A method for activation of stem cell function based on low intensity pulsed ultrasound

Abstract

This invention discloses a method for activating stem cell function based on low-intensity pulsed ultrasound, belonging to the field of stem cell technology. This invention uses low-intensity pulsed ultrasound for activation, achieving the activation effect on stem cells solely through an ultrasound therapy device. The ultrasound generator is reusable, resulting in extremely low long-term costs and significantly reducing treatment costs. Given the scarcity and high price of stem cells, this method significantly increases stem cell proliferation efficiency, leading to a substantial increase in yield. Experimental data demonstrates that using low-intensity pulsed ultrasound to stimulate various types of stem cells can enhance their proliferation, migration, and paracrine cytokine effects. It also significantly strengthens the positive influence of stem cells on other tissue cells through paracrine effects, thus achieving a good activation effect.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology, specifically relating to a method for activating stem cell function based on low-intensity pulsed ultrasound. Background Technology

[0002] In recent years, a growing body of research has confirmed that several "regenerative therapies" can improve various pathophysiological changes, including stem cell therapy, platelet-rich plasma (PRP), and micro-energy medicine, and can even control or reverse disease progression. Stem cells from various sources, such as bone marrow-derived stem cells, adipose-derived stem cells (ADSCs), urinary stem cells, and their derivatives, have been used in research on chronic disease treatment, yielding varying degrees of positive results. However, despite their regenerative effects and the hope of reversing or even curing diseases, their effectiveness remains limited. Further improvements in efficiency are needed to achieve satisfactory clinical outcomes before practical translational applications.

[0003] Therefore, how to provide new strategies to enhance the benefits of these regenerative therapies has become an important issue that urgently needs to be addressed. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a method for activating stem cell function based on low-intensity pulsed ultrasound, which uses low-intensity pulsed ultrasound treatment for activation, reduces activation costs, and increases stem cell proliferation efficiency.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for activating stem cell function based on low-intensity pulsed ultrasound, comprising the following steps: Passaged stem cells were treated with low-intensity pulsed ultrasound to obtain activated passaged stem cells. The low-intensity pulsed ultrasound treatment in S2 is performed 1-3 times. The first treatment is performed on day 0 after obtaining the passaged stem cells, the second treatment is performed on day 4 after obtaining the passaged stem cells, and the third treatment is performed on day 7 after obtaining the passaged stem cells.

[0007] The LIPUS device used was a low-intensity pulsed ultrasound therapy instrument (Beijing Wanboli) for low-intensity pulsed ultrasound (LIPUS) treatment.

[0008] Based on the above technical solution, the passaged stem cells are further obtained through the following method: Stem cells are cultured, and when the cell fusion rate reaches 80%, the cells are passaged using 0.25% trypsin to obtain the passaged stem cells.

[0009] Among them, the SD rat adipose-derived stem cell (ADSC) line, representing stem cell research, was purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd. (batch number 211123H61). This is because ADSC is recognized as having excellent paracrine function. The ADSC cell line of this batch has been identified as qualified stem cells by surface marker molecules (including CD90, CD34, CD45, CD44, CD11b / c, and CD29), and its pluripotent differentiation capacity has been confirmed by adipogenic induction differentiation, osteogenic induction differentiation, and chondrogenic induction differentiation experiments.

[0010] Based on the above technical solution, the stem cells are SD rat adipose-mesenchymal stem cells, and the culture and passage in S1 are carried out using a complete culture medium for SD rat adipose-mesenchymal stem cells containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0011] Based on the above technical solution, the passage number of the SD rat adipose-metastatic stem cells is less than 5.

[0012] Among them, the number of passages of adipose-derived mesenchymal stem cells from SD rats was less than 5 to ensure stemness.

[0013] Based on the above technical solution, the stem cells are further seeded in 100 mm culture dishes and placed in an incubator at a constant temperature of 37°C, containing 5% CO2 and with a humidity of 70%-80% for cell culture, with the culture medium being changed every 3 days.

[0014] Based on the above technical solution, the low-intensity pulsed ultrasound processing conditions are: ultrasonic frequency 1.7 mHz and intensity 200 mW / cm². 2 Pulse interval ratio 1:4, exposure for 5 min.

[0015] Based on the above technical solution, further, the pulse duration in S2 is 200 μs and the pulse interval is 800 μs.

[0016] The day on which passaged stem cells are obtained is designated as day 0.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses low-intensity pulsed ultrasound treatment for activation, and only the ultrasound therapy device is used to activate stem cells. The ultrasound generator can be reused, and the long-term cost is extremely low, which greatly reduces the cost of treatment. Stem cells are scarce and expensive. The method of this invention significantly increases the proliferation efficiency of stem cells, which can greatly increase their yield.

[0018] 2. This invention uses low-intensity pulsed ultrasound treatment for activation. Experimental data demonstrates that using low-intensity pulsed ultrasound to intervene and stimulate various types of stem cells can enhance their proliferation, migration, and paracrine cytokine effects; it significantly strengthens the positive influence of stem cells on other tissue cells through paracrine effects, thus achieving a good activation effect.

[0019] 3. This invention does not involve direct contact with stem cells, but rather uses ultrasound intervention. The mechanical parameters of the low-intensity pulsed ultrasound can be changed to different mechanical and temporal parameters to meet the growth needs of different types of stem cells, aiming to achieve the best activation effect and activation maintenance time, so that the activated stem cells can be applied at the best time. It can also be easily combined with other future technologies to achieve a greater degree of activation effect on stem cell function.

[0020] 4. This invention demonstrates the effects of low-intensity pulsed ultrasound on the transcriptome of stem cells through experimental data, and proposes the concepts of stem cell activation maintenance and optimal application timing; Hippo and other signaling pathways of stem cells are activated, and cell proliferation, apoptosis, paracrine effects, etc. are positively regulated; activated stem cells can enhance their ability to influence endothelial cells, effectively promote their proliferation, migration, and vascularization functions of endothelial cells, regulate their oxidative stress level, and confirm that their functions are activated.

[0021] 5. This invention, through a comparison of different frequency schemes, confirms that higher frequency LIPUS intervention stimulation can achieve a better activation effect. Attached Figure Description To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0022] Figure 1 This is a schematic diagram of different time points of LIPUS intervention in Embodiment 1 of the present invention; Figure 2 The following is a heatmap of gene expression patterns of ADSC in Example 1 of the present invention: A is a schematic diagram of the differences between groups G2 and G1, B is a schematic diagram of the differences between groups G3 and G1, and C is a schematic diagram of the differences between groups G4 and G1. Figure 3 The following are gene differential expression volcano diagrams for Group G1 as the control and Groups G2-4 in Example 1 of the present invention: A is the gene differential expression volcano diagram for G2, B is the gene differential expression volcano diagram for G3, C is the gene differential expression volcano diagram for G4, and D is a schematic diagram of obtaining 63 common DEGs by taking the intersection of differential genes in each group through Venn diagram. Figure 4In Embodiment 1 of the present invention, the vertical axis represents the name of each function / pathway; the larger the bubble in the bubble chart, the more differentially expressed genes there are; and the color gradient represents... P The larger the value and the darker the color, the more significantly the function / pathway is enriched. G2-G4 vs. G1 differentially expressed gene function enrichment map: A is the result of biological process analysis, B is the result of analysis, C is the result of molecular function analysis, and D is the result of pathway enrichment analysis from the Kyoto Encyclopedia of Genes and Genomes. Figure 5 In Embodiment 1 of the present invention, orange-yellow nodes represent a KEGG term, gray nodes represent differentially expressed genes enriched in each term, and the size of orange-yellow nodes is proportional to the number of differentially expressed genes enriched in that pathway. A network diagram is drawn for the top 10 KEGG terms with the most significant KEGG enrichment results. Figure 6 This is a schematic diagram of the relative mRNA expression levels (lg FPKM) of some Hippo pathway, cytokines, and apoptosis-related molecules in Example 1 of the present invention. In the diagram: ns: P >0.05, P <0.05, P <0.01, P <0.001; Figure 7 This is a schematic diagram of the supernatant extracted from LIPUS-treated mesenchymal stem cells (MSCs) and incorporated into the culture system of HUVECs. Figure 8 The dashed line in Example 2 of this invention represents different oxygen concentration levels (20% normoxic concentration on the left and 5% hypoxic concentration on the right). The CCK-8 test, with the NC group and Hypoxia group as control groups, confirmed that LIPUS enhanced the effect of ADSC supernatant in promoting HUVEC proliferation. In the picture: P <0.01, P <0.0001; Figure 9The Transwell migration assay in Example 2 of this invention, where the dashed lines represent different oxygen concentration levels (left side is normoxic concentration 20%, right side is hypoxic concentration 5%), confirms that LIPUS enhances the effect of ADSC supernatant in promoting HUVEC migration. The results are shown in Figure A, a representative figure of HUVEC cell migration in different treatment groups under a 200x light microscope, and Figure B, a statistical graph of the number of HUVEC cells that migrated in different treatment groups. In the picture: P <0.05, P <0.001, P <0.0001; Figure 10 The results of TUNEL fluorescence analysis on both sides of the dashed line in Example 2 of the present invention (left side is normoxic concentration 20%, right side is hypoxic concentration 5%) confirm that LIPUS enhances the effect of ADSC supernatant in inhibiting HUVEC apoptosis. Figure A is a representative image of TUNEL apoptosis marker fluorescence staining of HUVEC cells in different treatment groups, and B is a statistical graph of the TUNEL fluorescence positive signal level of HUVEC cells in different treatment groups. In the picture: P <0.05, P <0.01, P <0.0001; Figure 11 The dashed lines in Example 2 of this invention represent different oxygen concentration levels (left side is normoxic concentration 20%, right side is hypoxic concentration 5%). The results of flow cytometry analysis of ROS generation in HUVECs and the confirmation of the effect of LIPUS in enhancing ADSC supernatant to improve the oxidative stress level of HUVECs are shown in the figure: A is a representative graph of ROS flow cytometry analysis of HUVECs in different treatment groups, and B is a statistical graph of ROS signal levels of HUVECs in different treatment groups. In the picture: P <0.05, P <0.01; Figure 12The following figures illustrate the tubule formation experiment of Example 2 of this invention, which confirms that LIPUS enhances the effect of ADSC supernatant in promoting HUVEC vascularization: A is a representative figure of the tubule formation experiment of HUVEC cells in different treatment groups under dark field with a 40x light microscope; B is a statistical diagram of the number of vascular nodes in HUVEC cells in different treatment groups; C is a statistical diagram of the number of vascular branches in HUVEC cells in different treatment groups; and D is a statistical diagram of the number of vascular connections in HUVEC cells in different treatment groups. In the picture: P <0.05, P <0.01. Detailed Implementation

[0023] Numerous studies have confirmed that LIPUS can produce positive biological effects on various cells, such as promoting the proliferation and differentiation of satellite cells and osteoblasts, promoting Schwann cell proliferation, and promoting myelin gene expression. Therefore, this invention aims to utilize LIPUS to stimulate stem cells through mechano-biological signal coupling, thereby activating their therapeutic functions, maximizing tissue repair, and subsequently generating biological effects such as promoting angiogenesis and neurogenesis, thereby treating chronic diseases.

[0024] Therefore, this invention has demonstrated the following questions through a series of experiments: ① Does LIPUS produce biological effects on ADSCs, and what are those effects? ② Can LIPUS activation of ADSCs have a positive effect on other tissue cells through paracrine effects? ③ How can LIPUS stimulation better activate ADSCs? Through our experimental demonstration, we can propose the core content of our invention—a method for activating stem cell function based on low-intensity pulsed ultrasound.

[0025] To achieve the best activation effect, functional verification experiments were all conducted within 24 hours of the last stimulation.

[0026] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0027] Example 1 This embodiment verifies that LIPUS intervention produces a wide range of biological effects on ADSC.

[0028] To explore the effects and molecular mechanisms of LIPUS intervention on ADSC, we will use transcriptome sequencing to analyze the biological effects of LIPUS at different frequencies on ADSC, identify key pathways from differentially expressed genes, and analyze the molecular basis of LIPUS's effects.

[0029] like Figure 1 As shown, groups G2, 3, and 4 received 1, 2, and 3 LIPUS treatments, respectively. The day of the last subculture was designated as day 0, and the 1st to 3rd stimuli were performed on days 7, 4&7, and 0&4&7, respectively, while group G1 received no treatment.

[0030] 24 h after the last LIPUS stimulation, cells were digested with trypsin and collected, and transcriptome sequencing was performed on ADSCs from each group.

[0031] RNA extraction, library preparation, and sequencing Total RNA was extracted from ADSCs using Trizol, and the A260 / A280 ratio was measured using a nanospectrophotometer to reflect RNA purity. RNA integrity and the presence of DNA contamination were assessed using agarose gel electrophoresis. Preliminary library quantification was performed using a Qubit 2.0 Fluorometer, diluting the library to 1.5 ng / μL. Subsequently, the insert size of the library was determined using an Agilent 2100 bioanalyzer. Once the expected size was met, the effective concentration of the library was accurately quantified by qRT-PCR (effective concentration greater than 2 nM) to ensure library quality.

[0032] We use Trussq TM The RNA Sample Prep Kit is a kit for mRNA library preparation. Library preparation is performed according to the instructions. To eliminate potential repetitive errors during PCR and sequencing, cDNA molecules are pre-labeled with a specific molecular identifier (consisting of 8 random bases) before amplification. Finally, the library product corresponding to 200-500 base pairs is fed into the HiSeq sequencing platform for sequencing-while-synthesizing.

[0033] Quality control of transcriptome sequencing data To obtain high-quality sequencing fragments, the raw sequencing data needs to be quality filtered. The specific steps are as follows: ① Remove the adapter sequence from the reads; ② Remove non-AGCT bases at the 5' end before splicing; ③ Trim the ends of reads with low sequencing quality (sequencing quality value less than Q20); ④ Remove reads containing N up to 10%; ⑤ Discard adapters and small fragments less than 25 bp in length after quality trimming. By performing these steps, we have essentially eliminated the bias and errors caused by PCR amplification and transcriptome sequencing. Subsequently, based on the HISAT2 algorithm...

[22] Clean sequencing fragments are mapped onto a rat reference genome. By calculating the number of clean reads mapped to the reference genome region, the "fragments per kilobase of exon model per million mapped fragments" (FPKM) for each gene in the sample can be obtained, which represents the expression level of each gene in the sample.

[0034] Bioinformatics Analysis The “DESeq2” software package was used to identify differentially expressed genes (DEGs) between groups, using |log2Fold Change|>= 1.00 and FDR<0.05 as criteria. Venn plots were used to calculate the intersection of differentially expressed DEGs. The “pheatmap” software package was used for visualization of pattern clustering heatmaps. Genetic functional analysis, including enrichment analysis of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), was constructed using the online tool “Database for Annotation, Visualization, and Integrated Discovery” (DAVID). The protein-protein interaction (PPI) network of differentially expressed DEGs was generated from the STRING database (version 11.0). Fisher tests were used to detect relationships between gene pairs.

[0035] The results for this section are as follows: 1. Differential gene identification To determine the role of LIPUS in ADSC culture, we performed transcriptome sequencing on four groups of ADSCs (n = 3 per group). Gene expression pattern clustering heatmap ( Figure 2 In the figure, each column represents a sample, each row represents a gene, and the color depth represents the expression level (logarithm). 10 (FPKM), red indicates high gene expression in the sample, green indicates low expression. The data shows that all three biological replicates within each group were clustered into the same cluster, and significant differential gene expression was observed even with G1 as the control group. This not only indicates good reproducibility of the cell culture procedure but also confirms that LIPUS can significantly affect the mRNA expression profile of ADSCs. Furthermore, group G4 showed a wider range of gene expression differences compared to group G1. Figure 2 C) indicates that higher frequency LIPUS processing has a more significant impact on ADSCs.

[0036] We used R software to identify the DEGs between groups. Using group G1 as the control group, 705 DEGs were identified in group G2, of which 187 mRNAs were upregulated and 518 mRNAs were downregulated; 429 DEGs were identified in group G3, of which 208 mRNAs were upregulated and 221 mRNAs were downregulated; and 5067 DEGs were identified in group G4, of which 2502 mRNAs were upregulated and 2565 mRNAs were downregulated. Figure 3 AC). Venn diagram shows ( Figure 3 (D) Compared with the G1 control group, 63 common DEGs showed differential expression in all groups, suggesting that these genes are likely key molecules stably affected by LIPUS treatment and thus maintain differential expression patterns under different frequencies of stimulation.

[0037] 2. Functional enrichment analysis We performed functional enrichment analysis on the aforementioned common DEGs. For example... Figure 4As shown, in terms of biological processes (BP), terms such as "biosynthetic processes," "gene expression," "gene expression regulation," "metabolic processes," "regulation of metabolic processes," "positive regulation of biological processes," "negative regulation of biological processes," and "positive regulation of cellular processes," as well as many related terms, are significantly enriched. In terms of cell composition (CC), terms such as "nucleus," "nucleoplasm," "cytoplasm," "endomembrane system," "protein complexes," "organelles," "cytoplasm," and "cytoskeleton" are involved. In terms of molecular function (MF), terms such as "nucleic acid binding," "catalytic activity," "protein binding," "enzyme binding," "ion binding," "organocyclic compound binding," "ATP binding," "sugar derivative binding," "nucleotide binding," "transcription factor binding," and "transferase activity" are involved.

[0038] Furthermore, according to the KEGG pathway enrichment analysis, pathways such as "ribosomes," "glucagon signaling pathway," "endocytosis," "Hippo signaling pathway-multi-species," "cell cycle," "Hippo signaling pathway," "aminoacyl-tRNA biosynthesis," "apoptosis," "Hedgehog signaling pathway," "glutathione metabolism," and "phosphatidylinositol signaling system" were significantly enriched, suggesting that LIPUS can broadly influence multiple signaling pathways in ADSC. Figure 4 D). Both the Hippo and Hedgehog pathways have been shown to be modulated by mechanostimulation, consistent with the characteristics of LIPUS. Network mapping revealed a more significant enrichment of the Hippo pathway (see [link to network diagram]). Figure 5 Furthermore, the term "Hippo signaling pathway-multi-species" was also enriched. Therefore, this study will further explore this pathway and analyze the specific molecular mechanism by which LIPUS acts on ADSCs.

[0039] Figure 5 A network diagram was constructed for the top 10 KEGG terms with the most significant KEGG enrichment results, where each orange-yellow node represents a KEGG term. The gray nodes represent differentially expressed genes enriched in each term. The size of the orange-yellow nodes is proportional to the number of differentially expressed genes enriched in that pathway.

[0040] 3. Expression levels of the Hippo pathway, cytokines, and apoptosis-related molecules Furthermore, we present the expression of some key Hippo pathways, cytokines, and apoptosis-related molecules. For example... Figure 6 As shown, the ADSC values ​​for each group are... Yap1, Taz, Tead1, Tead2, Tead3, Casp3, Vegfa, VegfSignificant differential expression of genes ( P (All values ​​<0.05). Furthermore, the expression levels of these molecules mostly showed an increasing or decreasing trend across groups G1-G4. Meanwhile, in the post-hoc analysis of variance (see Table 1.3), the expression of these molecules in group G4 showed significant differences compared to group G1, indicating that the transcriptome of ADSCs under three LIPUS stimulations underwent more significant changes. These results suggest that LIPUS may, to some extent, affect the apoptosis level and cytokine secretion of ADSCs, and is involved in the regulation of the Hippo pathway, with a potential dose-response relationship between the frequency of LIPUS stimulation and the individual's response.

[0041] Table 1.3 Post-hoc analysis of expression levels of some key mRNAs from transcriptome sequencing

[0042] Example 2 This embodiment verifies that LIPUS enhances the effect of ADSC supernatant on HUVEC cells.

[0043] We incorporated the culture supernatant of ADSCs from various groups into the culture system of human umbilical vein endothelial cells (HUVECs) and observed their phenotypic changes under normal oxygen and hypoxic conditions to explore whether the survival and function of endothelial cells could be improved, thereby reflecting the application potential of the combined regimen for the treatment of erectile dysfunction (ED).

[0044] The HUVECs used in this study were purchased from Jinyuan Biotechnology Co., Ltd. (Shanghai, China). Endothelial cell medium (ECM) containing 10% fetal bovine serum and 1% penicillin / streptomycin was used. Cells were cultured in 100 mm tissue culture dishes at 37°C in a humid environment with 5% CO2. The medium was changed every 3 days. When cell confluence reached 80%, the cells were passaged once with 0.25% trypsin.

[0045] HUVECs were divided into 6 groups for culture. Based on ECM, supernatant from ADSCs of groups G1-G4 was added to the HUVEC culture system of 4 of the groups at a 1:1 ratio, designated as NC group, L1 group, L2 group, and L3 group, respectively. The remaining two groups were cultured in a tri-gas incubator under a low-oxygen concentration (5% O2) environment, and the supernatant from group G4 was added to group 5 at the above ratio (e.g., Figure 7(As shown in the image), Group 6 was supplemented with an equal volume of culture medium, and was designated as the L3+Hypoxia group and the Hypoxia group, respectively. Further experiments were conducted after 48 hours of treatment.

[0046] The results for this section are as follows: 1. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC proliferation. To observe the effects of ADSC paracrine factors on HUVEC cell behavior and survival, and the effect of LIPUS on ADSC, we first performed a CCK8 assay to observe the proliferation capacity of HUVECs. Figure 8 Under normoxic culture conditions, compared with the NC group, the survival rates of HUVECs in the L1-L3 groups increased by 1.26 times, 1.47 times, and 3.34 times, respectively (overall). P <0.0001); Under hypoxic conditions, the survival rate of HUVECs in the L3+Hypoxia group was upregulated to 3.43 times that in the Hypoxia group by ADSC supernatant, which was statistically significant. P <0.0001). This indicates that LIPUS can enhance the effect of ADSC supernatant in promoting HUVEC proliferation, and this effect is strengthened with increasing LIPUS treatment frequency. Under pathological conditions simulated by hypoxia, this proliferative effect reached the same level, suggesting that LIPUS-pretreated ADSCs still have good application potential under certain pathological conditions.

[0047] 2. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC migration. To observe the changes in the impact of ADSC supernatant on HUVEC migration ability under LIPUS, we conducted a Transwell migration experiment. Figure 9 The results showed that, compared with the NC group, the migration ability of HUVEC cells in the L1 group did not change significantly, while the migration ability of HUVEC cells in the L2 group was upregulated. P =0.0209), while the migration ability of the L3 group was significantly enhanced ( P <0.0001); Under hypoxic conditions, the ADSC supernatant of group G4 also enhanced the migration ability of HUVECs in group L3 ( P <0.0001). The above results suggest that LIPUS can enhance the effect of ADSC supernatant in promoting HUVEC migration, even under hypoxic conditions, and this effect is strengthened with increasing LIPUS treatment frequency.

[0048] 3. LIPUS enhances the inhibitory effect of ADSC supernatant on HUVEC apoptosis. Furthermore, we used the TUNEL assay to detect the apoptosis of HUVECs under supernatant incorporation. Figure 10 Overall, the results showed significant differences in apoptosis levels among the groups. P <0.0001). Compared with the NC group, there was no significant difference in the apoptosis level of HUVEC cells in the L1 and L2 groups, while the apoptosis fluorescence signal level in the L3 group was significantly downregulated ( P =0.0054). Under hypoxic conditions, the overall apoptosis level of HUVECs increased, while the ADSC supernatant in the G4 group did not significantly improve HUVEC apoptosis ( P> The concentration of HUVECs decreased by 0.05%, but showed a trend of improvement. These results indicate that more frequent LIPUS treatment significantly enhanced the effect of ADSC paracrine factors in reducing HUVEC apoptosis.

[0049] 4. LIPUS enhances the effect of ADSC supernatant in improving the oxidative stress level of HUVECs. To observe the effect of ADSC supernatant on the oxidative stress level of HUVECs under LIPUS treatment, we used flow cytometry to assess the ROS levels of different treatment groups to reflect changes in their oxidative stress levels. Figure 11 Overall, the results showed significant differences in ROS generation levels among the groups. P = 0.0010). Compared with the NC group, there was no significant difference in ROS signal intensity of HUVEC cells in the L1 group, while the ROS signal intensity in the L2 and L3 groups was significantly downregulated ( P All values ​​were <0.05. No significant differences were observed under hypoxic conditions. P> 0.05). The above results indicate that more frequent LIPUS treatment enhances the effect of ADSC supernatant in improving the oxidative stress level of HUVECs.

[0050] 5. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC vascularization. Vascularization is one of the functions of HUVECs as endothelial cells. Therefore, we used a tubule formation assay to observe the changes in the effect of ADSC supernatant on the vascularization function of HUVECs under the action of LIPUS. Figure 12 Overall, the analysis of variance results showed that the number of vascular nodes in each group ( P = 0.0028), number of branches ( P = 0.0107) and the number of connections ( P The level of HUVEC cells (= 0.0093) showed a significant difference. In particular, compared to the NC group, the number of vascular nodes in HUVEC cells in the L3 group ( P = 0.0034), number of branches ( P=0.0177) and the number of connections ( P = 0.0105) levels were significantly increased. These results indicate that more frequent LIPUS treatment enhances the effect of ADSC supernatant in improving the vascularization function of HUVECs.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for activating stem cell function based on low-intensity pulsed ultrasound, characterized in that, Includes the following steps: Passaged stem cells were treated with low-intensity pulsed ultrasound to obtain activated passaged stem cells. The low-intensity pulsed ultrasound treatment in S2 is performed 1-3 times. The first treatment is performed on day 0 after obtaining the passaged stem cells, the second treatment is performed on day 4 after obtaining the passaged stem cells, and the third treatment is performed on day 7 after obtaining the passaged stem cells.

2. The method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 1, characterized in that, The passaged stem cells were obtained through the following methods: Stem cells are cultured, and when the cell fusion rate reaches 80%, the cells are passaged using 0.25% trypsin to obtain the passaged stem cells.

3. A method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 2, characterized in that, The stem cells are SD rat adipose-metastatic stem cells. The culture and passage in S1 are carried out using a complete culture medium for SD rat adipose-metastatic stem cells containing 10% fetal bovine serum and 1% penicillin / streptomycin.

4. The method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 3, characterized in that, The number of passages of the adipose-derived mesenchymal stem cells from the SD rats was less than 5.

5. A method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 1, characterized in that, The stem cells were seeded in 100 mm culture dishes and placed in an incubator at a constant temperature of 37 ℃, containing 5% CO2 and with a humidity of 70%-80% for cell culture. The culture medium was changed every 3 days.

6. A method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 1, characterized in that, The low-intensity pulsed ultrasound treatment conditions are: ultrasonic frequency 1.7 mHz and intensity 200 mW / cm². 2 Pulse interval ratio 1:4, exposure for 5 min.

7. A method for activating stem cell function based on low-intensity pulsed ultrasound according to claim 6, characterized in that, In S2, the pulse duration is 200 μs and the pulse interval is 800 μs.

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