Application of PINCH2 protein as target spot in preparation of medicine for treating dynamic urinary retention

Abstract

The invention relates to an application of PINCH2 protein or gene as a target spot in a drug for treating dynamic urinary retention, belongs to the technical field of biomedical engineering, and particularly relates to an application of a substance for regulating PINCH2 protein expression or a substance for regulating PINCH2 protein activity in a drug for treating dynamic urinary retention. According to the invention, a target protein PINCH2 which plays a crucial role in regulation and control of the detrusor of the bladder is found for the first time, and by targeting the PINCH2 protein in the detrusor of the bladder through a drug, the specificity of the drug can be further enhanced, and side effects are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and particularly to the application of PINCH2 protein as a target in drugs for treating dynamic urinary retention. Background Art

[0002] Urinary retention refers to the situation where only part of the urine or even no urine can be discharged after urination, resulting in urine accumulation in the bladder. The typical manifestations are inability to urinate and a feeling of distension in the lower abdomen. The common causes of urinary retention are mainly divided into two categories. The first category is mechanical: that is, various reasons lead to stenosis or even complete blockage of the urine excretion channel. The second category is dynamic: the main driving force for bladder urine is the contraction of the detrusor muscle of the bladder, which is innervated by nerves. When various reasons cause poor contraction or inability to contract of the detrusor muscle of the bladder, it belongs to dynamic urinary retention; common ones include central and peripheral nervous system diseases, such as spinal cord injury and diabetes; surgical injury to the innervating nerves, such as rectal and gynecological surgeries; general anesthesia or lumbar anesthesia; the use of some drugs, such as atropine and 654-2. Currently, the treatment methods for urinary retention are mostly medical device-assisted treatments. For example, for acute urinary retention, indwelling a urinary catheter through the external urethral orifice is the first choice. However, when indwelling fails, bladder puncture drainage can be performed with a thick needle through the abdominal wall, or suprapubic cystostomy can be performed through the abdominal wall, or a urinary catheter or cystostomy tube can be indwelled for a long time. Another example is chronic dynamic urinary retention. When dynamic urinary retention causes hydronephrosis of both kidneys and impaired renal function, the urine in the bladder is drained first, and then the cause is treated after the hydronephrosis and renal function are improved. When the cause cannot be treated, the urine in the bladder needs to be drained for a long time. The methods include catheterization, cystostomy, and clean intermittent self-catheterization. Currently, there is still a lack of effective drugs and treatment methods for treating dynamic urinary retention in clinical practice.

[0003] PINCH is a LIM domain protein. There are two PINCH proteins in mammals, namely PINCH1 protein and PINCH2 protein. PINCH1 (also known as LIMS1) is the earliest discovered member of the PINCH family. After the discovery of PINCH1, a new PINCH family was discovered in 2003 and named PINCH2. Studies have found that the overall similarity between PINCH2 and PINCH1 is 92%, and it contains 5 LIM domains similar to PINCH1. PINCH1 and PINCH2 interact with ILK through the LIM domain and play a key role in cell shape regulation, proliferation, survival and differentiation, while also affecting cell spreading and migration. In addition, PINCH family proteins are closely related to the organization of the cytoskeleton. They affect the assembly and stability of actin stress fibers by interacting with actin-binding proteins such as α-actin. Therefore, the study of PINCH proteins helps us understand how cells respond to external signals and their functional changes in disease states, especially in cancers, metabolic diseases and muscle diseases, etc., providing new strategies and targets for the diagnosis and treatment of related diseases. The prior art CN115820656A uses PINCH proteins (including PINCH1 protein and PINCH2 protein) or genes (including PINCH1 gene and PINCH2 gene) to prepare signal pathway activators or signal pathway inhibitors, which play an important role in the development and function regulation of embryonic kidneys. The deletion of the PINCH gene will lead to abnormal kidney development, accompanied by abnormal development of glomeruli and ureteric buds. By measuring the expression of the PINCH gene or PINCH protein to detect or predict diseases related to abnormal embryonic kidney development, that is, abnormal expression levels of PINCH proteins or genes predict abnormal kidney development. Diagnostic products that detect the abnormal expression site and expression level of PINCH proteins or genes can be used for prenatal screening, diagnosis and treatment of diseases related to abnormal embryonic kidney development.

[0004] The present invention discovers that PINCH2 protein is generally expressed in the detrusor of the bladder, but its role and molecular mechanism in the regulation of the detrusor of the bladder are still unclear.

[0005] To deeply understand the pathogenesis of dynamic urinary retention and provide relevant means for the prevention and treatment of related dynamic urinary retention diseases, there is an urgent need in the art to study genes related to the regulation and function of the detrusor of the bladder and their regulatory pathways. Summary of the Invention

[0006] For the clinical development of effective drugs and treatment methods for dynamic urinary retention, the present invention proposes the application of PINCH2 protein as a target in drugs for dynamic urinary retention. The present invention discovers for the first time a target protein PINCH2 that plays a crucial role in the regulation of the detrusor muscle of the bladder. By targeting the PINCH2 protein in the detrusor muscle with drugs, the specificity of the drugs can be further enhanced and side effects can be reduced.

[0007] In a first aspect, the present invention proposes the application of PINCH2 protein in drugs for dynamic urinary retention, and the drugs target the PINCH2 protein.

[0008] Preferably, the drugs target the PINCH2 protein in the detrusor muscle of the bladder.

[0009] Preferably, the drugs can regulate the expression level of the PINCH2 protein.

[0010] Preferably, the drugs can regulate the expression level of the PINCH2 protein in the detrusor muscle of the bladder.

[0011] Preferably, the drugs can increase the expression level of the PINCH2 protein.

[0012] Preferably, the drugs can increase the expression level of the PINCH2 protein in the detrusor muscle of the bladder.

[0013] In a second aspect, the present invention proposes a pharmaceutical composition for treating dynamic urinary retention, which includes a PINCH2 protein promoter and a targeting agent. The PINCH2 protein promoter is loaded on the targeting agent, and the targeting agent targets the PINCH2 protein.

[0014] Preferably, the targeting agent includes AAV virus (Adeno-associated virus), miRNA (MicroRNA), and polymeric micelles.

[0015] In a third aspect, the present invention proposes the application of PINCH2 protein in the preparation of a detection reagent for dynamic urinary retention, and the detection reagent uses the PINCH2 protein as a biomarker for dynamic urinary retention.

[0016] Compared with the prior art, the present invention has the following beneficial effects: In the application of the present invention, after the systemic knockout of the PINCH2 gene, the PINCH2 protein in mice is significantly reduced, and thus the phenotype of dynamic urinary retention appears in the bladder of mice. Microscopically, it can be seen that the detrusor cells proliferate and hypertrophy; the muscle bundles are disorderly arranged, and the interstitial components are loose, indicating that the PINCH2 gene has a significant impact on the development of detrusor cells. The present invention discovers for the first time a target protein PINCH2 that plays a crucial role in the regulation of dynamic urinary retention. By regulating the expression level of the PINCH2 protein or its activity through targeted drugs, the contraction function of the bladder detrusor can be enhanced by increasing the expression level of the PINCH2 protein, thereby effectively improving or treating the symptoms of dynamic urinary retention. Description of the Drawings

[0017] Figure 1-1 It is a phenotypic diagram of bladder urinary retention in 1-month-old mice in the experimental group; Figure 1-2 It is a phenotypic diagram of bladder urinary retention in 4-month-old mice in the experimental group; Figure 2 It is a HE staining result diagram of the bladder detrusor cells of control group and experimental group mice; Figure 3 It is a statistical chart of the muscle wall thickness of the bladder detrusor in HE staining of control group and experimental group mice; Figure 4 It is a VG staining result diagram of the bladder detrusor cells of control group and experimental group mice; Figure 5 It is a statistical chart of the ratio of detrusor to collagen fiber in VG staining of the bladder of control group and experimental group mice; Figure 6 It is a culture result diagram of primary bladder detrusor cells of PINCH2 knockout mice; Figure 7 It is an identification diagram of α-SMA of primary bladder detrusor cells of PINCH2 knockout mice; Figure 8 It is a WB verification comparison diagram of two viruses with knocked-down PINCH2; Figure 9 It is an effect diagram of the collagen gel contraction experiment of bladder detrusor cells; Figure 10 It is a statistical chart of the collagen diameters of each group in the collagen gel contraction experiment. Detailed Implementation Modes

[0018] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following specifically describes the specific embodiments of the present invention in conjunction with specific examples and the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0019] Referring to FIGS. 1-10, the present invention provides an application of PINCH2 protein in a drug for treating dynamic urinary retention, wherein the drug targets the PINCH2 protein.

[0020] PINCH2 is a protein with five LIM domains and is involved in the regulation of integrin-mediated cell adhesion. Research shows that PINCH2 interacts with integrin-linked kinase and Nck2. However, there are few studies on PINCH2 protein as a therapeutic target, and there is no research report on its application in dynamic urinary retention. The inventors of the present invention unexpectedly found through experimental research that the expression of PINCH2 protein is closely related to dynamic urinary retention. Under microscopic observation, the detrusor cells of the bladder in PINCH2 knockout mice showed hyperplasia and hypertrophy; the muscle bundles were disorderly arranged, and the interstitial components were loose, resulting in difficulty in urination and dynamic urinary retention. The experimental results of this study provide a new target for drugs treating dynamic urinary retention. The drug targets the PINCH2 protein, thereby regulating the expression level of PINCH2 protein in the body and providing treatment for dynamic urinary retention.

[0021] Specifically, the "drug for treating dynamic urinary retention" mentioned herein can be a drug prepared for treating patients already diagnosed with dynamic urinary retention or a drug prepared for preventing dynamic urinary retention.

[0022] In an embodiment of the application of PINCH2 protein in a drug for treating dynamic urinary retention, the drug targets the PINCH2 protein in the detrusor muscle of the bladder. The detrusor muscle of the bladder is closely related to dynamic urinary retention. By targeting the PINCH2 protein in the detrusor muscle of the bladder with the drug, the specificity of the drug can be further enhanced, and side effects can be reduced.

[0023] In an embodiment of the application of PINCH2 protein in a drug for treating dynamic urinary retention, the drug can regulate the expression level of PINCH2 protein. As a target, PINCH2 regulates the expression level of PINCH2 protein in the body according to the conditions of different patients, so that the expression and secretion of PINCH2 protein in the body are normal.

[0024] Specifically, in the application of PINCH2 protein in drugs for dynamic urinary retention, the drug can increase the expression level of PINCH2 protein. Referring to Appendix Figure 1-1 and Appendix Figure 1-2 , the research results show that after PINCH2 gene knockout in mice, the detrusor muscle cells of the bladder proliferate and hypertrophy, the muscle bundles are arranged disorderly, and the interstitial muscle components are loose. Experiments have proved that when the secretion level of PINCH2 protein decreases, the contraction function of the detrusor muscle of the bladder is damaged, resulting in symptoms such as dysuria and dynamic urinary retention. Therefore, the contraction function of the detrusor muscle of the bladder can be enhanced by increasing the expression level of PINCH2 protein through drugs, and symptoms such as dynamic urinary retention can be improved.

[0025] Furthermore, in the application of PINCH2 protein in drugs for dynamic urinary retention, the drug can increase the expression level of PINCH2 protein in the detrusor muscle of the bladder. By increasing the expression level of PINCH2 protein in the detrusor muscle of the bladder through drugs, the specificity of the drug is enhanced, and then the function of the detrusor muscle of the bladder is improved, further preventing or treating dynamic urinary retention.

[0026] In an embodiment of the application of PINCH2 protein in drugs for dynamic urinary retention, the drug targets PINCH2 protein. After experimental research, it is found that after systemic knockout of PINCH2, mice show symptoms of low-activity detrusor such as dysuria and dynamic urinary retention, suggesting that PINCH2 protein can be used as a new target for drugs to treat dynamic urinary retention of the detrusor muscle of the bladder.

[0027] Furthermore, in the application of PINCH2 protein in the preparation of drugs for treating dynamic urinary retention of the detrusor muscle of the bladder, the drug can target PINCH2 protein in the detrusor muscle of the bladder.

[0028] In one embodiment, the drug specifically targets PINCH2 protein in detrusor muscle cells of the bladder.

[0029] In one embodiment, the drug can increase the expression level of PINCH2 protein, specifically, it can increase the expression level of PINCH2 protein in the detrusor muscle of the bladder.

[0030] In summary, the research results show that there is a close correlation between the expression level of PINCH2 protein and dynamic urinary retention. PINCH2 protein can be used as a new target for the preparation of drugs for treating dynamic urinary retention and has good application prospects in the application of the preparation of drugs for preventing or treating dynamic urinary retention.

[0031] In addition, the present application also provides a drug for treating dynamic urinary retention, wherein the drug can increase the expression level of PINCH2 protein.

[0032] In one embodiment, the drug for treating dynamic urinary retention targets the PINCH2 protein in the detrusor muscle of the bladder and can increase the expression level of the PINCH2 protein in the detrusor muscle of the bladder.

[0033] The above-mentioned drug uses the PINCH2 protein as a therapeutic target and treats dynamic urinary retention by increasing the expression level of the PINCH2 protein. The research results show that when the whole body PINCH2 gene is knocked out, the expression level of the PINCH2 protein decreases, the detrusor muscle cells of the mouse bladder proliferate and hypertrophy, the arrangement of the detrusor muscle bundles is disordered, the muscle interstitium is fibrotic, and the contraction function of the detrusor muscle of the bladder is reduced. Therefore, the expression level of the PINCH2 protein can be increased by drugs to prevent detrusor muscle fibrosis, improve the contraction function of the detrusor muscle of the bladder, and thus treat dynamic urinary retention.

[0034] Furthermore, the present application also provides a drug composition for treating dynamic urinary retention. The composition includes a PINCH2 protein promoter and a targeting agent. The PINCH2 protein promoter is loaded on the targeting agent, and the targeting agent targets the PINCH2 protein.

[0035] Among them, the PINCH2 protein promoter is a drug or reagent that can increase the expression level of the PINCH2 protein, and the targeting agent is an agent that can target the PINCH2 protein; the PINCH2 protein promoter can be loaded on the targeting agent by chemical bond connection or physical methods such as coating.

[0036] In one embodiment, the targeting agent includes an AAV virus (Adeno-associated virus), miRNA (MicroRNA), and polymeric micelles.

[0037] Specifically, the AAV virus, miRNA, or polymeric micelles serve as carriers to carry the PINCH2 protein promoter to a specific site in the body. The release of the PINCH2 protein promoter promotes the expression of the PINCH2 protein in the body, thereby increasing the expression level of the PINCH2 protein, preventing sclerosis and interstitial fibrosis of the detrusor muscle of the bladder, improving the function of the detrusor muscle of the bladder, and thus preventing or treating dynamic urinary retention.

[0038] In one embodiment, the AAV virus can also carry a gene that promotes the PINCH2 protein, so as to express the PINCH2 protein in the body and increase the expression level of the PINCH2 protein.

[0039] The above-mentioned drug composition for treating dynamic urinary retention includes a PINCH2 protein promoter and a targeting agent. The two work synergistically to increase the expression level of the PINCH2 protein, and further prevent or treat dynamic urinary retention.

[0040] It should be noted that according to the treatment needs, different targeting agents can be selected in the present invention, and the targeting agent can effectively carry a PINCH2 protein promoter. Other embodiments in which the targeting agent carries a PINCH2 protein promoter to achieve the expression or overexpression of the PINCH2 protein are within the protection scope of the present invention.

[0041] In addition, the present application also provides an application of the PINCH2 protein in a specific embodiment in the preparation of a detection reagent for dynamic urinary retention, wherein the detection reagent uses the PINCH2 protein as a biomarker for dynamic urinary retention.

[0042] Research shows that there is a close correlation between the expression level of the PINCH2 protein and dynamic urinary retention. Therefore, the PINCH2 protein can be used as a biomarker for dynamic urinary retention. Specifically, the detection reagent can determine the condition of dynamic urinary retention based on the expression level of the PINCH2 protein.

[0043] In summary, through experimental research, it was unexpectedly found that there is a close correlation between the PINCH2 protein and dynamic urinary retention. After systemic knockout of the PINCH2 gene, the expression level of the PINCH2 protein decreased, and the structure of the detrusor muscle of the mouse bladder was severely damaged, with obvious sclerosis of the detrusor muscle and interstitial fibrosis. The experimental results suggest that the PINCH2 protein can be used as a new target for the treatment of dynamic urinary retention. The PINCH2 protein has good application prospects in the preparation of drugs for the treatment of dynamic urinary retention or the preparation of detection reagents for dynamic urinary retention.

[0044] The following are specific examples.

[0045] Unless otherwise specified, the drugs and instruments used in the examples are all conventional selections in the art. For the experimental methods without specific conditions indicated in the examples, they are usually carried out under conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturers.

[0046] Unless otherwise specified, in the following examples, WT represents wild-type control mice, and P2- / - represents mice with systemic knockout of the PINCH2 gene. Example

[0047] Study the effect of knocking out the PINCH2 gene on the bladder of mice The PINCH2- / - mice were constructed using gene knockout technology. The mice were bred in a standard mouse breeding device in the Animal Center of Southern University of Science and Technology, and were given a 12:12 h light condition and free diet and water intake. As shown in Appendix Figure 1-1 and Appendix Figure 1-2 It was observed in the present invention that 1 month after knocking out the PINCH2 gene, the mice showed manifestations of urinary retention in the bladder, and the urinary retention was more severe after 4 months.

[0048] The above results indicate that knocking out PINCH2 leads to a decrease in the bladder contraction function of mice, and the mice show manifestations of urinary retention. Example

[0049] Study on HE staining and VG staining of the bladder smooth muscle of PINCH2 gene knockout mice The results of HE staining are as shown in the appendix Figure 2-3 As shown, the muscle bundles of the detrusor cells in the bladder of the control group mice run orderly and are closely arranged between the muscle bundles; the bladder muscle wall of the experimental group mice becomes thinner, the muscle bundles become thinner and the alignment is disordered, the arrangement is loose, the loose fibrous connective tissue between the muscle bundles increases, and the muscle fiber gap increases.

[0050] The results of VG staining are as Figure 4-5 shown. Collagen fibers are mainly deposited in the detrusor interstitial tissue. The tissue space of the bladder of the experimental group mice increases, fibroblast proliferation is significant, collagen fiber deposition occurs, and the proportion of the detrusor muscle to collagen fibers in the mice bladder is reduced compared with the control group. Example

[0051] Study on the effect of knocking out PINCH2 in bladder smooth muscle on contraction function First step, the present invention uses the enzyme digestion method to isolate detrusor cells for primary cell culture. Specifically, collagen I 4mg / ml and bovine serum albumin (BSA) 4mg / ml are used to digest for 40 - 60 minutes under the conditions of 37.0 °C and 5% CO2. Then, the cell morphology and growth mode of the obtained cells are observed.

[0052] The present invention uses the enzyme digestion method to isolate and culture primary bladder detrusor cells. Smooth muscle cells can adhere to the wall after 24 hours, and the cells are spindle-shaped. After 3 - 5 days, the cells gradually fuse into a sheet-like growth, and the cell fusion reaches more than 80% in about 7 days. The cells grow in a typical "peak-valley" pattern of smooth muscle cells, as shown in the appendix Figure 6 as shown.

[0053] Second step, the present invention uses immunofluorescence staining to identify bladder detrusor cells. The detailed steps are as follows: The cultured cells were digested with 0.125% trypsin at 37.0 °C for 30 s, 1 ml of culture medium was added, and the cells were gently pipetted with a Pasteur pipette. Then, 400 μL of the cell suspension was inoculated into a confocal dish, fixed with 4% paraformaldehyde at room temperature for 30 minutes, then perforated with 1% triton for 15 minutes, and 3% BSA was added dropwise and reacted at 37.0 °C for 1 hour. Next, the excess liquid was discarded without washing. Then, the mouse anti-a-SMA monoclonal antibody was added dropwise at a concentration of 1:200 according to the antibody instruction manual and incubated overnight at 4 °C. The next day, the primary antibody solution was removed and washed twice with PBS for 20 min each time; FITC-labeled goat anti-mouse IgG was added at 1:200 and placed at 37.0 °C for 30 minutes; then washed three times with PBS for 1 min each time. Then DAPI was used to stain the nuclei. After immunofluorescence staining, a small amount of DAPI staining solution was added to the confocal dish to cover the sample, incubated at room temperature for 10 minutes, the DAPI staining solution was aspirated, and washed three times with PBS for 5 min each time. Then, observation and photographing were carried out under a confocal microscope.

[0054] a-SMA antibody and FITC-IgG were used for immunofluorescence identification of cells, and DAPI was used to stain the nuclei. Under the confocal microscope, the cell nuclei were blue-stained and showed oval shapes with blunt ends at both ends. The a-SMA in the cells was excited by the excitation light and showed red fluorescence, which was identified as smooth muscle cells. As shown Figure 7 in the figure, the expression level of a-SMA in the bladder detrusor muscle cells of the control group was relatively high, and the red fluorescence was relatively strong, indicating that the cells had a relatively high degree of smooth muscle differentiation; the expression level of a-SMA in the bladder detrusor muscle cells of the PINCH2 knockout group was relatively low, and the red fluorescence was relatively weak, indicating that the cells had a relatively low degree of smooth muscle differentiation.

[0055] In the third step, PINCH2 was knocked out in the bladder detrusor muscle cells. In the construction of the PINCH2 knockdown vector, two different interference sequences were used for PINCH2 in the present invention. The nucleotide sequences of the interference sequences are as follows: shPINCH2-1 (5’-TCACCCTGAAGAACAAGTTTG-3’); shPINCH2-2 (5’-CTGCGAACACGACTTCCAAAT-3); Among them, the interference vector was purchased from Addgene, and the catalog number was pLKO.1-TRC cloning vectorPlasmid#10878.

[0056] The shNC vector of the control group is pLKO.1-TRC control Plasmid #10879; two interference sequences were respectively ligated to the pLKO.1-TRC cloning vector through the standard procedure provided by the official website to obtain two interference vectors, shP2-1 and shP2-2; The steps for knocking out PINCH2 in bladder detrusor cells further include: Packaging of lentivirus: Step 1. Culture 293T cells in a 10 cm cell culture dish until 70%-80% confluent. For each group of virus packaging, take two 1.5 ml centrifuge tubes. Add 500 μl of Opti-MEM medium to one tube and 20 μl of lipo3000 reagent.

[0057] Step 2. Add 500 μl of Opti-MEM medium, 20 μl of p3000 reagent, 10 μg of the target plasmid (shP1 / shP2 / shCN, prepare one tube for each group), 7.5 μg of the shuttle plasmid psPAX2, and 2.5 μg of the shuttle plasmid pMD2.G to the other tube. Incubate at room temperature for 10 min.

[0058] Step 3. Then mix the reagents in tube 1 into tube 2, and after mixing, incubate at room temperature for 10 min.

[0059] Step 4. Replace the medium of 293T cells with 10 ml of fresh medium and slowly drop the reagent after incubation in Step 3. Shake well and return to the incubator.

[0060] Step 5. After 48 hours, collect the supernatant of the 293T cell medium. Centrifuge at 3000 rpm for 10 min and filter through a 0.45 μm filter to obtain the virus.

[0061] Step 6. Add 10 ml of fresh medium to 293T cells, and the virus can be collected for the second time 24 hours later according to the above steps.

[0062] Next, culture bladder detrusor cells. After digestion, collection, and counting, re-seed them into a 10-cm cell culture dish. Add 10 ml of fresh medium to each dish and seed 500,000 cells. After shaking well, place them in an incubator at 37 °C for culture. After 24 hours, remove 5 ml of the medium from each dish of cells. Then, add 5 ml of shP2-1 / shP2-2 / shCN virus to 3 of the dishes of cells respectively, and at the same time add 10 μl of 1000x Polybrane to help the virus enter the cells. Twenty-four hours after dropping the virus, remove the virus. Add 10 ml of fresh medium and return it to the incubator for continued culture. Twenty-four hours after changing the medium, collect the cells in groups and count them. Re-seed the cells into new 10-cm cell culture dishes, with 500,000 cells in each dish. Seventy-two hours later, collect the cell protein samples, detect the expression levels of each protein in the protein samples by Western blotting and analyze them. As shown in Figure 9 the figure, the collagen gel contraction degree of the WT group and the shNC group is relatively high, indicating that under normal circumstances, the contraction ability of bladder detrusor cells is relatively strong; the collagen gel contraction degree of the shP2-1 and shP2-2 groups is significantly lower than that of the WT group and the shNC group, indicating that after PINCH2 is knocked down, the contraction ability of bladder detrusor cells is significantly reduced.

[0063] It should be noted that the specific data of the time and dose in the above experiments can be adjusted adaptively according to actual applications. Conventional adjustments based on this principle are all within the scope of protection of this application.

[0064] In the present invention, a collagen gel contraction experiment is used to compare and verify the contraction function of bladder detrusor cells in the control group and the experimental group of mice. Digest and centrifuge the bladder smooth muscle cells cultured to 80%, resuspend them with complete culture medium for cell counting, with 5 × 10^5 cells in each well of a 24-well plate. Prepare a 600-μl system (400 μl of cell culture medium, 200 μl of collagen, 2 μl of NaOH). After mixing the cell suspension evenly, slowly add it to the 24-well plate, avoiding the generation of bubbles, 500 μl per well. Incubate at 37 °C for 30 minutes until the collagen solidifies. Then, slowly add 500 μl of complete culture medium along the well wall and continue to culture in an incubator for 24 hours. Observe and take pictures under a microscope, measure the diameter of the collagen, and repeat the experiment 3 times. The results are as shown in Figure 9-10 the figure, the collagen diameter of the WT group and the shNC group is smaller, while the collagen diameter of the shP2-1 and shP2-2 groups is significantly larger; this indicates that after PINCH2 is knocked down, the contraction degree of the collagen gel decreases, that is, the contraction ability of bladder detrusor cells is significantly weakened.

[0065] The above results show that after knocking out the PINCH2 protein, the contraction function of mouse bladder detrusor cells is significantly reduced.

[0066] All of the above experimental results indicate that after PINCH2 knockout, the structure of the detrusor muscle in mice is severely damaged, with obvious detrusor sclerosis and interstitial fibrosis of the bladder, the contraction function of the detrusor muscle is significantly reduced, and the mice show manifestations of dysuria and dynamic urinary retention.

Claims

1. Use of PINCH2 protein in the preparation of a drug for treating dynamic urinary retention, characterized in that, The drug targets the PINCH2 protein.

2. Use of the PINCH2 protein according to claim 1 in a medicament for dynamic urinary retention, characterized in that, The drug targets the PINCH2 protein in the detrusor muscle of the bladder.

3. Use of the PINCH2 protein according to claim 1 in a medicament for dynamic urinary retention, characterized in that, The drug can regulate the expression level of the PINCH2 protein.

4. Use of the PINCH2 protein according to claim 1 or 3 in a medicament for dynamic urinary retention, characterized in that, The drug can increase the expression level of the PINCH2 protein.

5. Use of the PINCH2 protein according to claim 1 in a medicament for dynamic urinary retention, characterized in that, The drug can regulate the expression level of the PINCH2 protein in the detrusor muscle of the bladder.

6. Use of the PINCH2 protein according to claim 1 or 2 in a medicament for dynamic urinary retention, characterized in that, The drug can increase the expression level of the PINCH2 protein in the detrusor muscle of the bladder.

7. A pharmaceutical composition for treating dynamic urinary retention, characterized in that, It includes a PINCH2 protein promoter and a targeting agent. The PINCH2 protein promoter is loaded on the targeting agent, and the targeting agent targets the PINCH2 protein.

8. The pharmaceutical composition for treating dynamic urinary retention according to claim 7, characterized in that, The targeting agent includes an AAV virus, miRNA, and a polymeric micelle.

9. Use of PINCH2 protein in the preparation of a detection reagent for dynamic urinary retention, characterized in that, The detection reagent uses the PINCH2 protein as a biomarker for detrusor areflexia.

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