Adipose tissue active material as well as preparation method and application thereof

Abstract

The invention relates to an adipose tissue active material as well as a preparation method and application thereof, and the adipose tissue active material is prepared by the following method: (1) taking adipose tissue and cutting into pieces; (2) adding 0.05 wt%-0.1 wt% of a type I collagenase solution, uniformly mixing, and digesting; (3) centrifuging a digestion product at 1000g-3000g for 5-15 minutes, and taking a middle-layer clear liquid 1; and (4) centrifuging the middle-layer clear liquid 1 at 2000g-3000g for 5-10 minutes, taking middle-layer clear liquid 2, filtering, and taking filtrate, so as to obtain the traditional Chinese medicine composition. The adipose tissue active material retains more bioactive components, has better anti-oxidation, anti-apoptosis and inflammation regulation effects, and has great application prospects in treatment of tissue repair related diseases including myocardial infarction, acute kidney injury, acute liver injury and the like; and the preparation method is efficient, simple and short in time consumption.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to adipose tissue active materials, in particular to adipose tissue active materials and a preparation method and application thereof. Background Art

[0002] For a long time, adipose tissue has been regarded as medical waste and its physiological functions have not been taken seriously; however, in recent years, adipose tissue has been found to have powerful endocrine functions and can secrete a series of biologically active substances, called "adipokines". Adipokines including adiponectin, resistin, chemokines and other cytokines (growth factors, inflammatory-related factors) have been found to have tissue repair effects such as antioxidant, anti-apoptotic and inflammatory regulation. A large number of clinical and basic medical studies have shown that common human diseases such as cardiovascular disease, inflammation, malignant tumors, diabetes and arteriosclerosis are all related to oxidative damage caused by reactive oxygen species. Excessive reactive oxygen species can damage proteins, lipids, and nucleic acids in cells, leading to cell apoptosis and activating inflammation, all of which can cause or aggravate tissue damage. Therefore, antioxidant, anti-apoptotic and inflammatory regulatory substances can be used for tissue repair. In addition, adipose tissue is rich in sources and easy to obtain, making it an ideal source of tissue repair materials. It can be seen that it has great application potential in the field of tissue repair.

[0003] At present, there are many forms of adipose tissue-derived biomaterials used for tissue repair, which can be mainly divided into two categories. One is to use adipose tissue-derived cells for treatment, such as adipose mesenchymal stem cells, stromal vascular fraction (SVF) and micro-fragmented adipose tissue. This category mainly uses the cell paracrine effect for tissue repair, but there are problems such as low cell retention rate, poor vitality, unstable treatment effect and immunogenicity. The other is to use adipose tissue-derived extracellular matrix and / or its secreted bioactive components for treatment, such as adipose decellularized extracellular matrix and adipose-derived extracellular vesicles. However, the preparation process of adipose decellularized extracellular matrix is ​​cumbersome, time-consuming and prone to loss of bioactive components. Adipose-derived extracellular vesicles have problems such as low yield and high preparation cost.

[0004] Therefore, in order to more fully exert and utilize the tissue repair function of adipose tissue, it is necessary to develop an adipose tissue-derived biomaterial that is efficient, simple, and can fully retain the bioactive components in fat. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an adipose tissue active material (ATAM), which has a higher content of bioactive components, better tissue repair effect, and is simple and fast to prepare.

[0006] The first aspect of the present invention is to provide a method for preparing an adipose tissue active material, comprising the following steps:

[0007] (1) Take adipose tissue and cut it into pieces;

[0008] (2) adding 0.05 wt% to 0.1 wt% type I collagenase solution, mixing, and digesting;

[0009] (3) The digestion product is centrifuged at 1000g to 3000g for 5 min to 15 min, and the middle layer of supernatant 1 is collected;

[0010] (4) Centrifuge the middle layer clear liquid 1 at 2000g-3000g for 5min-10min, take the middle layer clear liquid 2, filter it, and take the filtrate to obtain the product.

[0011] In some embodiments, in step (2), the digestion is carried out at 35-45°C for 0.5-3 h; preferably, the digestion is carried out at 36-40°C for 1-2 h.

[0012] In some embodiments, the centrifugal force in step (3) is less than that in step (4).

[0013] In some embodiments, step (3) is centrifuged at 1000 g to 2000 g for 5 min to 10 min.

[0014] In some embodiments, step (4) is centrifuged at 2500 g to 3000 g for 5 min to 10 min.

[0015] In some embodiments, the volume of the type I collagenase solution is 1 to 2 times the volume of the adipose tissue.

[0016] In some embodiments, the pore size of the filter material used in the filtration in step (4) is 100 μm to 200 μm, preferably 150 μm to 200 μm.

[0017] In some embodiments, step (4) further comprises freeze-drying the filtrate.

[0018] The second aspect of the present invention is to provide an adipose tissue active material prepared by the preparation method as described above.

[0019] The third aspect of the present invention is to provide the use of the above-mentioned adipose tissue active material in the preparation of anti-oxidation, anti-apoptosis and / or anti-inflammatory drugs.

[0020] The fourth aspect of the present invention is to provide the use of the above-mentioned adipose tissue active material in the preparation of tissue repair medicines or products, which can be used in various forms such as direct vascular injection and compounding with hydrogel.

[0021] In some of the embodiments, the application includes preparing a drug or product for treating myocardial infarction, acute kidney injury and / or acute liver injury.

[0022] The fifth aspect of the present invention is to provide a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient includes the adipose tissue active material as described above.

[0023] The present invention can effectively extract and separate the bioactive components in the adipose tissue by digesting the adipose tissue with a type I collagenase solution of a suitable concentration (0.05wt% to 0.1wt%), and then centrifuging twice under specific conditions, thereby removing components such as oil and cells, and preparing the high-purity adipose tissue active material of the present invention, which can retain about 94% of the protein substances in natural adipose tissue. Compared with the adipose decellularized extracellular matrix, the adipose tissue active material of the present invention retains more bioactive components, has better antioxidant, anti-apoptosis and inflammation regulation effects, and has great application prospects in the treatment of tissue repair-related diseases, including myocardial infarction, acute kidney injury and acute liver injury.

[0024] The preparation method of the adipose tissue active material of the present invention is simple and time-saving; the raw material adipose tissue is abundant and easily obtained, and large-scale preparation can be achieved in a short time. Compared with the preparation of adipose decellularized extracellular matrix and adipose-derived extracellular vesicles, the preparation is simpler and more efficient.

[0025] The adipose tissue active material of the present invention removes cellular components from the material and can be stored for a long time under low-temperature conditions after freeze-drying. Compared with therapies based on adipose tissue-derived cells, the adipose tissue active material has lower immunogenicity and more stable therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preparation and characterization diagram of ATAM; A is the prepared powdered ATAM; B is the TEM result; C is the particle size result of ATAM detected by nanoparticle size analyzer.

[0027] Figure 2 These are the proteomic results of adipose tissue active material (ATAM), natural adipose tissue (NAT), and adipose decellularized extracellular matrix (AECM); A is the Venn diagram of protein identification results; B is the clustering heat map of protein expression levels; and C is the volcano map of differential proteins between ATAM and AECM.

[0028] Figure 3 Results of GO function enrichment analysis and KEGG pathway enrichment analysis of proteins upregulated in ATAM relative to AECM expression levels.

[0029] Figure 4The figure shows the cell compatibility results of ATAM at different concentrations; A is the result of cytotoxicity test; B is the result of cell live-dead staining; the Contro1 group is cells cultured in complete medium; 0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL represent cells cultured in different concentrations of ATAM.

[0030] Figure 5 The results are the characterization of the protective effect of ATAM on cellular oxidative stress; wherein, A is the result of ATAM in vitro hydroxyl radical (·OH) scavenging experiment; B is the result of ATAM in vitro DPPH radical scavenging experiment; C is the protective effect of ATAM on H2O2-induced oxidative stress in H9C2 cells; D is the inhibitory effect of ATAM on H2O2-induced apoptosis of H9C2 cells; B1ank group is healthy cells without treatment; Contro1 group is cells induced with oxidative stress using H2O2.

[0031] Figure 6 To characterize the regulatory effect of ATAM on macrophage polarization, flow cytometry was used to detect the expression of CD86 (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages).

[0032] Figure 7 Typical echocardiographic images and important cardiac function parameters of rats with myocardial infarction 28 days after treatment; among them, the Sham group was a sham operation group, used as a healthy control; the MI group was a negative control group without treatment after myocardial infarction surgery modeling; the CP group was a heart patch treatment group without ATAM; and the CP-A group was a heart patch treatment group with ATAM.

[0033] Figure 8 Representative images of Masson staining of rat heart tissue after 28 days of treatment and statistical results of myocardial infarction area and left ventricular wall thickness; among them, the Sham group was a sham operation group, used as a healthy control; the MI group was a negative control group without treatment after myocardial infarction surgery; the CP group was a heart patch treatment group without ATAM; and the CP-A group was a heart patch treatment group with ATAM.

[0034] Fig. 9Representative optical images of the kidneys on the 8th day of treatment; the Healthy group was the healthy control; the AKI+PBS group was the negative control group with cisplatin-induced renal injury and no treatment; the AKI+ATAM5 group was the treatment group with cisplatin-induced renal injury and injected with 5 mg / mL ATAM for 6 days; the AKI+ATAM10 group was the treatment group with cisplatin-induced renal injury and injected with 10 mg / mL ATAM for 6 days; the AKI+NAC group was the positive control group with cisplatin-induced renal injury and injected with 160 mg / kg N-acetylcysteine ​​(NAC) for 6 days.

[0035] Fig.10 These are the test results of creatinine (CREA) in the serum biochemical indicators of mice on the 8th day of treatment; among them, the Healthy group was the healthy control; the AKI+PBS group was the negative control group with cisplatin-induced renal injury and no treatment; the AKI+ATAM5 group was the treatment group with cisplatin-induced renal injury and injected with 5 mg / mL ATAM for 6 days; the AKI+ATAM10 group was the treatment group with cisplatin-induced renal injury and injected with 10 mg / mL ATAM for 6 days; the AKI+NAC group was the positive control group with cisplatin-induced renal injury and injected with 160 mg / kg N-acetylcysteine ​​(NAC) for 6 days. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0039] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0040] In some embodiments of the present invention, a method for preparing an adipose tissue active material is provided, comprising the following steps:

[0041] (1) Take adipose tissue and cut it into pieces;

[0042] (2) adding 0.05 wt% to 0.1 wt% type I collagenase solution, mixing, and digesting;

[0043] (3) The digestion product is centrifuged at 1000g to 3000g for 5 min to 15 min, and the middle layer of supernatant 1 is collected;

[0044] (4) Centrifuge the middle layer clear liquid 1 at 2000g-3000g for 5min-10min, take the middle layer clear liquid 2, filter it, and take the filtrate to obtain the product.

[0045] The preparation method of the active material of adipose tissue of the present invention can effectively extract the bioactive components in adipose tissue, especially the active components related to anti-oxidation, anti-apoptosis and inflammation regulation, by first digesting the adipose tissue with type I collagenase of appropriate concentration (0.05wt% to 0.1wt%); and then further combine the centrifugation under two specific conditions, the first centrifugation (1000g to 3000g centrifugation for 5min to 15min) to remove the grease and undigested fat first, and the second centrifugation with a larger centrifugal force (2000g to 3000g centrifugation for 5min to 10min) to remove the remaining grease and cell components. Through two differential centrifugations, the grease, undigested fat and cell components in the digestion product can be effectively removed to obtain a high-purity active material of adipose tissue and reduce immunogenicity.

[0046] The adipose tissue active material prepared by the method of the present invention can retain about 94% of the protein substances in natural adipose tissue. Compared with adipose decellularized extracellular matrix, the expression of multiple proteins related to cell redox homeostasis, reaction with hydrogen peroxide, regulation of endothelial barrier establishment, endothelial cell activation, cell apoptosis process, oxidoreductase activity, cell proliferation promotion, actin skeleton organization and intracellular protein transport in the adipose tissue active material is upregulated. The upregulated proteins are mainly involved in the biosynthesis of amino acids, cell apoptosis, actin skeleton regulation, cell-cell (or ECM) interaction (tight junction, cell adhesion molecules, focal adhesion) and metabolic pathways related to mitochondrial function and sugar metabolism (such as oxidative phosphorylation, TCA cycle, glycolysis / gluconeogenesis). Therefore, the adipose tissue active material of the present invention has better anti-oxidation, anti-apoptosis and inflammation regulation effects.

[0047] The present invention is further described in detail below with reference to specific embodiments.

[0048] In the following examples and comparative examples, a PBS buffer having a pH value of 7.2 to 7.4 was used to prepare a type I collagenase solution, wherein the PBS buffer contained Ca2 + (the concentration may be 0.3mmol / L to 0.5mmol / L, preferably 0.45mmol / L in the following examples and comparative examples) and Mg 2+ (the concentration can be 0.4mmol / L to 0.6mmol / L, preferably 0.5mmol / L in the following examples and comparative examples), Ca2+ can be derived from CaCl2, Mg 2+ Can be derived from MgCl2.

[0049] Example 1 Preparation of adipose tissue active material (ATAM)

[0050] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0051] 2) preparing a 0.05 wt % type I collagenase solution;

[0052] 3) Add 1.5 times the volume of the type I collagenase solution to the centrifuge tube containing the adipose tissue, mix well and digest at 37° C. for 2 h;

[0053] 4) After digestion, centrifuge at 1500g for 5 min, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid;

[0054] 5) Centrifuge at 2500 g for 10 min, take the middle layer of supernatant, and filter it using a filter with a pore size of 200 μm;

[0055] 6) After freeze drying for 2 days, grind to obtain powdered ATAM ( Figure 1 Store at -20℃ for later use.

[0056] Example 2 Characterization of ATAM

[0057] The ATAM prepared in Example 1 was characterized.

[0058] (1) Microscopic morphology characterization

[0059] The microscopic morphology of ATAM was observed using a transmission electron microscope (TEM). ATAM was fully dispersed in deionized water and then dropped onto a copper grid. It was then negatively stained with a phosphotungstic acid solution and allowed to air dry before being observed using a TEM. Figure 1 As shown in B, ATAM is aggregated in the form of spherical particles with a particle size of about 150-200 nm. It has high purity and no obvious impurities remain.

[0060] (2) Particle size characterization

[0061] The particle size of ATAM was further measured using a nanoparticle size analyzer. Figure 1 As shown in middle C, the particle size of ATAM is about 200.2 nm, which is consistent with the TEM results.

[0062] (3) Proteomic analysis

[0063] The protein composition of ATAM was analyzed by label free proteomics, with native adipose tissue (NAT) from the same source as in Example 1 and adipose decellularized extracellular matrix (AECM) prepared from native adipose tissue from the same source as in Example 1 as controls.

[0064] Among them, the preparation of the AECM uses the currently commonly used method, that is, decellularization with sodium dodecyl sulfate and peracetic acid, and isopropanol is used to remove lipids, and is prepared, including the following steps: the adipose tissue is immersed in a 0.5% SDS solution and stirred to decellularize for 48 hours (the volume of the solution used (ml) is twice the mass of fat (g)), the solution is replaced every 12 hours, and then washed twice with deionized water. After that, it is treated with isopropanol for 48 hours, the isopropanol is replaced every 12 hours, and then washed twice with deionized water. Next, it is treated with a 4% ethanol solution containing 0.1% peracetic acid for 4 hours, and then soaked in a PBS solution for 2 hours and washed twice with deionized water. The obtained AECM is freeze-dried and stored at -20°C for use.

[0065] The ATAM, NAT and AECM were sent to Shanghai GeneCare Gene Medicine Technology Co., Ltd. for label-free proteomics detection, and the detection results were analyzed by the following method: the mass spectrometry files were processed using the search software PaSER2023, the mass spectrometry data were used for protein qualitative analysis using the Uniprot protein database, and the target protein set was annotated with GO, analyzed by GO enrichment, and analyzed by KEGG enrichment using DAVID software.

[0066] Proteomic identification results Figure 2 As shown, Figure 2 Figure A shows that ATAM contains 3684 proteins, 3639 of which are shared with NAT, and the prepared ATAM retains 93.91% of the proteins in NAT; AECM contains 3315 proteins, 3281 of which are shared with NAT, and 84.67% of the proteins in NAT are retained. Protein expression level clustering heat map ( Figure 2 B) and differential protein volcano plot ( Figure 2 The results in middle C) also showed that compared with AECM, ATAM better retained the bioactive components in NAT.

[0067] Figure 3 The protein components in ATAM and AECM were compared. The GO analysis and KEGG analysis results of the upregulated proteins in ATAM compared with AECM were as follows: Figure 3 As shown. GO analysis results showed that upregulated proteins in ATAM were related to functions such as cellular redox homeostasis, reaction with hydrogen peroxide, regulation of endothelial barrier establishment, endothelial cell activation, cell apoptosis process, oxidoreductase activity, cell proliferation promotion, actin skeleton organization and intracellular protein transport. KEGG analysis results showed that upregulated proteins in ATAM were mainly involved in amino acid biosynthesis, cell apoptosis, actin skeleton regulation, cell-cell (or ECM) interactions (tight junctions, cell adhesion molecules, focal adhesion) and metabolic pathways related to mitochondrial function and sugar metabolism (such as oxidative phosphorylation, TCA cycle, glycolysis / gluconeogenesis).

[0068] The above results show that compared with AECM, ATAM contains more bioactive ingredients that are beneficial to tissue repair, has better antioxidant, anti-apoptotic and inflammatory regulatory effects, and has better therapeutic effects on tissue repair-related diseases, including myocardial infarction, acute kidney injury and acute liver injury.

[0069] Example 3 In vitro biological properties of ATAM

[0070] This example studies the in vitro biological properties of ATAM prepared in Example 1.

[0071] (1) Cytocompatibility

[0072] H9C2 cardiomyocytes were used as an example to detect the cell compatibility of ATAM. ATAM solutions of 0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL were prepared using DMEM medium and co-incubated with H9C2 cells. CCK8 kit (Biyuntian, C0038) and Calcein / PI cell activity and cytotoxicity detection kit (Biyuntian, C2015S) were used to detect cell survival and proliferation on days 1, 3, and 5. The detection steps were strictly carried out according to the instructions.

[0073] Figure 4 The results showed that compared with the Control1 group, there was no significant difference in cell viability in the 0.01mg / mL-1mg / mLATAM group on days 1, 3, and 5 ( Figure 4 Middle A), live-dead staining results ( Figure 4 Middle B) shows that the cell survival rate of each group is close to 100%, indicating that ATAM has good cell compatibility.

[0074] (2) Protective effect of ATAM on cellular oxidative stress

[0075] Taking ·OH and DPPH as examples, the in vitro free radical scavenging ability of ATAM was verified. ATAM solutions of different concentrations (0.01 mg / mL, 0.1 mg / mL, 1 mg / mL) were added to ·OH (0.5 M) and DPPH (0.1 mM) solutions (volume ratio of 1:1), and after sufficient reaction (OH: 50℃-55℃ reaction for 30min-60min; DPPH: room temperature, incubate in the dark for 6h-12h), the absorbance of the corresponding wavelength (·OH: 492nm; DPPH: 517nm) was detected using an ELISA instrument to calculate the free radical scavenging rate of ATAM. The calculation formula is as follows: ·OH scavenging rate = (A sample -A control ) / (A blank -A control );DPPH clearance rate=(A control -A sample ) / A control The results are as follows Figure 5 As shown in A and B, 0.01 mg / mL-1 mg / mL ATAM has a certain free radical scavenging ability, which is concentration-dependent. As the ATAM concentration increases from 0.01 mg / mL to 1 mg / mL, the ·OH scavenging rate increases from 20% to 70%, and the DPPH· scavenging rate increases from 18% to 32%.

[0076] H9C2 cells (cell density 60%-80%) were treated with 600 μM H202 for 1 h-2 h to induce oxidative stress, and different concentrations of ATAM were added for treatment. After DCFH-DA probe was loaded into the cells (co-incubated for 30-60 min), the intracellular ROS content was observed using a fluorescence microscope to evaluate the protective effect of ATAM on cardiomyocytes. Figure 5 As shown in C, the DCF fluorescence intensity of the experimental group containing ATAM was significantly lower than that of the control group, indicating that ATAM can reduce the oxidative stress damage of H2O2 to cells.

[0077] In addition, the cells were stained with Annexin V-FITC apoptosis detection kit (Biyuntian, C1062M), and the cell apoptosis caused by oxidative stress was detected by cell flow cytometry. The experimental operation was carried out strictly according to the instructions. Figure 5 As shown in D, after the addition of three different final concentrations of ATAM (0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL), the apoptosis rate of cells in each group decreased, indicating that ATAM can reduce oxidative stress-induced apoptosis.

[0078] The above results show that ATAM has a significant antioxidant effect, can scavenge free radicals, reduce the oxidative stress damage of H2O2 to cells, and reduce oxidative stress-induced cell apoptosis. It can be used to prepare antioxidant and anti-apoptotic drugs, as well as tissue repair drugs.

[0079] (3) Inflammatory regulation of ATAM

[0080] In order to study the regulatory ability of ATAM on the repolarization of M1 macrophages to M2, 100 ng / mL of LPS and 20 ng / mL of IFN-γ were used to induce M0 macrophages to polarize to M1, and ATAM was added to co-incubate with macrophages for 12h-24h. The cells were stained with CD206 (Invitrogen, 17-2061-82) and CD86 (Invitrogen, 11-0862-82) antibodies. The staining steps were strictly carried out according to the instructions. Finally, the polarization of macrophages was detected by flow cytometry. The results are shown in Figure 2. Figure 6 As shown in the figure, after LPS and IFN-γ induction, M0 macrophages transformed into M1 macrophages (CD86 + The proportion of M1 macrophages increased, while the proportion of M2 macrophages (CD206 +After treatment with ATAM at three different final concentrations, 0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL, the proportion of M1 macrophages was significantly reduced, and the proportion of M2 macrophages was significantly increased, indicating that ATAM can regulate the repolarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, has good inflammation regulation ability, can inhibit inflammatory response, and is used to prepare anti-inflammatory drugs.

[0081] Example 4 The repair effect of ATAM on myocardial infarction

[0082] This example studies the repairing effect of ATAM prepared in Example 1 on myocardial infarction.

[0083] ROS clearance and inflammation inhibition at the site of tissue damage are key to the treatment of many diseases. For example, the death of myocardial cells in the infarct area after myocardial infarction (MI) activates inflammatory responses and stimulates the recruitment and proliferation of macrophages. At the same time, the large amount of ROS produced after MI can activate multiple pathways to produce proinflammatory cytokines. Excessive ROS and long-term inflammation will eventually lead to left ventricular dilation and systolic dysfunction. We established a rat myocardial infarction model by permanently ligating the left anterior descending coronary artery to study the repair effect of ATAM on damaged myocardium.

[0084] The method for constructing a rat myocardial infarction model is as follows: male SD rats (7-8 weeks old, weighing 200g-250g) were used to construct a myocardial infarction model by ligating the left anterior descending artery. Specifically, the rats were anesthetized (2% isoflurane) and the hair on the chest was shaved. The rats were then placed in a supine position, intubated and connected to a small animal ventilator. After disinfection with iodine, the thorax was opened to expose the anterior surface of the rat heart, and the pericardium was gently torn open with forceps. The left anterior descending coronary artery was located 1 min below the intersection of the pulmonary artery cone and the left atrial appendage, and was permanently ligated using 7-0 surgical sutures. The whitening of the distal anterior ventricular area of ​​the ligation was a successful ligation. Subsequently, the treatment group used 7-0 surgical sutures to suture the heart patch to the whitened area, and the chest cavity, muscle and skin layers were closed with 7-0 surgical sutures. Heart patch is currently a commonly used method for treating MI, so we used a hydrogel heart patch (CP-A) encapsulating ATAM for treatment, and a heart patch (CP) without ATAM encapsulating ATAM was used as a control. Among them, CP and CP-A were prepared by the following method: 9% methacrylic anhydride modified gelatin (GelMA), 6% polyethylene glycol diacrylate (PEGDA), 0.15% dopamine modified polypyrrole (DA-PPy), and 0.2% LAP photoinitiator were dissolved in PBS, 1 mg / mL ATAM was additionally added to CP-A, and gelation was carried out under ultraviolet light to obtain CP and CP-A.

[0085] The animal experiment was divided into 4 groups: Sham group was a sham operation group, used as a healthy control; MI group was a negative control group without treatment after myocardial infarction surgery; CP group was a heart patch treatment group without ATAM; CP-A group was a heart patch treatment group with ATAM; each group had 5 rats. Echocardiographic characterization and histochemical staining were used to evaluate the repair of infarcted hearts.

[0086] The results are as follows Figure 7 , Figure 8 As shown, Figure 7 Typical echocardiographic images and important cardiac function parameters of rats with myocardial infarction after 28 days of treatment: Figure 8 The representative images of Masson staining of rat heart tissue after 28 days of treatment and the statistical results of myocardial infarction area and left ventricular wall thickness. Figure 7 The results of echocardiography showed that compared with the Sham group, the cardiac function parameters EF and FS of the rats in the MI group decreased significantly, while the EF and FS of the rats in the CP group and CP-A group increased significantly, and the EF and FS of the rats in the CP-A group were significantly higher than those in the CP group. Figure 8 It can be seen that compared with the Sham group, the myocardial infarction area of ​​rats in the MI group was significantly increased, while CP and CP-A treatment could significantly reduce the myocardial infarction area of ​​rats, and the myocardial infarction area of ​​the CP-A group was significantly lower than that of the CP group. Compared with the Sham group, the left ventricular wall thickness of rats in the MI group was significantly reduced, and CP and CP-A treatment could increase the left ventricular wall thickness of rats, and the left ventricular wall thickness of the CP-A group was greater than that of the CP group. This shows that compared with the MI group, the myocardial infarction of rats in the CP group and the CP-A group was improved: However, compared with the CP group, the treatment effect of the CP-A group was more significant, and the cardiac function and cardiac physiological structure were better restored, which was specifically manifested in the significant improvement of the cardiac function parameters EF and FS, the reduction of infarction area, and the myocardial wall thickness closer to the Sham group.

[0087] The above results indicate that ATAM plays an important role in the treatment of MI, has a good effect on repairing damaged myocardial tissue, and can be used to prepare drugs for the treatment of myocardial infarction.

[0088] Example 5 Repair effect of ATAM on kidney injury

[0089] This example studies the repairing effect of ATAM prepared in Example 1 on kidney damage.

[0090] Chemotherapy is the fastest-growing treatment strategy in anti-tumor drugs in recent years. The kidney, as an important organ for drug metabolism and excretion, is often affected, and cisplatin, a platinum preparation, is one of the most common chemotherapy drugs with nephrotoxicity. The mechanisms of cisplatin-induced acute kidney injury (AKI) include oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress, which lead to apoptosis, necrosis, and iron loss of renal tubular epithelial cells, resulting in rapid loss of renal function. We established a mouse renal injury model by intraperitoneal injection of cisplatin to study the repair effect of ATAM on damaged kidneys.

[0091] The method for constructing a mouse renal injury model is as follows: male C57BL / 6 mice (8-10 weeks old, weighing 20 g) were used to construct a renal injury model by intraperitoneal injection of 20 mg / kg cisplatin (concentration 1 mg / mL). Specifically, the mice were deprived of water 15 hours in advance, followed by intraperitoneal injection of 20 mg / kg cisplatin (dissolved in PBS buffer, final concentration of 1 mg / mL, pay attention to avoid light), and water was restored. We administered ATAM (100 μL, 10 mg / mL) into the tail vein for 3 days before and after injury induction, for a total of 6 days.

[0092] The animal experiment was divided into 5 groups: Healthy group was the healthy control; AKI+PBS group was the negative control group with cisplatin-induced renal injury and no treatment; AKI+ATAM5 group was the treatment group with cisplatin-induced renal injury and injected with 5 mg / mL ATAM for 6 days; AKI+ATAM10 group was the treatment group with cisplatin-induced renal injury and injected with 10 mg / mL ATAM for 6 days; AKI+NAC group was the positive control group with cisplatin-induced renal injury and injected with 160 mg / kg N-acetylcysteine ​​(NAC) for 6 days.

[0093] The results are as follows Fig. 9 and Fig.10 As shown: Fig. 9 This is an optical image of the kidney on day 8. Fig.10 This is the test result of creatinine (CREA) in the serum biochemical indicators of mice on the 8th day.

[0094] Depend on Fig. 9 It can be seen that the kidneys of mice in the Healthy group were dark red, indicating that they were in a healthy state. In contrast, the kidneys of mice in the AKI+PBS group were reduced in size and lacked luster, indicating kidney damage. After treatment with 10 mg / mL ATAM or 160 mg / kg NAC, the kidneys showed no signs of volume reduction and showed a dark red color similar to that of the healthy group.

[0095] Creatinine is a product of muscle metabolism in the human body. It comes from food intake and self-synthesis in the body and is mainly excreted from the body by glomerular filtration. Clinically, detecting creatinine levels is one of the main methods to understand renal function. Fig.10 It can be seen that compared with the Healthy group, the CREA level in the AKI+PBS group was significantly increased, and the ATAM treatment could significantly reduce the CREA level, among which the treatment effect of 10mg / mL ATAM was better than that of 5mg / mL, and was basically the same as that of the NAC treatment group.

[0096] The above results indicate that ATAM plays an important role in the treatment of AKI, has a good effect on repairing damaged renal tissue, and can be used to prepare drugs for the treatment of acute kidney injury.

[0097] Example 6 Preparation of ATAM

[0098] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0099] 2) preparing a 0.075 wt % type I collagenase solution;

[0100] 3) Add the type I collagenase solution in a volume twice the volume of the adipose tissue to the centrifuge tube containing the adipose tissue, mix well and digest at 37° C. for 3 h;

[0101] 4) After digestion, centrifuge at 1500g for 15 min, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid;

[0102] 5) Centrifuge at 2000 g for 10 min, take the middle layer of supernatant, and filter it using a filter with a pore size of 150 μm;

[0103] 6) After freeze drying in a freeze dryer for 2 days, grind to obtain powdered ATAM and store at -20°C for future use.

[0104] Example 7 Preparation of ATAM

[0105] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0106] 2) preparing a 0.1 wt% type I collagenase solution;

[0107] 3) Adding 1 times the volume of the type I collagenase solution to the centrifuge tube containing the adipose tissue, mixing well and digesting at 37° C. for 1 h;

[0108] 4) After digestion, centrifuge at 2000g for 10 min, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid;

[0109] 5) Centrifuge at 3000 g for 5 min, take the middle layer of supernatant, and filter it using a filter with a pore size of 100 μm;

[0110] 6) After freeze drying in a freeze dryer for 2 days, grind to obtain powdered ATAM and store at -80°C for future use.

[0111] According to label free proteomics analysis (same method as Example 2), the ATAM prepared in Examples 6 and 7 effectively retained the proteins in NAT, which were significantly higher than AECM prepared from natural pig subcutaneous adipose tissue from the same source. The GO analysis and KEGG analysis results of the upregulated proteins expressed by ATAM compared with AECM were similar to those of the ATAM prepared in Example 1. The upregulated proteins were related to functions such as cellular redox homeostasis, reaction with hydrogen peroxide, regulation of endothelial barrier establishment, endothelial cell activation, cell apoptosis process, oxidoreductase activity, cell proliferation promotion, actin skeleton organization and intracellular protein transport; they were mainly involved in the biosynthesis of amino acids, cell apoptosis, actin skeleton regulation, cell-cell (or ECM) interactions (tight junctions, cell adhesion molecules, focal adhesion) and metabolic pathways related to mitochondrial function and sugar metabolism (such as oxidative phosphorylation, TCA cycle, glycolysis / gluconeogenesis).

[0112] Comparative Example 1 Preparation of ATAM

[0113] The preparation method of ATAM in this comparative example is the same as that in Example 1 except that type I collagenase is replaced by type IV collagenase, and the other aspects (including the source of subcutaneous adipose tissue) are as follows:

[0114] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0115] 2) preparing a 0.05 wt % type IV collagenase solution;

[0116] 3) adding 1.5 times the volume of the type IV collagenase solution to the centrifuge tube containing the adipose tissue, mixing well and digesting at 37° C. for 2 h;

[0117] 4) After digestion, centrifuge at 3000g for 5 min, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid;

[0118] 5) Centrifuge at 2500 g for 10 min, take the middle layer of supernatant, and filter it using a filter with a pore size of 200 μm;

[0119] 6) After freeze drying in a freeze dryer for 2 days, grind to obtain powdered ATAM and store at -20°C for future use.

[0120] According to label free proteomics analysis (same method as Example 2), the number of shared proteins between ATAM and NAT prepared in this comparative example is significantly lower than that in Example 1, and the protein content related to antioxidant, anti-apoptotic and inflammatory regulation is reduced. This indicates that the use of type IV collagenase to digest adipose tissue cannot well extract the bioactive components therein, especially the bioactive components related to antioxidant, anti-apoptotic and inflammatory regulation. However, the use of type I collagenase to digest adipose tissue can effectively extract and retain the bioactive components in adipose tissue, and improve the antioxidant, anti-apoptotic and inflammatory regulation effects of the prepared ATAM.

[0121] Comparative Example 2 Preparation of ATAM

[0122] The preparation method of ATAM in this comparative example is the same as that in Example 1 except that the concentration of type I collagenase is adjusted to 0.02wt%, and the other steps (including the source of subcutaneous adipose tissue) are as follows:

[0123] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0124] 2) preparing a 0.02 wt % type I collagenase solution;

[0125] 3) Add 1.5 times the volume of the type I collagenase solution to the centrifuge tube containing the adipose tissue, mix well and digest at 37° C. for 2 h;

[0126] 4) After digestion, centrifuge at 3000g for 5 minutes, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid:

[0127] 5) Centrifuge at 2500g for 10 min, take the middle layer of the clear liquid, and filter it using a filter with a pore size of 200 μm:

[0128] 6) After freeze drying for 2 days, grind to obtain powdered ATAM ( Figure 1 Store at -20℃ for later use.

[0129] According to label free proteomics analysis (the method is the same as that of Example 2), the number of shared proteins between ATAM and NAT in the comparative example prepared in this comparative example is significantly lower than that in Example 1. This indicates that the concentration of type I collagenase will affect the extraction and retention of bioactive components. The concentration of 0.05wt% to 0.1wt% is an appropriate digestion concentration. When the concentration of type I collagenase is too low (e.g., 0.02wt%), the bioactive components therein cannot be extracted well.

[0130] Comparative Example 3 Preparation of ATAM

[0131] The preparation method of ATAM in this comparative example is the same as that in Example 1 except that the concentration of type I collagenase is adjusted to 0.15wt%, and the other factors (including the source of subcutaneous adipose tissue) are as follows:

[0132] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0133] 2) preparing a 0.15 wt % type I collagenase solution;

[0134] 3) Add 1.5 times the volume of the type I collagenase solution to the centrifuge tube containing the adipose tissue, mix well and digest at 37° C. for 2 h;

[0135] 4) After digestion, centrifuge at 3000g for 5 min, discard the precipitate and the uppermost layer of fat and tissue, and take the middle layer of clear liquid;

[0136] 5) Centrifuge at 2500 g for 10 min, take the middle layer of supernatant, and filter it using a filter with a pore size of 200 μm;

[0137] 6) After freeze drying in a freeze dryer for 2 days, grind to obtain powdered ATAM ( Figure 1 Store at -20℃ for later use.

[0138] According to label free proteomics analysis (the same method as in Example 2), the number of shared proteins with NAT in the ATAM prepared in this comparative example decreased, and the types of bioactive components also changed, and the content of proteins related to antioxidant, anti-apoptotic and inflammatory regulation decreased compared with Example 1. This shows that the concentration of type I collagenase will affect the extraction and retention of bioactive components, and the concentration of 0.05wt% to 0.1wt% is an appropriate digestion concentration. When the concentration of type I collagenase is too high (for example, 0.15wt%), it is not conducive to the extraction and retention of bioactive components in adipose tissue.

[0139] Comparative Example 4 Preparation of ATAM

[0140] The preparation method of ATAM in this comparative example is the same as that in Example 1 except for the centrifugation conditions, and other conditions (including the source of subcutaneous adipose tissue) are as follows:

[0141] 1) Take subcutaneous adipose tissue from pigs, remove all tissues except fat with scissors and tweezers, cut the adipose tissue into pieces with scissors and put them into a centrifuge tube, and wash them with PBS several times;

[0142] 2) preparing a 0.05 wt % type I collagenase solution;

[0143] 3) Add 1.5 times the volume of the type I collagenase solution to the centrifuge tube containing the adipose tissue, mix well and digest at 37° C. for 2 h;

[0144] 4) After digestion, centrifuge at 2500g for 15 min, take the middle layer of the supernatant, and filter it using a filter with a pore size of 200 μm;

[0145] 5) After freeze drying in a freeze dryer for 2 days, grind to obtain powdered ATAM and store at -20°C for future use.

[0146] After testing, the ATAM prepared in this comparative example still has other components such as cells and oils, and the purity is significantly reduced, indicating that only one centrifugation cannot effectively remove components such as cells and oils. The results show that the digestion product is first centrifuged at 1000g to 3000g to remove oil and undigested fat, and then centrifuged at 2000g to 3000g to remove oil and cell components. Such two differential centrifugations can effectively separate undigested fat, oil and cell components, leaving no residue in ATAM, and obtain high-purity ATAM.

[0147] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing an adipose tissue active material, characterized in that: The following steps are involved: (1) Take adipose tissue and cut it into pieces; (2) adding 0.05 wt% to 0.1 wt% type I collagenase solution, mixing, and digesting; (3) The digestion product is centrifuged at 1000g to 3000g for 5 min to 15 min, and the middle layer of supernatant 1 is collected; (4) Centrifuge the middle layer clear liquid 1 at 2000g-3000g for 5min-10min, take the middle layer clear liquid 2, filter it, and take the filtrate to obtain the product.

2. The preparation method according to claim 1, characterized in that In step (2), digestion is carried out at 35°C to 45°C for 0.5h to 3h; preferably, digestion is carried out at 36°C to 40°C for 1h to 2h.

3. The preparation method according to claim 1, characterized in that: The centrifugal force of the centrifugation in step (3) is smaller than that in step (4).

4. The preparation method according to claim 1 or 3, characterized in that: Step (3) centrifuging at 1000 g to 2000 g for 5 min to 10 min; and / or, Step (4) is centrifuged at 2500 g to 3000 g for 5 min to 10 min.

5. The preparation method according to claim 1, characterized in that: The volume of the type I collagenase solution is 1 to 2 times the volume of the adipose tissue; and / or, The pore size of the filter material used in the filtration in step (4) is 100 μm to 200 μm; and / or, Step (4) also includes freeze-drying the filtrate.

6. The adipose tissue active material obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the adipose tissue active material according to claim 6 in the preparation of anti-oxidation, anti-apoptosis and / or anti-inflammatory drugs or products.

8. Use of the adipose tissue active material according to claim 6 in the preparation of tissue repair drugs or products.

9. The use according to claim 8, characterized in that The application includes preparing medicines or products for treating myocardial infarction, acute kidney injury and / or acute liver injury.

10. A pharmaceutical composition, characterized in that It comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the adipose tissue active material as claimed in claim 6.

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