Methods and applications of targeting and activating PIEZO1 in DPP4-positive cells to promote liver regeneration

Abstract

This invention discloses a method and its application for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells. This invention is the first to discover that DPP4-positive cells exhibit significant mechanosensitivity, with significantly higher expression levels of mechanosensitive genes compared to DPP4-negative cells. Based on this, a method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells is provided: specifically activating the PIEZO1 mechanosensitive channel within DPP4-positive cells in the liver of non-human mammals. This invention also provides a PIEZO1 gain-of-function transgenic mouse model and its application in screening drugs that promote liver regeneration. Experiments have demonstrated that in hepatectomy and induced acute liver injury models, activation of PIEZO1 in DPP4-positive cells significantly increases hepatocyte proliferation, reduces serum transaminase levels, and alleviates liver injury. This invention achieves precise regulation of key regenerative cell subsets, providing a new strategy for liver regeneration research and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine and liver surgery, specifically relating to a method and application of targeting and activating PIEZO1 in DPP4-positive cells to promote liver regeneration. Background Technology

[0002] The liver possesses a powerful regenerative capacity, playing a crucial role in maintaining homeostasis and responding to external damage. However, effectively promoting liver regeneration remains a significant challenge in clinical practice. Current treatment strategies for acute large-area liver injury, complex hepatectomy, and advanced chronic liver disease mainly include surgical procedures, drug intervention, and cell transplantation. Surgical procedures such as liver transplantation, portal vein ligation, or embolization can restore liver function to some extent, but they suffer from significant trauma, substantial individual variability, and a severe shortage of donors. Drug interventions, such as cytokines, growth factors, and DPP4 inhibitors, can activate liver regeneration-related pathways, but they lack selectivity for specific cell subpopulations, making systemic side effects difficult to control. Cell transplantation, such as stem cell and hepatic progenitor cell transplantation, faces obstacles such as high cellular heterogeneity and a high risk of immune rejection. Overall, existing strategies represent a "broad-brush, non-specific" liver regeneration activation model, lacking precise regulation of key hepatocyte subpopulations, necessitating the development of novel intervention strategies with clear targets and well-defined mechanisms.

[0003] Dipeptidyl peptidase 4 (DPP4) is a widely expressed serine protease involved in glucose metabolism, immune regulation, and inflammatory responses in the liver. Current research on the role of DPP4 in the liver primarily focuses on its function in chronic liver disease, viewing it as a pathological marker of liver fibrosis and steatosis. However, whether DPP4-positive cells possess unique mechanosensitivity and whether their regenerative function can be directly regulated through mechanosignal pathways remain crucial questions for further investigation.

[0004] Mechanotransmission plays a crucial role in tissue development, repair, and regeneration. PIEZO1, as a mechanosensitive ion channel, can sense mechanical stimuli such as blood flow shear force, tissue tension, and matrix stiffness, inducing calcium ion influx and activating downstream signaling pathways such as MAPK and YAP / TAZ. Current research on PIEZO1 in the liver primarily focuses on its paracrine mechanisms in non-hepatic parenchymal cells (such as endothelial cells, macrophages, and stellate cells), lacking studies on direct targeting of PIEZO1 within hepatic parenchymal cells. In particular, the functional role of PIEZO1 in the specific DPP4-positive hepatocyte subset, and its direct driving mechanism for the autonomous regeneration cycle of this cell subset, remain unexplored in current technology.

[0005] In summary, existing technologies for promoting liver regeneration have the following shortcomings: First, poor selectivity, as current intervention methods are difficult to precisely target key regenerative cell populations; second, unclear mechanisms, as the mechanosensitivity of DPP4-positive cells and the mechanism by which they regulate regeneration have not yet been elucidated; and third, lack of systematic solutions, as no technical solution has yet combined "DPP4-positive cell subsets" with "PIEZO1 mechanosensitivity regulation" for promoting liver regeneration. Summary of the Invention

[0006] This invention aims to provide a method and application for enhancing liver regeneration and reducing liver damage by targeting and activating the mechanosensitive ion channel PIEZO1 in a specific hepatocyte subset (DPP4-positive cells). The core technical problem this invention addresses is how to precisely amplify the mechanosensitivity and proliferative activity of this key cell subset by specifically activating the Piezo1 channel activity in DPP4-positive hepatocytes.

[0007] The technical solution adopted is to provide a method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells, which includes specifically activating the PIEZO1 mechanosensitive channel in DPP4-positive cells in non-human mammalian livers.

[0008] In a preferred embodiment, the specific activation is achieved through the following steps:

[0009] (a) Provide Dpp4-CreER T2 Piezo1-GOF transgenic mice;

[0010] (b) Tamoxifen was administered to the mice to induce the specific expression of the PIEZO1 gain-of-function mutant in DPP4 positive cells.

[0011] The preferred gain-of-function mutation in PIEZO1 is the R2481H point mutation (corresponding to R2482H in human PIEZO1 and R2481H in mice). This mutation exhibits significantly slowed inactivation kinetics and enhanced mechanosensitive channel activity.

[0012] The Dpp4- CreER T2 Piezo1-GOF transgenic mice were created by crossing mice carrying the inducible R2481H Piezo1 allele with Dpp4-CreER. T2 The mice were obtained through hybridization. The specific construction method was as follows: using CRISPR-Cas9 technology, the R2481H mutation (in mice) was introduced into exon 51 of the Piezo1 locus, and loxP sites were introduced into exons 45-51 flanking it to construct a Piezo1 gain-of-function mouse; then this mouse was crossed with Dpp4-CreER...T2 Mouse hybridization was performed to obtain offspring. Target activation of Piezo1 in DPP4-positive cells was achieved by injection of tamoxifen. Plasmids containing a gRNA expression cassette targeting exon 51 of the Piezo1 gene and a fragment of the wild-type Piezo1 gene are provided, as shown in SEQ ID NO:1; a single-stranded oligonucleotide (ssODN) repair template for introducing the R2481H mutation is provided, as shown in SEQ ID NO:2; and related vector plasmids (used as a backbone) are provided, as shown in SEQ ID NO:3.

[0013] The primer sequences used to identify the above mouse models are shown in Table 1:

[0014]

[0015] The method for promoting liver regeneration can be applied to the following models: (a) liver regeneration after partial hepatectomy; (b) liver regeneration after chemically induced acute liver injury. Preferably, the chemically induced acute liver injury is caused by carbon tetrachloride (…). Induced model, The application plan is as follows: Mix with corn oil at a volume ratio of 1:3 and administer once via intraperitoneal injection at a dose of 1 μL / g body weight.

[0016] The present invention also provides a PIEZO1 gain-of-function transgenic mouse model, wherein the mouse carries an R2481H point mutation (corresponding to human R2482H) in exon 51 of the Piezo1 locus, and the mutation is flanked by loxP sites, which can be conditionally activated under Cre recombinase mediation.

[0017] The present invention also provides the application of the above-mentioned mouse model in screening candidate drugs for promoting liver regeneration: the candidate drug is administered to the mouse model, and the regeneration effect of the drug is evaluated by detecting the enhancement of liver regeneration-related indicators (such as liver weight / body weight ratio, Ki67 positive cell rate, EdU positive cell rate, serum ALT / AST level).

[0018] The beneficial effects of this invention are as follows: This invention is the first to demonstrate that the expression level of mechanosensitive mRNA in DPP4-positive cells is significantly higher than that in DPP4-negative cells, revealing that this cell subpopulation possesses unique mechanoresponsive characteristics. It is the first to propose and validate an intervention strategy targeting the key regenerative subpopulation of "DPP4-positive cells," achieving precise regulation of liver regeneration by modulating its unique PIEZO1 mechanosensitive channel. It is the first to couple the "DPP4-positive cell subpopulation" with "PIEZO1 mechanosensitive regulation," revealing the specific mechanism by which the PIEZO1 channel regulates regeneration in DPP4-positive cells. This is achieved through the Dpp4-CreER pathway. T2The Piezo1-GOF mouse model confirmed that specific activation of PIEZO1 in DPP4-positive cells significantly enhanced hepatocyte proliferation and effectively reduced serum transaminase levels. In a liver resection model and... In induced acute liver injury models, they all showed the effects of promoting regeneration and reducing damage. Attached Figure Description

[0019] Figure 1 Schematic diagram of mouse model construction. A represents Dpp4-CreER. T2 R26-LSL-GFP mouse construction strategy; B represents Dpp4-CreER. T2 Piezo1GOF mouse construction strategy.

[0020] Figure 2 Comparison of the mechanosensitivity of DPP4 positive cells and DPP4 negative cells.

[0021] Figure 3 :Dpp4- CreER T2 Comparison of liver tissue morphology after hepatectomy between Piezo1GOF mice and control mice.

[0022] Figure 4 :Dpp4- CreER T2 Changes in liver injury markers (serum ALT / AST) in Piezo1GOF mice and control mice after surgery.

[0023] Figure 5 :Dpp4- CreER T2 Comparison of liver regeneration capacity between Piezo1GOF mice and control mice after hepatectomy.

[0024] Figure 6 :Dpp4- CreER T2 Comparison of liver tissue morphology between Piezo1GOF mice and control mice after carbon tetrachloride injury.

[0025] Figure 7 :Dpp4- CreER T2 Changes in liver injury markers (serum ALT / AST) in Piezo1GOF mice and control mice after carbon tetrachloride injury.

[0026] Figure 8 :Dpp4- CreER T2 Comparison of liver regeneration capacity between Piezo1GOF mice and control mice after carbon tetrachloride injury. Detailed Implementation

[0027] Example 1

[0028] Mouse liver resection

[0029] (1) Anesthesia and skin preparation: After weighing the mice, they were anesthetized by intraperitoneal injection. They were then fixed on the operating table, and the skin on their abdomen was shaved and disinfected.

[0030] (2) Opening the abdomen: Make a longitudinal incision along the midline of the abdomen below the xiphoid process, and cut through the skin and muscles layer by layer to expose the abdominal cavity. Gently separate the intestines with a cotton swab moistened with physiological saline to expose the liver.

[0031] (3) Hepatic lobe dissection and resection: Identify the anatomical structure of the liver and select the middle lobe and the left lateral lobe for resection. Gently separate the ligaments and tissues around the selected liver lobe with cotton swabs or microsurgical instruments and bring it out of the abdominal cavity. Ligate the base of the liver lobe with sterile surgical sutures, and then remove the liver tissue distal to the ligament.

[0032] (4) Closure of the abdomen: After confirming that there is no active bleeding in the remaining liver, the muscles and skin are sutured layer by layer with absorbable sutures.

[0033] (5) Postoperative care: Place the mouse on a heating pad until it wakes up, and closely monitor its vital signs after the operation.

[0034] Example 2

[0035] Tissue sampling and embedding

[0036] (1) Blood and liver tissue collection: After the experiment, whole blood was collected from mice by orbital blood collection. After standing at room temperature for 2 hours, serum was separated by centrifugation at 3000 rpm for 15 minutes and stored at -80℃ for later use. Mice were euthanized by cervical dislocation, and the abdomen was quickly dissected and perfused with PBS. Liver tissue was separated and removed. Part of the liver tissue was fixed in pre-cooled 4% paraformaldehyde solution and incubated overnight at 4℃. The other part was stored at -80℃.

[0037] (2) Paraffin embedding and sectioning: The fixed liver tissue was removed, washed three times with PBS, and trimmed into appropriately sized tissue blocks. The tissue blocks were dehydrated by a gradient of ethanol (70% ethanol overnight, 80%, 90%, and 95% ethanol for 2 hours each, and anhydrous ethanol at 4°C overnight). After clearing with xylene, the tissue blocks were immersed in molten paraffin for paraffin permeation treatment. The paraffin-impregnated tissue blocks were then placed in an embedding frame, injected with paraffin for embedding, and cooled and solidified at room temperature to form paraffin blocks. The paraffin blocks were trimmed and fixed on a paraffin microtome and cut into continuous sections with a thickness of 5 μm. After flattening the sections in 45°C warm water, they were lifted with a slide to prevent detachment and placed in a 55°C constant temperature oven for 2 hours. They were then stored at room temperature for later use.

[0038] (3) Cryopreservation and sectioning: Wash the fixed tissue (samples used for microsurgery are not fixed) three times with PBS and aspirate the surface moisture. Trim the tissue to a suitable size (usually no more than 1 cm in length and width, and no more than 0.5 cm in thickness). Place the tissue on the embedding holder, add OCT embedding agent to completely cover the tissue. Quickly pour in liquid nitrogen for flash freezing. Once the OCT solidifies and turns white, store at -80°C. Fix the tissue block on the microtome sample holder and adjust the angle so that the tissue section is parallel to the blade. Adjust to the desired section thickness (6 μm). Adjust the position of the anti-roll plate, and crank the handwheel at a uniform speed to cut a complete tissue section. Store at -80°C.

[0039] Example 3

[0040] Construction of mouse model

[0041] All mice used in the experiments were raised in an SPF-grade environment at the Experimental Animal Center of Xi'an Jiaotong University. All animal experiments were conducted with the permission of the Experimental Animal Ethics Committee of the School of Medicine of Xi'an Jiaotong University and met ethical requirements.

[0042] (1) Constructing Dpp4-CreER T2 R26-LSL-GFP mice: Using R26-LSL-GFP mice and Dpp4-CreER T2 The tool mouse was mated to produce the F1 generation Dpp4- CreER T2 R26-LSL-GFP heterozygous mice were then crossbred with R26-LSL-GFP mice to obtain the F2 generation Dpp4-CreER. T2 R26-LSL-GFP homozygous mice. Among them, Dpp4-CreER... T2 R26-LSL-GFP mice were used as the experimental group, and R26-LSL-GFP mice were used as the control group. Eight-week-old Dpp4-CreER mice were used. T2 R26-LSL-GFP mice and their littermate control mice were intraperitoneally injected with 50 μl of 20 mg / ml tamoxifen for five consecutive days. (Dpp4- CreER) T2 In R26-LSL-GFP mice, DPP4-positive cells can be labeled with GFP. (See schematic diagram.) Figure 1 A.

[0043] (2) Construct Dpp4-CreER T2 Piezo1GOF mice: using Piezo1GOF flox / flox Mice (exon 45-51 mutation) are similar to Dpp4-CreER T2 Mice were mated to obtain the Piezo1 GOF genotype. flox / - Dpp4- CreERT2 F1 generation heterozygous mice were then bred and bred with Piezo1 GOF mice after reaching adulthood. flox / flox Mice were co-cultured to obtain the Piezo1GOF genotype. flox / flox Dpp4- CreER T2 The F2 generation mice were named DPP4-positive cell-specific Piezo1 overexpressing mice. The control group consisted of littermate Piezo1 GOF mice. flox / flox Dpp4- CreER T2 Negative mice. Among them, DPP4-positive cell-specific Piezo1 overexpression mice showed enhanced Piezo1 function after 5 days of tamoxifen administration. See schematic diagram. Figure 1 B.

[0044] Example 4

[0045] Verification of the mechanosensitivity of DPP4-positive cells

[0046] (1) Sample preparation: Using Dpp4-CreER from Example 3 T2 Four days after hepatectomy in R26-LSL-GFP mice, livers were harvested in the dark and samples were prepared using the cryopreservation method described in Example 2. Dpp4-CreER livers were then removed from the liver after hepatectomy at -80°C. T2 R26-LSL-GFP mouse liver OCT embedding blocks were placed in a microtome and equilibrated to -20°C. The sample blocks were trimmed to the area to be cut, and the slice thickness was set to 15 μm. Tissue slices were cut and attached to PEN membrane slides. The PEN membrane slides containing the slices were removed from -80°C and quickly immersed in pre-cooled 75% ethanol (prepared with DEPC water) for 30-60 seconds for fixation / dehydration. Then, the slides were sequentially immersed in 75% ethanol → 95% ethanol → 100% ethanol, each step approximately 30 seconds. The slides were placed in a fume hood or air-dried at room temperature for 2-5 minutes, followed by laser microdissection. A collection tube was placed in the stage, and 50 μl of lysis buffer was added to the cap. The air-dried slides were placed in the instrument stage, the fluorescence channel was switched, and the GFP-positive region (cells expressing DPP4) was located and labeled. The cutting area was drawn, and laser cutting was initiated. The cut fragments fell into the PCR tube cap below. After collecting positive cells, replace the tube with a new PCR tube containing lysis buffer and cut and collect negative cells. After collection, briefly centrifuge the PCR tube to allow the lysis buffer in the cap to collect at the bottom of the tube. Immediately place the sample tube on ice, or proceed directly with RNA extraction.

[0047] (2) RNA extraction: Add 1 ml Trizol to each sample, incubate at room temperature for 5 minutes, add 200 μL chloroform, shake vigorously for 15 seconds, incubate at room temperature for 3 minutes, and then centrifuge at 12000 rpm for 15 minutes at 4℃. Carefully aspirate approximately 400 μL of the colorless aqueous phase from the top to a new EP tube, add an equal volume of isopropanol, mix by inverting, incubate at room temperature for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4℃. A white precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water), gently wash the precipitate, and centrifuge at 7500 rpm for 5 minutes at 4℃. Repeat twice. Discard the supernatant, dry the precipitate at room temperature for 5-10 minutes, and add an appropriate amount of DEPC water to dissolve the RNA. Use a spectrophotometer to determine the RNA concentration and purity (OD 260 / 280 should be between 1.8 and 2.0). Take 1 μg of total RNA, add reverse transcription premix according to the kit instructions, and incubate according to the program. cDNA was obtained after the reaction was completed.

[0048] (3) Real-time quantitative PCR: Add 10 μL of 2× qPCR premix (SYBR Green method), 0.4-1 μL each of forward and reverse primers (10 μM), and 1-2 μL of cDNA template to each well, and add enzyme-free water to a final volume of 20 μL. Reaction program: Pre-denaturation at 95℃ for 2-10 minutes (1 cycle); Cyclic reaction (40 cycles): denaturation at 95℃ for 15 seconds, annealing for 30 seconds, extension at 72℃ for 30 seconds; Melting curve was used to detect product specificity. Using 18s as an internal control, the relative expression level of the target gene was calculated. Results are as follows: Figure 2 As shown, the expression of mechanically related mRNAs in DPP4-positive cells was significantly higher than that in DPP4-negative cells, confirming that DPP4-positive cells have stronger mechanical sensitivity.

[0049] Example 5

[0050] Dpp4- CreER T2 Phenotypic analysis of Piezo1GOF mice after hepatectomy

[0051] (1) Mouse modeling and tissue collection: using Dpp4-CreER in Example 3 T2 ;Piezo1 GOF flox / flox Mice underwent liver resection as described in Example 1, and were sacrificed 4 days post-surgery. Liver weight and body weight were measured, and the liver weight / body weight ratio was calculated. Results are as follows... Figure 3 As shown in B, the liver weight / body weight ratio was significantly increased in the GOF group.

[0052] (2) H&E staining: Paraffin-embedded tissue sections were immersed in xylene I, II, and III for 7 min each, then in a gradient of ethanol (anhydrous ethanol I and II, 95%, 90%, 80%, and 70%) for 1 min each, and rinsed with running water for 2 min. The hydrated sections were immersed in a modified hematoxylin staining solution and incubated at room temperature for 7 min, then rinsed briefly with running water; then quickly immersed in hydrochloric acid differentiation solution for a few seconds, and immediately rinsed with running water; then placed in blueing solution for 5 min, and rinsed with running water for 10 min. The blued sections were immersed in eosin staining solution and incubated at room temperature for 4 min, then rinsed briefly with running water. Gradual dehydration was performed by immersing in 70%, 80%, 90%, 95%, and anhydrous ethanol I and II for 30 s each time. The sections were then immersed in xylene I and II for 3 min each to make them transparent. A suitable amount of neutral resin was dropped into the center of the tissue, and a coverslip was placed on top to seal the slide. The slide was photographed under a microscope. The results are as follows. Figure 3 As shown in C and 3D, the sinusoidal space of the liver in the GOF group mice was significantly increased compared with that in the control group, indicating that the GOF group mice had more proliferation.

[0053] (3) ALT activity assay: The assay was performed strictly according to the instructions of the Nanjing Jiancheng reagent kit. Serum was collected, and the alanine aminotransferase matrix solution, 2,4-dinitrophenylhydrazine solution, and 2 μmol / mL sodium pyruvate standard solution were prepared according to the instructions. The matrix solution and the sample to be tested were added to the test wells, mixed well, and reacted at 37°C for 30 minutes. Then, 2,4-dinitrophenylhydrazine solution was added, mixed well, and reacted at 37°C for 20 minutes. Finally, 0.4 mol / L sodium hydroxide solution was added, mixed well, and incubated at room temperature for 15 minutes. The absorbance of each well was measured at a wavelength of 505 nm, and the ALT activity was calculated according to the standard curve. The results are as follows: Figure 4 As shown in Figure A, ALT levels in the GOF group were significantly lower than those in the control group.

[0054] (4) AST activity assay: The assay was performed strictly according to the instructions of the Nanjing Jiancheng reagent kit. 20 μl of pre-warmed matrix solution (37℃) was accurately added to each well of a 96-well plate. 5 μl of the sample to be tested was added to the bottom of each well, gently shaken to mix, and incubated at 37℃ for 30 minutes. 20 μl of 2,4-dinitrophenylhydrazine solution was added to each well, mixed, and incubated at 37℃ for 20 minutes. 200 μl of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and incubated at room temperature for 15 minutes. The absorbance of each well was measured at 505 nm, and the AST activity was calculated based on the standard curve. The results are as follows: Figure 4 As shown in Figure B, the AST level in the GOF group was significantly lower than that in the control group.

[0055] (5) EdU detection: EdU (50 mg / kg / day) was injected intraperitoneally for 5 consecutive days after surgery. After euthanasia, the liver was harvested, and frozen sections were prepared according to the frozen embedding sectioning method described in Example 2. The sections were removed from -80℃ and brought to room temperature. The samples were washed three times with PBS. Permeabilization buffer was applied at room temperature for 10-15 minutes, followed by thorough rinsing with washing buffer. Click Reaction solution from the Beyotime EdU staining kit was prepared and added to the sections. The sections were incubated at room temperature in the dark for 30 minutes. The Click Reaction solution was removed, and the sections were rinsed with PBS. DAPI was added and incubated at room temperature in the dark for 10 minutes to label all cell nuclei. After rinsing with PBS, the sections were mounted with anti-fluorescence quenching mounting solution and observed and photographed under a fluorescence microscope. The results are as follows: Figure 5 As shown in A and 5B, the number of EdU-positive cells in the liver of the GOF group was significantly increased compared with that of the control group. Quantitative analysis showed that the hepatocyte proliferation index of the GOF group was approximately 23.10%, while that of the control group was 18.19%.

[0056] Example 6

[0057] Dpp4- CreER T2 Piezo1GOF mice in Phenotypic analysis in an induced acute liver injury model

[0058] (1) Carbon tetrachloride-induced acute liver injury model: using Dpp4-CreER from Example 3 T2 A carbon tetrachloride-induced acute liver injury model was established in Piezo1GOF mice on day 7 after the last tamoxifen injection. Carbon tetrachloride (… Mix with corn oil to prepare a 1:3 volume ratio solution (i.e., 25%). (Solution). Mice were administered the solution via intraperitoneal injection at a dose of 1 μL / g body weight. Mixed solution. To assess cell proliferation during liver injury repair, in EdU labeling began on the day of modeling at a dose of 50 mg / kg. For the next two days, the same dose of EdU was administered once daily at the same time, for a total of three injections. After modeling was completed, whole blood was collected from mice via orbital sampling, and serum was separated. Mice were euthanized by cervical dislocation after blood collection, and the liver was quickly removed, rinsed thoroughly with pre-cooled PBS, weighed, and the liver weight / body weight ratio was calculated. Results are as follows: Figure 6 As shown in B, the liver weight / body weight ratio in the GOF group was significantly higher than that in the control group.

[0059] (2) HE staining: HE staining was performed according to the method in Example 5. The results are as follows: Figure 6 As shown in Figure C, the GOF group showed better preservation of liver lobule structure, significantly reduced necrosis area, and markedly reduced hepatocyte ballooning degeneration and fatty degeneration.

[0060] (3) Serum ALT and AST detection: Serum ALT and AST levels were detected according to the method described in Example 5. The results are as follows: Figure 7 As shown in A and 7B, serum ALT and AST levels in the GOF group were significantly lower than those in the control group.

[0061] (4) EdU detection: Performed according to the EdU detection method in Example 5. The results are as follows: Figure 8 As shown in A and B, the number of EdU-positive cells in the liver of the GOF group was significantly increased compared with that of the control group. Quantitative analysis showed that the hepatocyte proliferation index of the GOF group was approximately 30.87%, while that of the control group was 24.69%.

[0062] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.

Claims

1. A method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells, characterized in that, It specifically activates the PIEZO1 mechanosensitive channel in DPP4-positive cells in the liver of non-human mammals.

2. The method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells according to claim 1, characterized in that, The specific activation is achieved through the following steps: (a) Provide Dpp4-CreER T2 Piezo1-GOF transgenic mice; (b) Tamoxifen was administered to the mice to induce the specific expression of the PIEZO1 gain-of-function mutant in DPP4 positive cells.

3. The method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells according to claim 2, characterized in that, The gain-of-function mutation of PIEZO1 is the R2481H point mutation.

4. The method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells according to claim 2, characterized in that, The Dpp4- CreER T2 Piezo1-GOF transgenic mice were created by crossing mice carrying the inducible R2481H Piezo1 allele with Dpp4-CreER. T2 Obtained by hybridization of mice.

5. The method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells according to claim 1, characterized in that, The liver regeneration promotion method is applied to at least one of the following models: (a) Liver regeneration after partial hepatectomy; (b) Liver regeneration following chemically induced acute liver injury.

6. The method for promoting liver regeneration by targeting and activating PIEZO1 in DPP4-positive cells according to claim 5, characterized in that, The chemically induced acute liver injury is Induced acute liver injury, The application plan is as follows: Mix with corn oil at a volume ratio of 1:3 and administer once via intraperitoneal injection at a dose of 1 μL / g body weight.

7. A PIEZO1 gain-of-function transgenic mouse model, characterized in that, The mice have an R2481H point mutation in exon 51 of the Piezo1 locus, and this mutation is flanked by loxP sites, which can be conditionally activated under Cre recombinase-mediated activation.

8. The application of the mouse model according to claim 7 in screening candidate drugs for promoting liver regeneration, characterized in that, The candidate drug was administered to the mouse model, and the regenerative effect of the drug was evaluated by detecting the enhancement of liver regeneration-related indicators.

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