YAP gene inhibitor and application thereof in preparation of medicine for improving fat transplantation

Abstract

The YAP gene inhibitor is used for inhibiting transcription of expression of a YAP gene, especially siRNA inhibits expression of the YAP gene, and the YAP gene inhibitor is used as an active component to prepare drugs or medical instruments for improving fat transplantation effects. Through verification, after the YAP gene in the ADSC is knocked down, the expression of adipogenic related genes and proteins of the ADSC is remarkably promoted, the adipogenic differentiation of the ADSC is remarkably promoted, and the lipid droplet content and the triglyceride content in cells are remarkably improved. Besides, after the YAP gene is inhibited, tissue inflammation, fibrosis and oil sac level are improved, the effect of transplanting fat in a body is remarkable, the retention period of the implanted fat is prolonged, and the transplantation effect of the fat is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a gene expression inhibitor for improving the effect of fat transplantation, such as increasing the retention rate or retention period, and reducing tissue inflammation and fibrosis. Background Art

[0002] Adipose tissue is a commonly used autologous soft tissue repair and reconstruction material in clinical practice. Due to its advantages such as abundant sources, easy material collection, simple operation, good tissue compatibility and hidden scars, free fat transplantation technology has become one of the most commonly used soft tissue defect repair technologies in plastic surgery. However, with the increase in clinical applications, the problems brought about by fat transplantation have gradually become prominent. First, the retention rate after autologous fat transplantation is difficult to predict. In clinical and experimental studies, the long-term retention rate of fat transplantation ranges from 34% to 82%. Secondly, autologous fat transplantation can also cause complications such as calcified fibrous tissue replacement, cyst formation, post-transplant fat liquefaction and necrosis, and the appearance of calcification points. The total probability of occurrence ranges from 7% to 10.5%. These shortcomings greatly limit the wider application of autologous fat transplantation in clinical practice.

[0003] CN102245765B discloses a cell transplantation composition for preventing cell membrane damage in fat transplants. The composition mixes triblock copolymers such as poloxamer P188 with fat cells or fat tissue to be transplanted into an individual to stabilize the cell membrane, thereby achieving a more successful fat transplantation in soft tissue reconstruction or filling.

[0004] In order to improve the survival rate of autologous fat transplantation and reduce the occurrence of complications, researchers have conducted a lot of research to try to accelerate the reconstruction of blood supply to transplanted fat, thereby shortening the ischemic period after fat transplantation, such as the use of cytokines (such as VEGF, etc.), stem cell exosomes and platelet-derived plasma (PRP).

[0005] CN104984399A discloses a method for preparing adipose tissue assisted by a biological scaffold material and SVF, wherein the biological scaffold material, vascular matrix components and purified fat particles are mixed evenly in a certain proportion to prepare a fat graft for filling. The vascular matrix components are mixed with autologous fat particles and then transplanted, and can continue to develop into mature adipose tissue in the body. The biological scaffold material can provide support for the transplanted fat, facilitate the revascularization of the transplanted tissue, and improve the survival rate of the transplanted tissue.

[0006] Compared with simple autologous fat transplantation, the use of the above methods has improved the effect of autologous fat transplantation to a certain extent and has been applied clinically. However, these methods have the problems of cumbersome operation, easy contamination, and the need to consume a large amount of adipose tissue. In addition, due to the presence of inflammation, there are still phenomena such as necrosis and fibrosis of fat cells after transplantation, so the improvement of transplanted fat effect is still very limited. Summary of the invention

[0007] One object of the present invention is to provide a YAP gene inhibitor for inhibiting the expression and transcription of the YAP gene, thereby facilitating increasing the amount of lipid droplets in cells.

[0008] Another object of the present invention is to provide a YAP gene inhibitor for inhibiting the expression and transcription of the YAP gene, thereby facilitating increasing the triglyceride content of cells.

[0009] Another object of the present invention is to provide a YAP gene inhibitor for inhibiting the expression and transcription of the YAP gene, thereby facilitating the adipogenic differentiation of adipose stem cells.

[0010] Another object of the present invention is to provide a YAP gene inhibitor for inhibiting the expression and transcription of the YAP gene, thereby improving the fat transplantation effect, such as the retention rate after fat transplantation, the retention period after fat transplantation, and reducing tissue inflammation and fibrosis.

[0011] The YAP gene referred to in the present invention is Yes1 associated transcriptional regulator (Yes-associated protein). YAP plays an important role in regulating tissue development. Previous studies have shown that the YAP pathway plays an important role in regulating stem cell differentiation.

[0012] The YAP inhibitor of the present invention is specifically siRNA, which specifically binds to and degrades the mRNA corresponding to the YAP gene based on base complementary pairing, thereby inhibiting the expression of YAP.

[0013] The YAP inhibitors of the present invention also include antibodies against YAP gene expression proteins.

[0014] It has been verified that knocking down the YAP gene in ADSCs significantly promoted the expression of adipogenesis-related genes and proteins in ADSCs, significantly promoted the adipogenic differentiation of ADSCs, and significantly increased the lipid droplet content and triglyceride content in the cells.

[0015] In addition, inhibiting the YAP gene improved tissue inflammation, fibrosis and oil cyst levels, which had a significant effect on transplanted body fat, prolonged the retention period of implanted fat, and improved the effect of fat transplantation.

[0016] A YAP gene inhibitor, comprising one or more of the following siRNAs:

[0017] GUCAGAGAUACUUCUUAAATT;

[0018] UUUAAGAAGUAUCUCUGACTT;

[0019] CAGGAAUUGAGAACAAUGATT;

[0020] UCAUUGUUCUCAAUUCCUGTT;

[0021] CCACCAAGCUAGAUAAAGATT; and

[0022] UCUUUAUCUAGCUUGGUGGTT.

[0023] Another YAP gene inhibitor includes one or more groups of siRNAs as follows:

[0024] Group 1: GUCAGAGAUACUUCUUAAATT and UUUAAGAAGUAUCUCUGACTT;

[0025] Group 2: CAGGAAUUGAGAACAAUGATT and UCAUUGUUCUCAAUUCCUGTT; and

[0026] Group 3: CCACCAAGCUAGAUAAAGATT and UCUUUAUCUAGCUUGGUGGTT.

[0027] The YAP inhibitor provided by the present invention is used as an active ingredient in the preparation of a drug for improving the retention rate of transplanted fat.

[0028] The YAP inhibitor provided by the present invention is used as an active ingredient in the preparation of medical devices for improving the retention rate of transplanted fat. These medical devices are

[0029] The YAP inhibitor of the present invention is applied to cells in vitro or in vivo to achieve the purpose of promoting cell (such as ADSC) adipogenesis or increasing the lipid droplet content of adipocytes or reducing tissue inflammation and fibrosis, which should be understood as the use of the YAP inhibitor of the present invention as a drug.

[0030] The cytokines of the present invention act on cells in vitro or in vivo to achieve the purpose of improving the effect of fat transplantation (such as increasing the retention rate), which should be understood as using the YAP inhibitor of the present invention as a drug.

[0031] The YAP inhibitor of the present invention is mixed with other auxiliary materials to prepare a medicine (preparation) for improving the retention rate of transplanted fat.

[0032] These pharmaceutical excipients can be those conventionally used in various preparations, such as but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they can also be selected for use in order to be compatible with the substances, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the administration effect of the composition.

[0033] In aqueous solution injections, excipients generally include isotonic agents and buffers, as well as necessary emulsifiers (such as Tweeen-80, Pluronic and Poloxamer), solubilizers and antibacterial agents, etc. In addition, other pharmaceutically acceptable excipients are also included, such as antioxidants, pH regulators and analgesics.

[0034] Excipients used to prepare oral liquid preparations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers and colorants.

[0035] The excipients used to make tablets generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and mannitol), binders (such as ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution and aqueous or alcoholic solution of polyvinyl pyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone and cross-linked sodium carboxymethyl cellulose) and lubricants (such as magnesium stearate, micro-powdered silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000 and magnesium lauryl sulfate, etc.).

[0036] The auxiliary materials used to prepare emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0037] The excipients used to prepare granules are similar to those used to prepare tablets, but the granulation process is different. The prepared granules are mixed with a glidant as needed and then filled into capsules to obtain capsules.

[0038] Various excipients and compounds are made into dosage forms that are beneficial to drug delivery, such as but not limited to aqueous solution injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder aerosols, sustained-release agents and controlled-release agents. In addition, excipients such as but not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials, such as but not limited to polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc., can be used to achieve specific drug delivery purposes or methods, such as sustained-release drug delivery, controlled-release drug delivery and pulse drug delivery. The main manifestations of the so-called "beneficial to drug delivery" include but not limited to improving therapeutic effects, improving bioavailability, reducing toxic side effects and improving patient compliance.

[0039] The YAP gene inhibitor of the present invention (such as an antibody) is combined with other excipients, such as chemical coupling, to further improve the efficacy of the compound, reduce toxic effects, and extend the administration cycle, etc. These excipients are usually polymers, such as polyesters, polyethers, and polyamides.

[0040] Drug-containing medical devices made by combining drugs and medical devices are also common, such as stents containing YAP gene inhibitors, with YAP inhibitors as active ingredients loaded or coated on stent materials, used to make medical devices to improve the effect of fat transplantation. Common stent materials include PLA, PLL, PGA, PLGA and metals. And mixed with biocompatible degradable materials to make microneedles and microneedle arrays, or loaded in metal microneedles to make microneedle chips. When the microneedle penetrates the skin, the YAP inhibitor is released in the tissue to improve the effect of fat transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a statistical diagram of the results of Edu staining of each group of cells in the present invention;

[0042] Figure 2A This is a statistical chart of the oil red staining results of each group of the present invention on the 4th day of inducing ADSC differentiation;

[0043] Figure 2B This is a statistical chart of the oil red staining results of each group of the present invention on the 8th day of inducing ADSC differentiation;

[0044] Figure 3A This is a statistical graph of triglyceride (TG) detection results on the 4th day of ADSC differentiation induction in each group of the present invention;

[0045] Figure 3B This is a statistical graph of triglyceride (TG) detection results on the 8th day of ADSC differentiation induction in each group of the present invention;

[0046] Figure 4AThe statistical graphs of the expression results of adipogenic differentiation-related genes of each group of ADSC differentiation induced on the 4th day (4 days) of the present invention;

[0047] Figure 4B The statistical graphs of the expression results of adipogenic differentiation-related genes of each group of ADSC differentiation induced on day 8 (8 days) of the present invention;

[0048] Figure 5A The statistical graphs of the expression results of adipogenic differentiation-related proteins of each group of ADSC differentiation induced on the 4th day (4 days) of the present invention;

[0049] Figure 5B The statistical graphs of the expression results of adipogenic differentiation-related proteins of each group of ADSC differentiation induced on day 8 (8 days) of the present invention;

[0050] Fig. 6A The morphological photos of the fat masses of each group after 1 week (1 week), 2 weeks (2 weeks) and 4 weeks (4 weeks) of implantation in animals of the present invention;

[0051] Figure 6B The weight and volume statistics of each group of fat blocks after 1 week, 2 weeks and 4 weeks of implantation in animals according to the present invention;

[0052] Fig. 7A The CT images of each group of fat masses implanted in the animal body for 4 weeks are shown in the figure.

[0053] Figure 7B The figure is a statistical diagram of the volume of survival fat in each group after the implantation of the present invention into the animal body for 4 weeks;

[0054] Fig. 8A The CT images of the animal tissues of each group after the implantation of the present invention into the animal body for 1 week (1 week), 2 weeks (2 weeks) and 4 weeks (4 weeks);

[0055] Figure 8B The figures are the H&E staining scoring results of each group of animal tissues for the structural integrity, inflammation, oil cysts and fibrosis of cells implanted in animals for 1 week (1w), 2 weeks (2w) and 4 weeks (4w) according to the present invention. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0057] The various test methods used in the following examples of the present invention are specifically described as follows:

[0058] 1) siRNA

[0059] The siRNA sequences used in each YAP knockdown group involved in each embodiment are as follows:

[0060]

[0061] 2) Oil red staining

[0062] ADSCs were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with fresh Oil Red O solution (Solarbio, China) for 30 min. Then, the stained cells were washed twice with PBS. Adipogenic differentiation was observed using an inverted fluorescence microscope (Nikon, Japan).

[0063] 3) Triglyceride determination

[0064] On the 4th and 8th day of differentiation, the triglyceride levels of cells were measured by a triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, China). In short, the assay started with enzymatic hydrolysis of triglycerides by lipase to produce glycerol and free fatty acids. The released glycerol was subsequently measured at a wavelength of 510 nm by an ELISA reader. Protein concentration was measured using a BCA kit (Boster, China).

[0065] Triglyceride content (mmol / gprot) = triglyceride concentration (mmol / L) / protein concentration (gprot / L).

[0066] 4) PCR detection

[0067] The steps for extracting total RNA from cells are as follows:

[0068] (1) Discard the cell culture medium and wash the cells three times with pre-cooled PBS.

[0069] (2) Place the cell culture dish on ice, add 1 ml of Trizol Reagent to each well, let it stand on ice for 5 minutes, then transfer the cells to a 1.5 ml sterile enzyme-free EP tube by pipetting for subsequent extraction or place in a -80°C refrigerator for future use.

[0070] (3) Add one fifth volume (0.2 ml) of chloroform to each of the above EP tubes and shake thoroughly on a vortex shaker for 15 seconds until stratification is visible, with the pink layer at the bottom and the white layer at the top. After standing on ice for 5 minutes, place in a precooled centrifuge at 4°C and centrifuge at 12,000 rpm for 15 minutes.

[0071] (4) After centrifugation, the sample can be seen to be divided into three layers. The upper layer is a transparent water layer containing RNA, the middle layer is white, containing protein, and the lower layer is pink, containing DNA. Carefully aspirate 400 μl of the upper layer of liquid and transfer it to a new 1.5 ml sterile enzyme-free EP tube. Be careful not to aspirate the lower two layers of liquid.

[0072] (5) Add an equal volume of isopropanol to each tube, mix thoroughly by inverting, and place the EP tube at -20°C for 1 hour. Centrifuge at 4°C, 12,000 rpm for 10 minutes. After taking out the EP tube, a flake-like white precipitate can be seen at the bottom of the tube.

[0073] (6) Discard the supernatant, prepare 75% ethanol with DEPC water and anhydrous ethanol, add 1 ml of the solution to each EP tube, mix thoroughly, and centrifuge at 4°C, 7500 rpm for 10 minutes.

[0074] (7) After centrifugation, discard the supernatant and invert the EP tube to air dry for 5 minutes to allow the RNA to dry and precipitate. Ensure that the sample is not completely dry to prevent the solubility of RNA from decreasing. Add 10ul of DEPC water to each tube to fully dissolve the RNA, and use a nucleic acid quantifier to detect the RNA concentration and A260 / 280 ratio. The A260 / 280 of normal RNA should be between 1.8-2.0. If the ratio is too low, it may be due to the presence of DNA and protein. If the ratio is too high, it may be due to RNA degradation. According to the obtained RNA concentration, reverse transcription is performed to obtain cDNA

[0075] Reverse transcription PCR, the steps are as follows:

[0076] (1) Prepare reagents: RNA sample, PrimeScript RT Reagent Kit (Takara), DEPC water.

[0077] (2) Prepare the transcription system: RNA X μl (X = 2000 ng / RNA concentration); 5× PrimeScript Buffer 4 ul; DEPC water (16-X μl).

[0078] (3) Reverse transcription: Mix the above liquids, centrifuge briefly, and perform reverse transcription. Set the reverse transcription conditions to 37°C for 15 minutes and 85°C for 5 seconds. After reverse transcription into cDNA, store it at -20°C and dilute the cDNA 10 times before use.

[0079] Real-time PCR (Real-time PCR), the steps are as follows:

[0080] (1) Primer preparation: Pipette 10ul of forward primer and 10ul of reverse primer, mix them together, add 180ul of ddH2O, and vortex to mix evenly.

[0081] (2) Prepare qPCR reaction system: ddH2O 3ul; SYBR Premix Ex Taq 5ul; ROX ReferenceDyeII 0.2ul; cDNA 1ul; primer 1ul.

[0082] (3) Set up three replicate wells for each group. After adding the samples, stick them on a PCR-specific membrane and vortex them slightly to mix them thoroughly. Then centrifuge them at 2000 rpm in a centrifuge precooled at 4°C for 2 minutes.

[0083] (4) Go to the machine, set the label information for each well, and set the real-time fluorescence quantitative program according to the Takara instruction manual. The program is as follows: 95°C for 30 seconds → 95°C for 5 seconds, 60°C for 30 seconds, a total of 50 cycles → 95°C for 30 seconds, 60°C for 1 minute.

[0084] (5) Analysis: GAPDH was used as the internal reference for all samples and 2^-△△Ct was calculated.

[0085] (6) Statistical analysis was performed using Graph Pad Prism6 software.

[0086] The expression of four genes including PPARγ, CEBPα, Adiponectin and FABP4 were detected.

[0087] 5) Western-blot

[0088] Cultured cells were lysed with RIPA buffer (Beyotime, China) supplemented with protease inhibitors (PMSF, Biosharp, China). In brief, 20 μg of protein was separated by 10% or 12% SDS-PAGE and blotted in a polyvinylidene difluoride membrane (Millipore Sigma, USA). The membrane was blocked with 5% skim milk for 1 hour at room temperature. The separated proteins were then immunoblotted and detected with anti-GAPDH antibody, anti-PPARγ antibody, CEBPα and FABP4 antibody at 4°C overnight. The next day, the secondary antibody was incubated at room temperature for 1 hour, followed by washing 3 times with TBST for 10 minutes. ImageJ software was used to quantify the immunoreactive bands.

[0089] 6) Construction of nude mouse fat transplantation model

[0090] All animal experiments were approved by the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Female nude mice (6 to 8 weeks old) were housed in individual cages with a 12-h light / dark cycle and provided with standard food and water. The mice were randomly divided into two groups (6 in each group): saline and siYAP groups. 0.2 ml of Coleman fat was injected subcutaneously on the left and right sides of the back of each mouse using a 1 ml syringe with a blunt infiltration cannula. The grafts were injected into a spherical shape. Every two days, mice were locally injected with 0.2 ml of saline or siYAP. On day 14, the grafts on the left back were harvested and carefully separated from the surrounding tissues, and their volume and weight were measured. The wound was closed with 6-0 nylon sutures, and antibiotic ointment was applied to the affected area for 1 week to prevent local infection. Histological evaluation was performed on each harvested sample. At 1, 2, and 4 weeks, the grafts were harvested and carefully separated from the surrounding tissues, and their volume and weight were measured. Histological evaluation was performed on each harvested sample.

[0091] 7) H&E staining and scoring

[0092] The differentiated cells were fixed with 4% paraformaldehyde for 24 hours, and then the cells were dehydrated, embedded in paraffin, and cut into 5 μm thick sections for H&E staining.

[0093] The stained cells were scored as follows:

[0094] Histological parameters, cellular integrity, tissue inflammation, presence of cysts / vacuoles, and degree of fibrosis were evaluated. Each parameter was scored as 0 = absent, 1 = mildly present, 2 = mild to moderately present, 3 = moderately present, 4 = moderate to extensively present, and 5 = extensively present. Scoring was performed independently by 3 peers who were unaware of the group assignments.

[0095] 9) CT

[0096] Preparation before scanning: 4 weeks after transplantation, mice were fully anesthetized using 0.6% sodium pentobarbital injected intraperitoneally.

[0097] Scanning: Place the nude mouse flat on the detection plate, set the voltage to 90 kV, reconstruct the pixel to 72 μm, scan time to 2 minutes, select the scanning area, and scan using Micro-CT.

[0098] ProPlan CMF 3.0 software was used to analyze the retained fat volume. By adjusting the grayscale value of the CT section, the subcutaneous transplanted fat area of ​​each section was identified, the fat retention volume was calculated, and three-dimensional reconstruction was performed.

[0099] 10) Statistical methods

[0100] Data were analyzed using SPSS software. One-Way ANOVA was used for comparison between groups. *p<0.05 indicates statistically significant difference, **p<0.01 indicates statistically significant difference, ns indicates no statistically significant difference. All values ​​are expressed as mean ± standard deviation.

[0101] Example 1 Investigating the effect of salvianolic acid B on the proliferation activity of ADSCs by Edu detection

[0102] Group treatment: control group (NC), siRNA knockdown group 1, siRNA knockdown group 2 and siRNA knockdown group 3.

[0103] Adipose-derived mesenchymal stem cells (ADSCs) in each knockdown group were treated with siRNA for 48 hours. ADSCs were seeded in 24-well plates. After 24 hours of incubation, cell proliferation was detected using an EdU cell proliferation detection kit (Invitrogen, USA). In brief, ADSCs were incubated with 50 μM EdU for 2 hours before fixation, permeabilization, and color development. Then, the cell nuclei were stained with Hoechst33342 (Invitrogen, USA) for 30 minutes. Cells incorporating EdU were determined by an inverted fluorescence microscope (Nikon, Japan). The results are shown in Figure 2. Figure 1 As shown, it can be seen that knocking down the YAP gene in ADSCs does not affect the proliferation level of ADSCs (from the data, there is no statistical difference).

[0104] Example 2 Oil red staining and triglyceride determination to detect the effect of inhibiting YAP on adipose stem cell differentiation. The patients were divided into groups: control group (NC), siRNA knockdown group 1 and siRNA knockdown group 3.

[0105] The statistical results of oil red staining of cells on the fourth and eighth days of ADSC differentiation in each group are shown in Figure 2A and Figure 2BAs shown in the figure, the accumulation of lipid droplets was observed. The amount of lipid droplets in each group on the 8th day was greater than that on the 4th day, and compared with the control group NC, knocking down the YAP gene in ADSCs significantly promoted the growth of lipid droplets in ADSCs.

[0106] The results of cell triglyceride detection on the 4th and 8th days of ADSC differentiation in each group are as follows Figure 3A and Figure 3B As shown, the triglyceride content of each group of cells at each time point was observed. The triglyceride content of each group of cells on the 8th day was greater than that on the 4th day, and the triglyceride content of the knockdown group was significantly higher than that of the control group.

[0107] It can be seen that knocking down the YAP gene in ADSCs significantly promotes the adipogenic differentiation of ADSCs.

[0108] Example 3 Effect on the expression of genes related to adipogenic differentiation of adipose stem cells

[0109] The cells were divided into control group (NC), siRNA knockdown group 1 and siRNA knockdown group 3. PCR detection was performed on the 4th and 8th days of differentiation (e.g. Figure 4A and 4B ) and Western-blot detection (as shown Figure 5A and 5B shown).

[0110] Figure 4A and 4B It can be seen that the expression levels of adipogenic differentiation-related genes such as PPARγ, CEBPα, Adiponectin and FABP4 in ADSCs of the YAP knockdown group were significantly higher than those in the control group. Figure 5A and 5B It can be seen that the expression levels of adipogenic differentiation-related proteins such as GAPDH, PPARγ, CEBPα and FABP4 in ADSCs of the YAP knockdown group were also significantly higher than those in the control group.

[0111] The results showed that knocking down the YAP gene in ADSCs significantly promoted the expression of adipogenesis-related genes and proteins in ADSCs.

[0112] Example 4 Histological evaluation of the effect of YAP inhibition on improving transplanted fat

[0113] Group treatment: control group (NC) and siRNA knockdown group 3.

[0114] The cells of each group were sampled 2 weeks and 4 weeks after transplantation into the model animals and stained with H&E. The results are shown in Fig. 6A and Figure 6B As shown. Fig. 6AIt can be seen intuitively that after 1 week of implantation in animals, the morphology of the fat mass in the knockdown group was similar to that in the control group. After 1, 2, and 4 weeks of implantation, the volume of the fat mass in the control group was significantly reduced, and the volume of the fat mass in the knockdown group was slightly reduced, but the degree of reduction in the volume of the fat mass after 2 and 4 weeks of implantation could not be distinguished by the naked eye. This can also be reflected in the data of the weight and volume of the fat mass. Figure 6B As shown, 1 week, 2 weeks and 4 weeks after implantation, the volume and weight of the fat mass in the control group were significantly reduced compared with those in the knockdown group, with statistically significant differences.

[0115] Furthermore, CT scans were performed on animals that had been implanted with fat for 4 weeks. From the CT images and statistical data, it can be seen that the amount of fat in the knockdown group was significantly higher than that in the control group, with a statistically significant difference. (See Fig. 7A and Figure 7B )

[0116] This shows that inhibiting YAP has a significant effect on fat transplantation in the body, prolonging the retention period of implanted fat and improving the effect of fat transplantation.

[0117] Example 5 Effects of YAP inhibition on tissue inflammation / fibrosis / oil cyst levels

[0118] Group treatment: control group (NC) and siRNA knockdown group 3.

[0119] The cells of each group were sampled 4 weeks after transplantation into the model animals, and CT observation and H&E staining were performed. Fig. 8A and Figure 8B As shown. Fig. 8A As shown, CT images indicate that inhibiting YAP effectively improved the survival rate of implanted fat, with more surviving fat tissue in the field of view.

[0120] The lower the level of inflammation / fibrosis in the fat, the less fat necrosis there is, and the fewer fat cysts (essentially necrotic fat), and the better the integrity of the fat. Inhibiting YAP significantly reduced the level of fibrosis and inflammation, resulting in fewer fat cysts and better integrity, indicating that the implanted fat is more active, see Figure 8B .

Claims

1. Use of a YAP gene inhibitor in the preparation of a drug for improving fat transplantation effects.

2. The use according to claim 1, characterized in that The improvement of fat transplantation effect is to increase the retention rate of implanted fat.

3. The use according to claim 1, characterized in that The improvement of fat transplantation effect is to prolong the retention period of implanted fat.

4. The use according to claim 1, characterized in that The improvement of fat transplantation effect is to reduce the degree of inflammation or fibrosis.

5. The use according to claim 1, characterized in that The YAP gene is derived from humans.

6. The use according to claim 1, characterized in that The YAP gene inhibitor includes one or more of the following siRNAs: GUCAGAGAUACUUCUUAAATT; UUUAAGAAGUAUCUCUGACTT; CAGGAAUUGAGAACAAUGATT; UCAUUGUUCUCAAUUCCUGTT; CCACCAAGCUAGAUAAAGATT; and UCUUUAUCUAGCUUGGUGGTT.

7. The use according to claim 1, characterized in that The YAP gene inhibitor comprises one or more groups of siRNA as follows: Group 1: GUCAGAGAUACUUCUUAAATT and UUUAAGAAGUAUCUCUGACTT; Group 2: CAGGAAUUGAGAACAAUGATT and UCAUUGUUCUCAAUUCCUGTT; and Group 3: CCACCAAGCUAGAUAAAGATT and UCUUUAUCUAGCUUGGUGGTT.

8. The use according to claim 1, characterized in that The YAP gene inhibitor is loaded or coated on a material as an active ingredient to make a medical device for improving the effect of fat transplantation.

9. The use according to claim 7, characterized in that The material is selected from the brackets made of PLA, PLL, PGA and PLGA.

10. The use according to claim 7, characterized in that The material is selected from microneedles made of PLA, PLL, PGA, PLGA or metal.

Images