) and treatment with common enzymes such as DNase, RNase, and proteinase K, ASCs-ApoVs still maintain their intact membrane structure and biological activity, with no significant attenuation in their ability to regulate the phenotype of proliferative scar fibroblasts. This characteristic overcomes the shortcomings of traditional extracellular vesicle preparations (such as exosomes) that require stringent storage and transportation conditions and are easily degraded by the in vivo environment, greatly facilitating clinical storage, transportation, and local drug delivery.

[0023] This invention discloses the application of adipose-derived stem cell apoptosis bodies in the preparation of products for treating hypertrophic scars. It is the first to reveal that adipose-derived stem cell apoptosis bodies specifically inhibit CDC20 expression, thereby blocking the abnormal activation of the downstream Wnt / β-catenin signaling pathway and exerting an anti-fibrotic effect. Furthermore, cytotoxicity assays (CCK-8) confirmed that ASCs-ApoVs exert their therapeutic effect without affecting normal cell activity, indicating that they exert their therapeutic effect through benign induction rather than cytotoxic killing, demonstrating extremely high biocompatibility.

[0024] The apoptotic bodies of adipose stem cells prepared in this invention have a significant inhibitory effect on the pathological phenotype and lateral migration of fibroblasts derived from proliferative scars. Attached Figure Description

[0025] Figure 1 A schematic diagram of the ASCs-ApoVs preparation process; Figure 2 Transmission electron microscopy image of ASCs-ApoVs; Figure 3 Fluorescent staining images of HS-fibroblasts uptake ASCs-ApoVs; where: Figure 3 a is a fluorescence staining image of the nuclear cells of HS-fibroblasts (blue); Figure 3 b is a fluorescent staining image of intracellular membranous organelles of HS-fibroblasts (red); Figure 3 c is the membrane fluorescence staining image of ASCs-ApoVs (green); Figure 3 d shows the fluorescence distribution after HS-fibroblasts take up ASCs-ApoVs; Figure 4 WB grayscale images of ASCs-ApoVs regulating the expression of COL1A1, COL3A1, and α-SMA in HS-fibroblasts; Figure 5 A bar chart showing the quantitative statistical analysis of Western blotting of scar phenotype-related markers (COL1A1, COL3A1, and α-SMA) expression in HS-fibroblasts by ASCs-ApoVs; where: Figure 5 a is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating COL1A1 expression; Figure 5 b is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating COL3A1 expression; Figure 5 c is a bar chart of Western blotting quantitative statistical analysis of the COL3A1 / COL1A1 expression ratio regulated by ASCs-ApoVs; Figure 5d is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating α-SMA expression; Figure 6 A bar chart showing the statistical analysis of RT-qPCR results for the regulation of the transcription of scar phenotype-related markers (COL1A1, COL3A1, and α-SMA) in HS-fibroblasts by ASCs-ApoVs; where: Figure 6 a is a bar chart showing the statistical analysis of RT-qPCR results of ASCs-ApoVs regulating COL1A1 transcription in COL1A1; Figure 6 b is a bar chart showing the statistical analysis of RT-qPCR on the regulation of COL3A1 transcription by ASCs-ApoVs; Figure 6 c is a bar chart of RT-qPCR statistical analysis of the COL3A1 / COL1A1 transcription ratio regulated by ASCs-ApoVs; Figure 6 d is a bar chart showing the statistical analysis of RT-qPCR on the regulation of α-SMA transcription by ASCs-ApoVs; Figure 7 WB grayscale image of ASCs-ApoVs regulating the expression of CDC20 and scar phenotype-related markers (Vimentin and TGF-β1) in HS-fibroblasts; Figure 8 A bar chart showing the quantitative statistical analysis of Western blotting of ASCs-ApoVs regulating the expression of CDC20 and scar phenotype-related markers (Vimentin and TGF-β1) in HS-fibroblasts; where: Figure 8 a is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating CDC20 expression; Figure 8 b is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating Vimentin expression; Figure 8 c is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating TGF-β1 expression; Figure 9 A bar chart showing the statistical analysis of RT-qPCR for the regulation of CDC20 and scar phenotype-related markers (Vimentin and TGF-β1) transcription in HS-fibroblasts by ASCs-ApoVs; Figure 9 a is a bar chart showing the statistical analysis of RT-qPCR results of ASCs-ApoVs regulating CDC20 transcription; Figure 9 b is a bar chart showing the statistical analysis of RT-qPCR in the regulation of Vimentin transcription by ASCs-ApoVs; Figure 9 c is a bar chart showing the statistical analysis of RT-qPCR on the regulation of TGF-β1 transcription by ASCs-ApoVs; Figure 10Visible light microscope image of ASCs-ApoVs regulating the horizontal migration of HS-fibroblasts (scratch assay); Figure 11 Line graph showing the statistical analysis of ASCs-ApoVs in regulating the scratch healing rate of HS-fibroblasts; Figure 12 Figure 72-hour CCK8 assay results for ASCs-ApoVs regulating HS-fibroblast cell viability; Figure 13 WB grayscale images of ASCs-ApoVs regulating the expression of CDC20 and scar phenotype-related markers in HS-fibroblasts after repeated freeze-thaw cycles and different enzyme treatments. Figure 14 A bar chart showing the quantitative statistical analysis of WB gray values ​​of ASCs-ApoVs regulating the expression of CDC20 and scar phenotype-related markers in HS-fibroblasts after repeated freeze-thaw cycles and different enzyme treatments; where: Figure 14 a is a bar chart showing the Western blotting statistical analysis of the regulation of COL1A1 expression by ASCs-ApoVs after repeated freeze-thaw cycles and different enzyme treatments; Figure 14 b is a bar chart showing the Western blotting statistical analysis of ASCs-ApoVs regulating COL3A1 expression after repeated freeze-thaw cycles and different enzyme treatments; Figure 14 c is a bar chart showing the Western blotting statistical analysis of the COL3A1 / COL1A1 expression ratio regulated by ASCs-ApoVs after repeated freeze-thaw cycles and different enzyme treatments; Figure 14 d is a bar chart showing the quantitative statistical analysis of ASCs-ApoVs regulating α-SMA expression after repeated freeze-thaw cycles and different enzyme treatments; Figure 14 e is a bar chart showing the Western blotting statistical analysis of ASCs-ApoVs regulating CDC20 expression after repeated freeze-thaw cycles and different enzyme treatments; Figure 14 f is a bar chart showing the Western blotting statistical analysis of the regulation of Vimentin expression by ASCs-ApoVs after repeated freeze-thaw cycles and different enzyme treatments. Figure 14 g is a bar chart showing the Western blotting statistical analysis of TGF-β1 expression regulated by ASCs-ApoVs after repeated freeze-thaw cycles and different enzyme treatments; Figure 15 A bar chart showing the statistical analysis of RT-qPCR transcription of CDC20 and scar phenotype-related markers in HS-fibroblasts by ASCs-ApoVs after repeated freeze-thaw cycles and different enzyme treatments; where: Figure 15 a is a bar chart showing the statistical analysis of RT-qPCR of ASCs-ApoVs regulating COL1A1 transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 15b is a bar chart showing the statistical analysis of RT-qPCR of ASCs-ApoVs regulating COL3A1 transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 15 c is a bar chart showing the statistical analysis of RT-qPCR of ASCs-ApoVs regulating COL3A1 / COL1A1 transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 15 d is a bar chart showing the statistical analysis of RT-qPCR of ASCs-ApoVs regulating α-SMA transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 15 e is a bar chart showing the statistical analysis of RT-qPCR results of ASCs-ApoVs regulating CDC20 transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 15 f is a bar chart showing the statistical analysis of RT-qPCR results of ASCs-ApoVs regulating Vimentin transcription after repeated freeze-thaw cycles and different enzyme treatments. Figure 15 g is a bar chart showing the statistical analysis of RT-qPCR of ASCs-ApoVs regulating TGF-β1 transcription after repeated freeze-thaw cycles and different enzyme treatments; Figure 16 WB grayscale images of markers related to the regulation of Wnt / β-catenin pathway expression in HS-fibroblasts by ASCs-ApoVs; Figure 17 Bar chart for Western blotting quantitative statistical analysis of biomarkers related to the Wnt / β-catenin pathway in HS-fibroblasts regulated by ASCs-ApoVs; where: Figure 17 a is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating p-β-catenin expression; Figure 17 b is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating β-catenin expression; Figure 17 c is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating AXIN2 expression; Figure 17 d is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating c-MYC expression; Figure 17 e is a bar chart of Western blotting quantitative statistical analysis of ASCs-ApoVs regulating Cyclin-D1 expression; Figure 18 A bar chart showing the statistical analysis of RT-qPCR for the regulation of Wnt / β-catenin pathway marker transcription in HS-fibroblasts by ASCs-ApoVs; where: Figure 18 a is a bar chart showing the statistical analysis of RT-qPCR results of ASCs-ApoVs regulating β-catenin transcription; Figure 18 b is a bar chart showing the statistical analysis of RT-qPCR on ASCs-ApoVs regulating AXIN2 transcription; Figure 18 c is a bar chart representing the statistical analysis of RT-qPCR on the regulation of c-MYC transcription by ASCs-ApoVs; Figure 18 d is a bar chart showing the statistical analysis of RT-qPCR on the regulation of Cyclin-D1 transcription by ASCs-ApoVs; Figure 19 Wb grayscale image of the regulation of Wnt / β-catenin pathway markers in HS-fibroblasts cells by Apcin-A (a CDC20 inhibitor); Figure 20 Bar chart for quantitative statistical analysis of Western blots on the expression of biomarkers related to the Wnt / β-catenin pathway in HS-fibroblasts regulated by Apcin-A; where: Figure 20 a is a bar chart of Western blotting quantitative statistical analysis of Apcin-A regulating p-β-catenin expression; Figure 20 b is a bar chart of Western blotting quantitative statistical analysis of Apcin-A regulating β-catenin expression; Figure 20 c is a bar chart of Western blotting quantitative statistical analysis of Apcin-A regulation of AXIN2 expression; Figure 20 d is a bar chart of Western blotting quantitative statistical analysis of Apcin-A regulating c-MYC expression; Figure 20 e is a bar chart of Western blotting quantitative statistical analysis of Apcin-A regulating Cyclin-D1 expression; Figure 21 A bar chart showing the statistical analysis of RT-qPCR for Apcin-A regulating the transcription of markers related to the Wnt / β-catenin pathway in HS-fibroblasts; where: Figure 21 a is a bar chart showing the statistical analysis of RT-qPCR results of Apcin-A regulating β-catenin transcription; Figure 21 b is a bar chart showing the statistical analysis of RT-qPCR on Apcin-A regulation of AXIN2 transcription; Figure 21 c is a bar chart representing the statistical analysis of RT-qPCR on Apcin-A regulation of c-MYC transcription; Figure 21 d is a bar chart showing the statistical analysis of RT-qPCR on Apcin-As regulating Cyclin-D1 transcription; Figure 22 A schematic diagram illustrating the mechanism by which ASCs-ApoVs regulate HS-fibroblasts. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments.

[0027] Example 1 This embodiment provides a method for preparing apoptotic bodies of adipose-derived stem cells.

[0028] like Figure 1 As shown, the method for preparing apoptotic bodies of adipose-derived stem cells provided in this embodiment includes the following steps: Step (1): Adipose-derived stem cells are obtained by digestion with type I collagenase and cultured in vitro to the induction phase, wherein the induction phase refers to the third generation of adipose-derived stem cells cultured in vitro.

[0029] Step (2): Take third-generation adipose-derived stem cells, induce apoptosis using the STS method, digest with trypsin, centrifuge at room temperature and differential speed, and the resulting precipitate is the apoptotic body of the adipose-derived stem cells.

[0030] In this embodiment, the specific operation of the type I collagenase digestion method is as follows: Take adipose tissue and add an equal volume of 1 mg / ml type I collagenase solution. After digestion in a 37°C water bath for 2 hours, take the non-oil layer liquid and filter it through a 70 μm filter to remove the incompletely digested tissue. Then, centrifuge the filtered collagenase digestion solution at 1000 rpm. The resulting cell precipitate is the adipose stem cell.

[0031] In this embodiment, the specific operation of in vitro culture of adipose stem cells is as follows: the adipose stem cells are resuspended in HD-DMEM medium, plated on 10cm cell culture dishes, named F0 (primary cells), and passaged to the adipose stem cell apoptosis body induction period (3rd generation).

[0032] In this embodiment, the STS induction procedure is as follows: 3rd generation adipose-derived stem cells cultured in vitro are taken, given 5 μM STS, and induced in a 37°C incubator for 12 h to induce concentrated apoptosis of the adipose-derived stem cells.

[0033] In this embodiment, the trypsin digestion procedure is as follows: take the STS-induced adipose stem cells, add 0.25% trypsin, digest at 37°C for 2-3 minutes, and finally add complete culture medium to end the digestion.

[0034] In this embodiment, the room temperature differential centrifugation operation includes the following steps: S1, take the trypsin digest solution from step (2), centrifuge at 300 rpm for 10 min, and collect the supernatant; S2, take the supernatant collected in S1, centrifuge at 3000 rpm for 10 min, collect the precipitate, and the precipitate obtained is the apoptotic body of adipose stem cells; S3: Take the precipitate collected in S2, resuspend it in PBS, centrifuge at 3000 rpm for 10 min, and collect the precipitate; repeat S3 twice. The resulting precipitate is the purified apoptotic body of adipose stem cells.

[0035] In addition, the apoptotic bodies of the obtained adipose stem cells were resuspended in PBS and stored at 4°C for short-term storage and at -80°C for long-term storage.

[0036] The apoptotic bodies of the purified adipose-derived stem cells from step S3 were taken and observed by transmission electron microscopy. The results are as follows: Figure 2 As shown, it is spherical in shape with a double-layered biomembrane structure on the outside, and its diameter ranges from 100 to 1000 nm.

[0037] Fluorescent labeling was performed on the nuclei of fibroblasts derived from hypertrophic scars, intracellular membranous organelles, and the membranes of apoptotic bodies in adipose-derived stem cells. The results are as follows: Figure 3 As shown, under a fluorescence microscope, the nuclei of fibroblasts originating from hypertrophic scars clearly show blue fluorescence. Figure 3 a); Intracellular membrane organelles exhibit red fluorescence ( Figure 3 b); while the membrane of apoptotic bodies in adipose-derived stem cells exhibits green fluorescence (b); Figure 3 c).

[0038] The state of hypertrophic scar-derived fibroblasts after engulfing apoptotic bodies of adipose stem cells was observed using laser confocal microscopy. The results are as follows: Figure 3 As shown in d, after apoptotic bodies of adipose stem cells are phagocytosed by fibroblasts derived from proliferative scars, they are mainly distributed in intracellular membranous organelles, with a small portion freely distributed in the cell lumen.

[0039] The hypertrophic scar-derived fibroblasts used in this invention are obtained from tissues collected during clinical hypertrophic scar excision surgery / extracted and prepared using type I collagenase digestion method, and the 3rd to 5th generations are used for cell experiments.

[0040] Example 2 This embodiment examines the regulatory effect of adipose-derived stem cell apoptosis bodies provided in Example 1 on scar phenotype-related markers in hypertrophic scar-derived fibroblasts.

[0041] Specifically, the following experiments are provided in this embodiment: This experiment consisted of two groups: fibroblasts derived from hypertrophic scars were treated with PBS (control group) and apoptotic bodies of 1.0 ug / ml adipose-derived stem cells (experimental group). The biomarkers related to scar phenotype in hypertrophic scar-derived fibroblasts used in this experiment included COL1A1, COL3A1, α-SMA, Vimentin, and TGF-β1.

[0042] Protein level detection: After treatment, cells were gently washed three times with PBS. Cellular proteins were extracted using Ripa with a protease inhibitor to prepare Western blot (WB) samples. The total protein concentration of the samples was determined using BCA, and the protein concentration of each sample was adjusted to the same level. Loading buffer was added, and the samples were boiled at 100°C for 10 min. 10-20 μg of total protein was loaded per well, followed by electrophoresis at 80V for 90 min and transfer at 300mA for 100 min. The PVDF membrane was thoroughly washed with TBST (5 min × 3 times) and then blocked with rapid sealing buffer on a shaker at room temperature for 20 min. The PVDF membrane was then thoroughly washed with TBST (5 min × 3 times) and incubated overnight at 4°C with specific COL1A1, COL3A1, α-SMA, Vimentin, and TGF-β1 antibodies (1:1000 dilution). Finally, the membrane was thoroughly washed with TBST (5 min × 3 times) and incubated with horseradish peroxidase-labeled secondary antibody on a shaker at room temperature for 1 h. After thorough washing with TBST (5 min × 3 times), add developing solution for development.

[0043] Following the steps above, the expression of scar-related phenotypic marker proteins in fibroblasts originating from hypertrophic scars in the control and experimental groups was detected, with three replicates for each group. Image-J software was used to analyze protein expression, and GAPDH was used as an internal reference for quantitative result normalization. Results are presented as grayscale images and bar charts with Wb grayscale values ​​(mean ± standard deviation).

[0044] Gene level detection: After grouping, cells were gently washed three times with PBS. Total RNA was extracted from each group of cells using Trizol reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNA were measured using a UV spectrophotometer (OD ratio between 2.0 and 2.2). Based on the measured concentration, 1 μg of RNA was used for reverse transcription according to the manufacturer's instructions to obtain total cDNA. The obtained cDNA was used as a template for RT-qPCR experiments according to the manufacturer's instructions to detect the transcriptional levels of COL1A1, COL3A1, α-SMA, Vimentin, and TGF-β1 genes in proliferative scar fibroblasts. GAPDH was used as an internal control for normalization.

[0045] Following the steps outlined above, the mRNA transcription levels of scar-related phenotypic markers in hypertrophic scar-derived fibroblasts of the control and experimental groups were detected, with three replicates for each group. Results were presented as follows: -△△Ct The statistical analysis data are presented in bar charts, and the results were normalized with respect to the control group and internal reference. Significant differences are expressed as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0046] The results of the WB experiment are as follows: Figure 4 and Figure 7 As shown, compared with the control group, the bands of scar-related phenotypic markers COL1A1, α-SMA, Vimentin and TGF-β1 in the hypertrophic scar-derived fibroblasts of the experimental group were lighter in color, indicating lower protein expression levels.

[0047] WB grayscale value quantitative results (mean ± standard deviation) and statistical analysis results are as follows: Figure 5 and Figure 8 As shown in the figures, adipose-derived stem cell apoptosis bodies regulate scar-related markers from fibroblasts originating from hypertrophic scars. Specifically, adipose-derived stem cell apoptosis bodies regulate the absolute expression level of COL1A1 and also alter the relative ratio of COL3A1 and COL1A1. Figure 5 a, Figure 5 b and Figure 5 c), which is beneficial for regulating the secretory profile of the extracellular matrix and promoting the formation of a softer scar phenotype that is closer to normal skin and has better mechanical properties. Simultaneously, adipose-derived stem cell apoptosis bodies also significantly inhibit the expression of α-SMA ( Figure 5 d) weakens myofibroblast-mediated collagen contraction, which helps alleviate scar contracture. Furthermore, adipose-derived stem cell apoptosis bodies downregulate vimentin expression ( Figure 8 (b) Inhibits epithelial-mesenchymal transition (EMT)-related fibrosis. Adipose-derived stem cell apoptosis bodies reduce TGF-β1 expression levels ( Figure 8 c), while TGF-β1 is one of the key regulatory factors in the development and progression of fibrotic diseases.

[0048] RT-qPCR experimental results are as follows Figure 6 and Figure 9 As shown, adipose-derived stem cell apoptosis bodies regulate the transcription of scar-related markers in fibroblasts derived from hypertrophic scars. Specifically, adipose-derived stem cell apoptosis bodies regulate the transcription of COL1A1 (… Figure 6 a) α-SMA ( Figure 6 d), and Vimentin (9b) mRNA transcription showed a significant inhibitory effect. At the same time, the apoptotic bodies of adipose stem cells significantly regulated the relative ratio of COL1A1 and COL3A1 gene transcription. Figure 6 c).

[0049] The above experimental results confirm that the adipose stem cell apoptosis body provided in Example 1 has the ability to regulate scar phenotype-related markers in fibroblasts derived from hypertrophic scars at multiple targets, demonstrating the significant therapeutic effect of the adipose stem cell apoptosis body provided in Example 1 on hypertrophic scars.

[0050] Example 3 This embodiment tested the biosafety of the adipose stem cell apoptosis bodies provided in Example 1 and their regulatory effect on the lateral migration of fibroblasts derived from hypertrophic scars.

[0051] Specifically, the following experiments are provided in this embodiment: This experiment set up three groups, which treated the proliferative scar-derived fibroblasts after scratch modeling in 6-well plates as follows: PBS (control group), 0.5 ug / ml adipose-derived stem cell apoptotic bodies, and 1.0 ug / ml adipose-derived stem cell apoptotic bodies.

[0052] Cell scratch articulation and result recording: Fibroblasts derived from hypertrophic scars were used to construct scratch art. The specific steps were as follows: Three equidistant straight lines parallel to the long axis of the plate were marked at the bottom of each well in a 6-well plate. The hypertrophic scar fibroblasts were digested, resuspended, and counted. The cells were then seeded into 3 × 106 cells per well. 5 Cells were cultured until 100% cell density was achieved. Vertical scratches were made along the central axis of the well plate using a 200µl sterile pipette tip. The cells were gently rinsed three times with PBS, then replaced with serum-free high-glucose DMEM medium, and different treatment groups were added. The cells were incubated at 37°C in a 5% CO2 incubator. Visible light microscopy was used to photograph the cells at 0, 24, and 48 hours, and the images were processed using Image-J software to obtain the scratch healing rate for each group.

[0053] Cell viability assay: A suitable number of proliferative scar fibroblasts were digested and seeded into 96-well plates. After incubation for 24 hours, the cells adhered to the plates. The medium was then changed, and different experimental treatments were performed. The cells were cultured for another 72 hours, and the absorbance (OD value) at 450 nm was measured in different wells using the CCK-8 assay kit according to the manufacturer's instructions. Cell-free wells were designated as the control group. Cell viability was calculated using the following formula: Relative cell viability (%) = (OD value of experimental group - OD value of control group / OD value of control group - OD value of control group) × 100%.

[0054] The results of the scratch test are as follows Figure 10 As shown in the figure. Statistical analysis results of the scratch healing rate of hypertrophic scars originating from fibroblasts, as shown in the figure. Figure 11 As shown in the figure, compared with the control group, treatment with 0.5 ug / ml and 1.0 ug / ml adipose-derived stem cell apoptotic bodies for 48 hours significantly inhibited the lateral migration ability of fibroblasts derived from hypertrophic scars (P<0.05). This indicates that adipose-derived stem cell apoptotic bodies, in addition to regulating the phenotype of fibroblasts derived from hypertrophic scars, also have the ability to inhibit the lateral migration of fibroblasts derived from hypertrophic scars, and can further inhibit the expansion of hypertrophic scars.

[0055] Cell viability test results of fibroblasts derived from hypertrophic scars, such as... Figure 12 As shown, compared with the control group, there was no statistically significant difference in the activity of hypertrophic scar-derived fibroblasts after treatment with 0.5 ug / ml and 1.0 ug / ml adipose-derived stem cell apoptosis bodies for 72 hours. This indicates that adipose-derived stem cell apoptosis bodies do not directly regulate hypertrophic scar-derived fibroblasts through cytotoxicity.

[0056] The above experimental results confirm the regulatory effect of the adipose stem cell apoptosis body provided in Example 1 on the lateral migration of fibroblasts derived from proliferative scars, and also confirm the biosafety of the adipose stem cell apoptosis body provided in Example 1.

[0057] Example 4 This embodiment verifies the stability of the adipose-derived stem cell apoptotic bodies provided in Example 1 in regulating the effects of fibroblasts derived from proliferative scars. To evaluate the stability of ASCs-ApoVs in in vitro storage and repeated clinical use, we subjected the adipose-derived stem cell apoptotic bodies to common environmental enzyme treatments and freeze-thaw tolerance tests.

[0058] Specifically, the following experiments are provided in this embodiment: Western blotting (WB) experiments were conducted in four groups, treating apoptotic bodies of adipose-derived stem cells (ASCs) using the following methods: PBS (control group), ASCs treated with three freeze-thaw cycles, ASCs treated with DNase after three freeze-thaw cycles, and ASCs treated with RNase after three freeze-thaw cycles. The specific steps included: ASCs-ApoVs suspended in PBS were subjected to three freeze-thaw cycles (-80°C for 20 minutes + 37°C for 10 minutes per cycle). The RNase group received 50 μg / mL RNase A; the DNase group received 8 μg / mL DNase I; all enzyme treatments were incubated at 37°C for 60 minutes. Hypertrophic scar-derived fibroblasts were treated with different apoptotic bodies for 24 hours. Following the aforementioned WB sample preparation and experimental methods, the expression of CDC20 and scar-related phenotypic markers in hypertrophic scar-derived fibroblasts was detected among different groups, using GAPDH as an internal control. Each group had three replicates. The results are presented as bar charts with grayscale images and quantitative results of WB grayscale values ​​(mean ± standard deviation).

[0059] RT-qPCR (transcriptional level detection): Five groups were set up, including PBS (control group), apoptotic bodies of adipose-derived stem cells after three freeze-thaw cycles, apoptotic bodies of adipose-derived stem cells after three freeze-thaw cycles and treated with DNase, apoptotic bodies of adipose-derived stem cells after three freeze-thaw cycles and treated with RNase, and apoptotic bodies of adipose-derived stem cells after three freeze-thaw cycles and treated with proteinase K. Most of the treatment steps were as described above, with proteinase K concentration at 50 μg / mL and incubation at 37°C for 60 minutes.

[0060] Hypertrophic scar-derived fibroblasts were grouped and treated with different apoptotic bodies for 24 h. Following the aforementioned RT-qPCR sample preparation and experimental procedures, the transcriptional status of CDC20 and scar-related phenotypic markers in hypertrophic scar-derived fibroblasts was detected among different groups, with three replicates for each group. Results were presented as follows: -△△Ct The statistical analysis is presented in bar chart format, with the control group and internal reference normalized. Significant differences are expressed as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0061] WB test results, such as Figure 13 and Figure 14 As shown, compared with the control group, the apoptotic bodies of adipose-derived stem cells, after repeated freeze-thaw cycles and enzyme treatment, still maintained a stable regulatory effect on various markers in fibroblasts derived from proliferative scars. Specifically, the apoptotic body treatment group significantly and continuously inhibited COL1A1 (P<0.0001). Figure 14 a) α-SMA (P<0.01, Figure 14 d) CDC20 (P<0.0001, Figure 14 e) and TGF-β1 (P<0.05, Figure 14 g), while maintaining a beneficial COL3A1 / COL1A1 ratio (P<0.0001, Figure 14 c).

[0062] Gene-level testing results, such as Figure 15 As shown, similarly, compared with the control group, the apoptotic bodies of adipose stem cells after repeated freeze-thaw cycles and enzyme treatment still had a stable regulatory effect on fibroblasts derived from hypertrophic scars. Specifically, they significantly inhibited COL1A1 (P<0.0001). Figure 15 a) α-SMA (P<0.0001, Figure 15 d) CDC20 (P<0.0001, Figure 15 e) and Vimentin (P<0.0001, Figure 15f) the transcriptional level, while the COL3A1 / COL1A1 ratio was maintained at a beneficial transcriptional level (P<0.001, Figure 15 c).

[0063] The above experimental results confirm the stability of the adipose-derived stem cell apoptosis bodies provided in Example 1 during in vitro storage. This excellent environmental resistance and functional stability highlight the clinical application potential of the adipose-derived stem cell apoptosis bodies provided in Example 1.

[0064] Example 5 This embodiment examined the regulatory effect of adipose-derived stem cell apoptosis bodies provided in Example 1 on the expression of related markers in the CDC20 and Wnt / β-catenin pathways in fibroblasts derived from hypertrophic scars, revealing the specific mechanism by which adipose-derived stem cell apoptosis bodies regulate hypertrophic scar fibroblasts.

[0065] Specifically, the following experiments are provided in this embodiment: To investigate the mechanism of action of adipose-derived stem cell apoptosis bodies and their relationship with the classic scar-promoting Wnt / β-catenin pathway, two groups were set up, with fibroblasts derived from hypertrophic scars treated as follows: PBS (control group) and 1.0 μg / ml adipose-derived stem cell apoptosis bodies (experimental group). The biomarkers related to the Wnt / β-catenin pathway in scar-derived fibroblasts involved in this experiment included p-β-catenin (protein-level detection limited), β-catenin, AXIN2, c-MYC, and Cyclin-D.

[0066] Protein level detection: Western blotting (WB) samples were prepared using the aforementioned method, and protein levels of Wnt / β-catenin pathway-related biomarkers were detected. GAPDH or β-actin were used as internal controls, with three replicates per group. Results are presented as grayscale images and bar charts of quantitative WB grayscale values ​​(mean ± standard deviation). Significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0067] Gene-level detection: RT-qPCR samples were prepared using the aforementioned methods, and transcriptional markers of the Wnt / β-catenin pathway were detected. Each group had three replicates. Results are presented as 2... -△△Ct Statistical analysis is presented in bar chart format, with normalization performed using the control group and GAPDH internal reference. Significant differences are represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0068] Western blot (WB) results after treatment with apoptotic bodies in adipose-derived stem cells are as follows: Figure 16 As shown, compared to the control group, the bands for β-catenin, AXIN2, c-MYC, and Cyclin-D in the Wnt / β-catenin pathway were lighter in the experimental group, indicating lower expression levels of these proteins. Conversely, the phosphorylated β-catenin (p-β-catenin) band was darker, suggesting increased β-catenin protein levels.

[0069] Statistical analysis results of WB grayscale value quantification (mean ± standard deviation) after treatment with adipose-derived stem cell apoptosis bodies, as follows: Figure 17 As shown, compared with the control group, the apoptosis body treatment group of adipose stem cells significantly reduced the activity of the Wnt / β-catenin signaling pathway in scar-derived fibroblasts, specifically by inhibiting β-catenin ( Figure 17 b) Promotes β-Catenin protein phosphorylation and subsequent degradation. Figure 17 a) Inhibit AXIN2 ( Figure 17 c) Inhibit c-Myc ( Figure 17 d) and inhibition of Cyclin D1 ( Figure 17 e) Protein expression.

[0070] The results of RT-qPCR experiments after treatment with adipose-derived stem cell apoptosis bodies are as follows: Figure 18 As shown, this further confirms that the apoptotic body of adipose-derived stem cells affects β-catenin in the Wnt / β-catenin pathway. Figure 18 a) AXIN2 ( Figure 18 b), c-Myc ( Figure 18 c) and Cyclin D1 ( Figure 18 d) has a significant inhibitory effect on transcription.

[0071] The results of the first set of experiments verified the inhibitory effect of adipose stem cell apoptosis bodies on the activity of the Wnt / β-catenin pathway and related markers in scar-derived fibroblasts.

[0072] In the previous embodiment 2, as Figure 7 , Figure 8 a, Figure 9 a) It has been confirmed that apoptotic bodies in adipose stem cells significantly inhibit CDC20 at the protein expression and gene transcription levels in fibroblasts derived from proliferative scars.

[0073] To further investigate the relationship between CDC20 inhibition and Wnt / β-catenin pathway inhibition mediated by apoptotic bodies in adipose-derived fibroblasts of hypertrophic scar origin, two groups were set up, and the hypertrophic scar-derived fibroblasts were treated as follows: PBS (control group) and 200uM Apcin-A: a CDC20 function inhibitor (experimental group).

[0074] Protein level detection: Western blotting (WB) samples were prepared using the aforementioned method, and protein levels of Wnt / β-catenin pathway-related biomarkers were detected. GAPDH or β-actin were used as internal controls, with three replicates per group. Results are presented as grayscale images and bar charts of quantitative WB grayscale values ​​(mean ± standard deviation). Significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0075] Gene-level detection: RT-qPCR samples were prepared using the aforementioned methods, and transcriptional markers of the Wnt / β-catenin pathway were detected. Each group had three replicates. Results are presented as 2... -△△Ct Statistical analysis is presented in bar chart format, with normalization performed using the control group and GAPDH internal reference. Significant differences are represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0076] The results of the Western blot experiment under 200 μM Apcin-A treatment are as follows: Figure 19 As shown, compared with the control group, the expression levels of proteins related to Wnt / β-catenin pathway markers in the experimental group were all reduced, except for the phosphorylated β-catenin (p-β-catenin) band, which was darker.

[0077] Statistical analysis results of Wb grayscale value quantification (mean ± standard deviation) under 200uM Apcin-A processing, as follows: Figure 20 As shown, compared to the control group, the experimental group had lower levels of β-catenin in the Wnt / β-catenin signaling pathway. Figure 20 b), AXIN2 ( Figure 20 c), c-MYC ( Figure 20 d) and Cyclin-D ( Figure 20 e) was significantly downregulated, while p-β-catenin levels were significantly increased ( Figure 20 a) indicates enhanced β-catenin degradation, suggesting weakened downstream signaling.

[0078] The results of RT-qPCR experiments under 200uM Apcin-A treatment are as follows: Figure 21 As shown, this indicates that the apoptotic body of adipose stem cells plays a role in the Wnt / β-catenin signaling pathway (β-catenin). Figure 21 a) AXIN2 ( Figure 21 b), c-MYC ( Figure 21 c) and Cyclin-D ( Figure 21 d) has a significant inhibitory effect on the mRNA transcription process.

[0079] The second set of experimental results verified the inhibitory effect of 200 uM Apcin-A on the activity of the Wnt / β-catenin pathway and related markers in scar-derived fibroblasts.

[0080] Comparing the results of the first and second groups of experiments, it can be seen that the effect of the adipose stem cell apoptosis body provided in Example 1 on the Wnt / β-catenin pathway in scar-derived fibroblasts is highly consistent with the effect of Apcin-A. This strongly demonstrates that the inhibitory effect of the adipose stem cell apoptosis body provided in Example 1 on CDC20 expression is one of the main mechanisms by which it regulates the Wnt / β-catenin pathway in scar-derived fibroblasts.

[0081] like Figure 22 As shown in Example 1, the mechanism of action of adipose-derived stem cell apoptosis bodies on fibrosis-related phenotypes in hypertrophic scar fibroblasts is as follows: Adipose-derived stem cell apoptosis bodies downregulate the expression of CDC20 in hypertrophic scar fibroblasts, thereby inhibiting the Wnt / β-catenin signaling pathway, ultimately suppressing fibrosis-related phenotypes (COL1A1, α-SMA, Vimentin, TGFβ1, etc.). This highlights the multi-targeting potential of apoptosis bodies in the treatment of hypertrophic scars and also reveals the important regulatory role of CDC20 in the fibrosis process.

[0082] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0083] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing a fat stem cell apoptosome, characterized by, Includes the following steps: Step (1): Take adipose tissue and add an equal volume of 1 mg / ml type I collagenase solution. After shaking and digesting in a water bath at 37°C for 2 hours, take the non-oil layer liquid and filter it through a 70 μm filter to remove the undigested tissue. Then, centrifuge the filtered collagenase digestion solution at 1000 rpm. The resulting cell pellet is adipose stem cells and is cultured in vitro to the induction phase. Step (2): Take adipose-derived stem cells in the induction phase, give them 5 μM STS, and incubate them in a 37℃ incubator for 12 h to induce apoptosis of adipose-derived stem cells. Then add 0.25% trypsin and digest them at 37℃ for 2-3 min. Add complete culture medium to finish digestion. Collect the precipitate by differential centrifugation at room temperature. The precipitate obtained is the apoptotic body of the adipose-derived stem cells. The room temperature differential centrifugation uses a non-refrigerated centrifuge and the operation includes the following steps: S1, take the trypsin digest solution from step (2), centrifuge at 300 rpm for 10-20 min, and collect the supernatant; S2, take the supernatant collected in S1, centrifuge at 3000 rpm for 10-20 min, collect the precipitate, the precipitate obtained is the apoptotic body of adipose stem cells; S3: Take the precipitate collected in S2, resuspend it in PBS, centrifuge at 3000 rpm for 10-20 min, and collect the precipitate. Repeat S3 twice. The resulting precipitate is the purified apoptotic body of adipose stem cells.

2. The method for preparing apoptotic bodies of adipose-derived stem cells as described in claim 1, characterized in that, The induced adipose stem cells mentioned are second- to fourth generation adipose stem cells cultured in vitro.

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