Application of ANG in preparation of medicine for preventing or treating diabetes

Abstract

The invention discloses application of ANG in preparation of a medicine for preventing or treating diabetes mellitus. The application finds that ANG can inhibit the apoptosis of islet cells during pancreatitis, enhance the ability of islet cells to resist inflammation damage, and inhibit the sugar tolerance damage of an organism, so that the ANG is expected to improve the clinical prevention and treatment effects of diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and in particular relates to the use of ANG in preparing medicines for preventing or treating diabetes. Background Art

[0002] Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia, and the core mechanisms of the disease are β-cell dysfunction and insulin resistance. It is mainly divided into type 1 diabetes and type 2 diabetes. In addition, there is a special type of diabetes secondary to pancreatic exocrine disease, usually called pancreatic diabetes or type 3c diabetes. Pathological pancreatic imaging (MRI, CT) shows abnormalities, accounting for 5%-10% of the incidence of diabetes. The symptoms of pancreatic diabetes are the same as other common symptoms of diabetes. They are not specific, such as increased water intake, increased urine output, weight loss, poor mental state, poor sleep quality, etc. If they are not actively and effectively controlled, they will also seriously endanger human health. Therefore, patients with pancreatic diabetes also need to actively receive treatment, eat a reasonable diet, avoid eating foods high in sugar, and control their weight.

[0003] Pancreatitis is a common digestive system disease worldwide. It refers to the abnormal activation of multiple digestive enzymes synthesized by acinar cells under special circumstances, which in turn causes an inflammatory reaction to the pancreas itself. Excessive drinking, gallstones and smoking are high-risk factors for the disease. In my country, with the development of social economy, people's living and eating habits have quietly changed, and the incidence of pancreatitis has also been rising. It has become one of the diseases that seriously endangers people's health and quality of life.

[0004] Islets are important endocrine tissues in the pancreas and play an important role in the body's blood sugar regulation. It is now clear that pancreatitis can cause damage to islets in the pancreas, and the risk of diabetes in patients with pancreatitis is more than twice that of the general population. However, due to the limited understanding of postpancreatitis diabetes mellitus (PPDM), there is currently a lack of specialized and recognized prevention, diagnosis and treatment methods for PPDM in clinical practice. In terms of blood sugar control alone, drugs used to treat type 1 and type 2 diabetes are also valuable in PPDM patients. Currently, insulin and metformin have been widely used to control blood sugar in PPDM patients; other drugs, such as sulfonylureas, thiazolidinediones, GLP-1 / GIP receptor agonists, DPP4 inhibitors, etc., are not currently recommended for the treatment of PPDM due to potential side effects, and their specific safety and therapeutic effects need to be further evaluated in clinical trials. The current problem facing the treatment of PPDM is not only whether the drug is applicable, but also that different PPDM patients and the same patient at different stages of disease progression often require more personalized treatment plans than type 1 and type 2 diabetes. Summary of the invention

[0005] The present invention aims to overcome some of the deficiencies of the prior art and provide a use of ANG in preparing a drug for preventing or treating diabetes.

[0006] In order to achieve the above-mentioned purpose and other related purposes, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention protects the use of ANG as a drug for the prevention, treatment or diagnosis of diabetes.

[0008] The second aspect of the present invention protects the use of ANG or ANG promoter in the preparation of a medicament for preventing or treating diabetes.

[0009] The third aspect of the present invention protects the use of a reagent for specifically detecting ANG expression levels in the preparation of a diabetes detection product.

[0010] The fourth aspect of the present invention protects a method for screening drugs for treating diabetes, comprising the following steps: screening drugs with ANG gene as a target, and taking drugs that can increase the expression level of ANG gene as candidate drugs.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The present application found that ANG and its preparations can inhibit pancreatic islet cell apoptosis, inhibit the body's glucose tolerance impairment, and enhance the ability of pancreatic islet cells to resist inflammatory damage, which is expected to improve the clinical prevention and treatment of pancreatitis-related diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the immunofluorescence result of the mice in Example 1.

[0014] Figure 2 This is the result of the ELISA experiment of pancreatic islets in Example 1.

[0015] Figure 3 These are the results of the glucose tolerance test in mice after pancreatitis induction in Example 2.

[0016] Figure 4 The following are the immunofluorescence staining images of pancreatic islets in mice with pancreatitis and the statistical analysis results of the average pancreatic islet area in Example 2.

[0017] Figure 5 The results of apoptosis staining and analysis of pancreatic islet cells in mice with pancreatitis in Example 2.

[0018] Figure 6 This is the flow cytometry analysis result of pancreatic islet cell apoptosis induced by inflammatory factors in Example 3.

[0019] Figure 7 These are the results of immunofluorescence detection and analysis of pancreatic islet cell apoptosis induced by inflammatory factors in Example 3. DETAILED DESCRIPTION

[0020] Angiogenin (ANG; also known as ribonuclease 5, RNASE5) is a secreted alkaline ribonuclease belonging to the ribonuclease A family. Its gene number in the Ensemble database is ENSG00000214274283.

[0021] Preliminary studies of this application have found that ANG levels in the blood or pancreatic islets of pancreatitis patients or pancreatitis mice are significantly increased, and ANG is correlated with the progression of pancreatitis. This application knocks out the ANG gene in mice and finds that the absence of the ANG gene can promote the loss and apoptosis of pancreatic islet cells, reduce the area of ​​the pancreatic islets, and aggravate impaired glucose tolerance; further, by treating the pancreatic islet cells with ANG gene knockout with ANG recombinant protein, it was found that it can effectively inhibit the apoptosis of pancreatic islet cells. Therefore, ANG can provide new potential targets and therapeutic strategies for the prevention and treatment of diabetes and pancreatic islet damage.

[0022] The first aspect of the present invention protects the use of ANG as a target in screening drugs for the prevention, treatment or diagnosis of diabetes.

[0023] In certain embodiments, the use of ANG in screening drugs for preventing or treating diabetes specifically refers to: taking the ANG gene or protein as the target of action, screening drugs to find drugs that can promote the expression level of the ANG gene, the expression or activity of the protein as alternative drugs for preventing and treating diabetes.

[0024] In certain embodiments, the use of ANG as a target in screening diabetes diagnostic drugs specifically refers to: using the ANG gene expression product as a diabetes diagnostic indicator for diabetes diagnostic drugs.

[0025] In certain embodiments, the drug includes a molecule that can specifically promote the transcription or translation of the ANG gene, or can specifically promote the expression or activity of the ANG protein, so that the drug can increase the expression level of the ANG gene, thereby inhibiting pancreatic cell apoptosis, increasing the islet area, and lowering blood glucose concentration.

[0026] In certain embodiments, the drug includes, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, peptides, proteins or interfering lentiviruses. The form of the drug is not particularly limited and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0027] The second aspect of the present invention protects the use of ANG or ANG promoter in the preparation of a medicament for preventing or treating diabetes.

[0028] The present invention finds that the use of ANG protein to treat pancreatic islet cells can inhibit the apoptosis of pancreatic islet cells induced by inflammatory factors, so ANG or ANG promoters can be used in the development of corresponding diabetes therapeutic drugs.

[0029] In certain embodiments, the ANG or the ANG agonist is the sole active ingredient or one of the active ingredients of the medicament.

[0030] In certain specific embodiments, the ANG promoter refers to a substance that can promote the transcription or translation of the ANG gene, or can promote the expression or activity of the ANG protein.

[0031] In certain more specific embodiments, the ANG promoter comprises a biological material related to the ANG gene, wherein the biological material is selected from one or both of a recombinant expression vector or a bioengineered bacterium.

[0032] In certain specific embodiments, the ANG promoter is selected from one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing tools, lentiviruses or adeno-associated viruses.

[0033] In certain embodiments, the ANG protein is a full-sequence ANG protein, a truncated ANG protein, or a mutant ANG protein isolated and purified from bacteria, fungi, or animal cells.

[0034] In certain embodiments, the inhibition of islet cell apoptosis refers to bringing the level of islet cells of the subject close to or reaching the number or activity of normal islet cells. For example, based on normal islet cells, the drug can restore the islet cells of the subject to at least 80%, 90%, 100% or 110% of the normal islet cell level.

[0035] In certain embodiments, the increase in islet area refers to making the islet area of ​​the patient close to or reach that of a normal islet. For example, based on a healthy islet, the drug can restore the area of ​​the target islet to at least 80%, 90%, 100% or 110% of that of a healthy islet.

[0036] In certain embodiments, the lowering of blood sugar concentration refers to bringing the blood sugar concentration of the subject close to or reaching a normal level. For example, based on healthy people, the drug can restore the blood sugar concentration of the subject to 90%, 100%, 110% or 120% of the normal level.

[0037] In certain embodiments, the pharmaceutically acceptable excipient is selected from one or more of a carrier, a diluent, a binder, a lubricant and a wetting agent.

[0038] In certain specific embodiments, specific examples of some substances that can be used as pharmaceutically acceptable carriers, diluents, binders, lubricants and wetting agents are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration.

[0039] In some specific embodiments, the drug is one or more of a solution, an injection, a spray, a nasal drop, an aerosol, a powder spray, a tablet, a capsule, and a granule. The drugs in the above various dosage forms can be prepared according to conventional methods in the pharmaceutical field. Preferably, it is a solution.

[0040] In certain specific embodiments, the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or wrapped with other substances. Preferably, it is introduced into the body subcutaneously. The pharmaceutical composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0041] The third aspect of the present invention protects the use of a reagent for specifically detecting the expression level of ANG for preparing a diabetes detection product.

[0042] In certain embodiments, the product is selected from one or more of a kit, a chip, a probe, and a membrane strip. The product comprises a substance for detecting the expression level of ANG.

[0043] In certain specific embodiments, the kit may contain one or more of a specific probe, a primer, and a specific antibody.

[0044] The fourth aspect of the present invention protects a method for screening drugs for treating diabetes, comprising the following steps: screening drugs with ANG gene as a target, and selecting drugs that can promote the expression level of ANG gene as candidate drugs.

[0045] In certain embodiments, the method comprises: applying the candidate drug to a cell model or an animal model in vitro, and detecting the content of ANG in the cell or animal after co-culture. The cell may be from a mammal.

[0046] In certain embodiments, an in vitro cell model or animal model with ANG gene knockout is constructed, the in vitro cell model or animal model is treated with a candidate drug, and then the ANG content is detected. - / - ) as an animal model, or Ang knockout mice (Ang - / - ) and then caerulein induction was used as the animal model.

[0047] The experimenter can determine whether the candidate drug is a drug with therapeutic significance by detecting the content of ANG. Generally speaking, compared with the control group, a drug that can increase the content of ANG by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% can be determined as a drug with therapeutic significance.

[0048] Another aspect of the present invention also protects a method of treatment, comprising: administering a therapeutically effective amount of ANG or an ANG promoter to an individual. The method of treatment provided by the present invention can be used to treat indications including but not limited to diabetes. The therapeutically effective amount generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period. The individual generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., who can benefit from treatment using the preparation, kit or combination preparation.

[0049] The diabetes described above in this application is selected from pancreatic diabetes. Preferably, it is selected from diabetes secondary to pancreatitis. The pancreatic diabetes is a type of diabetes secondary to pancreatic exocrine diseases, including diabetes caused by pancreatitis, pancreatic trauma, pancreatic tumors, pancreatic cystic fibrosis, hemochromatosis, etc. The diabetes secondary to pancreatitis is a type of diabetes secondary to pancreatitis. Due to the local inflammatory reaction of the pancreas, the pancreatic beta cells scattered in the pancreatic tissue are severely damaged, resulting in a decrease in insulin secretion, and ultimately causing an increase in blood sugar in the body. The following specific embodiments illustrate the implementation of the present invention, and those familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0050] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0051] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0052] Example 1

[0053] In this Example 1, Ang knockout mice were obtained from Professor Guofu Hu of Tufts University in the United States (see Angiogenin Promotes Hematopoietic Regeneration by Dichotomously Regulating Quiescence of Stem and Progenitor Cells), and wild-type (WT) mice and Ang knockout mice (Ang knockout mice) were obtained by mating. - / - ), and the knockout of ANG was verified at the protein level using immunofluorescence experiments on pancreatic tissue sections and enzyme-linked immunosorbent assays on pancreatic islets. The specific steps are as follows:

[0054] 1.1 Immunofluorescence validation of ANG knockout in the pancreas

[0055] Wild-type (WT) mice and Ang knockout mice (Ang - / - ) pancreatic tissue and fixed it in 4% paraformaldehyde overnight. The fixed tissue was immersed in 70%, 80%, 95%, and 100% ethanol for dehydration, and each concentration was immersed for 5-10 minutes. After dehydration, the tissue was immersed in xylene for 10 minutes, and repeated three times. The tissue was immersed in paraffin for curing for 4-24 hours. The paraffin-embedded tissue sections were cut into 4-6μm thick tissue sections using a microtome, and the tissue sections were used for subsequent immunofluorescence experiments.

[0056] The steps of immunofluorescence staining are as follows:

[0057] (1) Dewaxing and hydrating sections: Incubate the paraffin sections in a 60°C oven for 30 min. Then, in a fume hood, immerse the sections in xylene (10 min × 2 times), 100% ethanol (5 min × 2 times), 90% ethanol (3 min), 80% ethanol (3 min), 70% ethanol (3 min), deionized water (3 min), and PBS (5 min × 3 times).

[0058] (2) Antigen retrieval: Heat freshly prepared sodium citrate solution (10 mM, pH 6.0) in a microwave oven until boiling. Immerse the sections hydrated in step (1) in the boiling sodium citrate solution. Reduce the power and continue heating and boiling for 10 to 20 minutes. After completion, cool at room temperature and wash three times with PBS for 5 minutes each time.

[0059] (3) Blocking: Add 50-100 μL of sheep serum to each section and incubate at room temperature for 1 h.

[0060] (4) Primary antibody incubation: dilute the antibody with PBS at a certain ratio (ANG, 1:100; INS, 1:500), add 50 μL to each section, and incubate in a humidified chamber at 4°C overnight.

[0061] (5) Rinse: Wash three times with PBS, 5 min each time.

[0062] (6) Secondary antibody incubation: Add AF488 / AF555 fluorescently labeled anti-mouse / rabbit IgG antibody (1:500 dilution) working solution to each section and incubate at room temperature in the dark for 1 h.

[0063] (7) Rinse: Wash three times with PBS, 5 min each time.

[0064] (8) Nuclear staining: Use DAPI to stain the cell nucleus.

[0065] (9) Sealing and observation: Add 20 μL of anti-fluorescence quenching agent to each slide and cover with a coverslip; use an Olympus laser confocal microscope to image and take photos. Fluorescence imaging results are shown in Figure 1 .

[0066] 1.2 Isolation of pancreatic islets

[0067] (1) Preparation: Prepare 1 mg / mL type V collagenase solution and 10% FBS solution with HBSS and pre-cool on ice for later use; prepare cotton balls, syringes, 26G injection needles, ophthalmic scissors, ophthalmic forceps, and hemostatic forceps.

[0068] (2) Mouse treatment: The experimental mice were anesthetized by injection of 2% sodium pentobarbital solution at a dose of 50 mg / kg. After anesthesia, the mice were fixed on a sterile operating table, and their abdomens were disinfected with 75% ethanol. The abdominal and thoracic cavities were then opened to expose the heart and common bile duct, and the right atrial appendage of the mice was cut to bleed.

[0069] (3) Collagenase injection: Clean the blood of the mouse with a cotton ball, ligate the end of the common bile duct near the gallbladder with a hemostatic forceps, and then use a disposable 5 mL syringe to draw up 2 mL of type V collagenase solution; replace the syringe needle with a 26G needle, and then insert the needle through the duodenal papilla of the common bile duct and slowly inject the collagenase solution into the pancreas until the pancreas is completely filled (about 1.5 mL of enzyme solution is required).

[0070] (4) Pancreatic digestion: Carefully dissect out the full pancreas, place it in a 15-mL sterile centrifuge tube, add 3 mL of type V collagenase solution, and place it in a 37°C water bath for digestion for about 20 min. Invert and mix several times during the digestion. After digestion, vortex and oscillate 3 times for 3 to 5 seconds each time. At this time, the pancreas will appear like quicksand. Add twice the volume of pre-cooled 10% FBS solution to terminate the digestion reaction.

[0071] (5) Filtration: Filter the digested tissue fluid with a 30-mesh metal filter to remove undigested tissue pieces. Centrifuge at 50×g for 5 min, discard the supernatant, and resuspend the precipitate with 5 mL of 10% FBS. Pipette repeatedly for about 20 times, and then filter the solution with a 100 μm pore size filter. The islets will be intercepted on the filter. Rinse with HBSS several times, and finally rinse the islets with 10% FBS solution into a 10 cm culture dish.

[0072] (6) Islet selection: Place the culture dish under a stereoscope and adjust the magnification and focus. Islets with smooth edges and roughly oval or mulberry shapes can be observed. Use a pipette to suck up the islets and transfer them to another culture dish. When there are more acinar cells in the solution, repeat this step until no other types of tissues / cells are mixed in the islets.

[0073] (7) Islet culture: The selected islets were transferred to RPMI1640 culture medium containing 10% FBS and 1% PS and cultured in suspension at 37°C overnight before the next ELISA test.

[0074] 1.3 ELISA to verify ANG knockout in islets

[0075] (1) Islet protein extraction: The islet proteins of wild-type (WT) mice and Ang knockout mice (Ang knockout mice) were isolated by the above steps. - / - ) islets, and then the islet cells were lysed with 25 μL lysis buffer to obtain islet proteins.

[0076] (2) ELISA: The islet protein sample was diluted with sample dilution buffer at a ratio of 1:50, and the mouse ANG ELISA kit of Abcam was used, and the operation was performed according to the instructions; the ANG content in the sample was homogenized based on the concentration of the islet protein sample.

[0077] Immunofluorescence results of pancreatic tissue sections are shown in Figure 1 , where blue fluorescence marks the cell nucleus, green fluorescence marks the pancreatic islet, and red fluorescence marks the ANG.

[0078] from Figure 1 Compared with wild-type (WT) mice, Ang knockout mice (Ang - / - ) pancreatic tissue no longer expresses ANG.

[0079] The results of ELISA for pancreatic islets are shown in Figure 2 .

[0080] from Figure 2 It is known that Ang knockout mice (Ang - / -) showed a significant decrease in ANG protein levels in islets. Both results indicate that ANG has been successfully knocked out.

[0081] Example 2

[0082] In this Example 2, wild-type (WT) mice and Ang knockout mice (Ang knockout mice) obtained in Example 1 were injected intraperitoneally with caerulein. - / - ) were used to induce pancreatitis and study the damage to pancreatic islets during pancreatitis.

[0083] These include:

[0084] 2.1 Pancreatitis model

[0085] Wild-type (WT) mice and Ang knockout mice (Ang - / - ) After starvation overnight, caerulein was injected intraperitoneally at a dose of 50 μg / kg, once every hour for 6 consecutive injections; the mice were induced with caerulein once every two days for 30 days to obtain the induced wild-type (WT) mice and Ang knockout mice (Ang - / - After induction, glucose tolerance test (GTT) was performed, and pancreatic tissues of mice were obtained by dissection for immunofluorescence staining of tissue sections and detection of apoptotic cells to evaluate the differences between wild-type (WT) mice and Ang knockout mice (Ang - / - ) of pancreatic islet damage.

[0086] 2.2 Results

[0087] 2.2.1 Changes in glucose tolerance

[0088] First, wild-type mice (WT) and Ang knockout mice (Ang - / - ) Starve overnight, and prepare a 0.3g / mL sterile glucose solution. Assemble the blood glucose test strips onto the blood glucose meter for use; after alcohol disinfection, lift the mouse tail, cut off the tissue at the end of the mouse tail, and gently squeeze the tail to make the blood flow out; use a blood glucose meter to collect blood from the tip of the tail, and measure the blood glucose value of the mouse at 0min. Weigh and record the weight of the experimental mice, and calculate the injection volume of the glucose solution for each mouse according to the dose of 1.5g glucose per kilogram of body weight; after disinfection, inject the mouse with glucose solution intraperitoneally, and start timing at the same time. Use Yuyue 580 blood glucose meter to detect and record the blood glucose (bloodglucose) of the mice at 15, 30, 60, 90, and 120min after injection.

[0089] All the measured blood sugar data were summarized, a line graph was drawn, the area under the curve was calculated, and a difference analysis was performed. Figure 3 Among them, * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0090] from Figure 3 Compared with wild-type mice (WT), Ang knockout mice (Ang - / - ) had significantly increased blood glucose concentrations, with statistical differences at multiple time points, and glucose tolerance was significantly impaired; the results of area under the curve analysis also showed significant differences. Overall, ANG can protect pancreatic islet function in pancreatitis.

[0091] 2.2.2 Statistical analysis of changes in islet area

[0092] The collected pancreatic tissue was fixed overnight in 4% paraformaldehyde solution, and the fixed tissue was sequentially immersed in 70%, 80%, 95%, and 100% ethanol for dehydration, and each concentration was immersed for 5-10 minutes. After dehydration, the tissue was immersed in xylene for 10 minutes, and repeated three times. The tissue was immersed in paraffin for curing for 4-24 hours. The paraffin-embedded tissue sections were cut into 3-5 micron thick tissue sections using a microtome, and the tissue sections were used for subsequent immunofluorescence experiments.

[0093] The steps of immunofluorescence staining are as follows:

[0094] (1) Dewaxing and hydrating sections: Incubate the paraffin sections in a 60°C oven for 30 min. Then, in a fume hood, immerse the sections in xylene (10 min × 2 times), 100% ethanol (5 min × 2 times), 90% ethanol (3 min), 80% ethanol (3 min), 70% ethanol (3 min), deionized water (3 min), and PBS (5 min × 3 times).

[0095] (2) Antigen retrieval: Heat freshly prepared sodium citrate solution (10 mM, pH 6.0) in a microwave oven until boiling. Immerse the hydrated sections in the boiling sodium citrate solution, reduce the power and continue to boil for 10 to 20 minutes. After completion, let it cool at room temperature and wash it three times with PBS, each time for 5 minutes.

[0096] (3) Blocking: Add 50-100 μL of sheep serum to each section and incubate at room temperature for 1 h.

[0097] (4) Primary antibody incubation: dilute the antibody with PBS at a certain ratio (AMY2B, 1:100; INS, 1:500), add 50 μL to each section, and incubate in a humidified box at 4°C overnight.

[0098] (5) Rinse: Wash three times with PBS, 5 min each time.

[0099] (6) Secondary antibody incubation: Add AF488 / AF555 / AF647 fluorescently labeled anti-mouse / rabbit IgG antibody (1:500 dilution) working solution to each section and incubate at room temperature in the dark for 1 h.

[0100] (7) Rinse: Wash three times with PBS, 5 min each time.

[0101] (8) Nuclear staining: Use DAPI working solution to stain the cell nucleus.

[0102] (9) Sealing and observation: Add 20 μL of anti-fluorescence quencher to each slide and cover with a coverslip; scan and image using an Olympus VS200 scanner in fluorescence mode.

[0103] (10) Measurement and analysis: The average area of ​​the islets in each tissue section (the average area of ​​the insulin-positive area) was measured and compared with that of WT mice after pancreatitis induction and Ang II mice. - / - The size of pancreatic islets of mice was analyzed.

[0104] In pancreatitis, inflammation and fibrosis can damage islet cells, leading to decreased function and number. In addition, abnormal activation and secretion of pancreatic enzymes in pancreatic acinar cells can directly damage pancreatic tissue, accelerate the disease progression, induce severe acute pancreatitis, and further affect islet cells. Activation of pancreatic stellate cells (PSCs) and increased apoptosis of pancreatic β cells may also lead to islet cell loss.

[0105] Results Figure 4 ,In the fluorescence images, green fluorescence is used to mark the pancreatic islets, red fluorescence is used to mark the acinar tissue, and blue fluorescence is used to mark the cell nucleus.

[0106] from Figure 4 It can be seen that after pancreatitis induction, Ang - / - The average pancreatic islet area of ​​mice was significantly reduced, indicating that ANG can inhibit the loss of pancreatic islet cells during pancreatitis.

[0107] 2.2.3 Detection and analysis of pancreatic islet cell apoptosis

[0108] The apoptosis of pancreatic islet cells in pancreatitis after ANG knockout was analyzed by using terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling technique and immunofluorescence to detect the apoptosis level of pancreatic islet cells. The specific steps are as follows:

[0109] (1) Dewaxing and hydrating sections: The operation is the same as the corresponding steps in the immunofluorescence experiment and will not be repeated here.

[0110] (2) Tissue permeabilization: After shaking off excess water from the slide and absorbing the water around the sliced ​​tissue with absorbent paper, add 50-100 μL of proteinase K solution (0.1 mg / mL) to each sample. Place the slices in a humidified box and digest at 37°C for 20 min, then soak the slices in PBS and rinse three times, 5 min each time.

[0111] (3) Terminal fluorescent labeling: Use absorbent paper to absorb excess water around the tissue, add 100 μL labeling buffer (25 mM Tris-Cl, 200 mM sodium cacodylate, 2.5 mM CoCl2, 200 μM DTT, 250 μg / mL BSA) to each sample, and react at 37°C for 10 to 30 min. Remove the reaction buffer, add 50 μL labeling working solution (35 μL labeling buffer + 10 μL fluorescein EF555 labeled dUTP solution + 5 μL TdT enzyme solution), and place in a humidified box at 37°C in the dark for 1 to 4 h.

[0112] (4) Fluorescent staining of cell nuclei: After absorbing the water with absorbent paper, add 100 μL of DAPI staining solution and stain in a humidified box away from light for 5 min, and then re-stain the cell nuclei.

[0113] (5) Rinse: In the dark, rinse the sections in PBS three times, each time for at least 5 min.

[0114] (6) Blocking: Add 100 μL of sheep serum to each section and block for 1 h at room temperature in the dark.

[0115] (7) Primary antibody incubation: Dilute insulin antibody at 1:500 with PBS, add 50 μL to each section, and incubate overnight at 4°C in a humidified chamber.

[0116] (8) Rinse: In the dark, rinse the sections in PBS three times, each time for at least 5 min.

[0117] (9) Fluorescent secondary antibody incubation: dilute AF488-labeled secondary antibody at 1:500, add 100 μL to each section, and incubate in a humidified chamber at room temperature for 2 h.

[0118] (10) Rinse and mount: In the dark, rinse the sections in PBS for three times, each time for at least 5 min; add 20 μL of anti-fluorescence quenching mounting medium to each slide for mounting.

[0119] (11) Observation: Using an Olympus OSR laser confocal microscope, the sections were immersed in PBS and rinsed three times, each time for at least 5 min. 20 μL of anti-fluorescence quenching mounting medium was added to each slide for sealing.

[0120] The results of islet cell apoptosis are shown in Figure 5In the fluorescence image, green fluorescence was used to mark insulin-positive islet cells, red fluorescence was used to mark apoptotic islet cells, and blue fluorescence was used to mark cell nuclei; the ratio of the number of apoptotic cells to the green fluorescence area was used as the statistical result of apoptotic cell density for analysis.

[0121] from Figure 5 It can be seen that more cells in the pancreatic islets with ANG knockout underwent apoptosis, and the statistical analysis of the number of apoptotic cells per unit islet area showed that the number of apoptotic cells increased significantly after ANG loss. The above results indicate that ANG can inhibit the apoptosis of pancreatic islet cells during pancreatitis and play a protective role in the function of the pancreatic islets.

[0122] Example 3

[0123] In this Example 3, purified ANG recombinant protein was used to treat isolated Ang gene knockout islets, and after treatment with inflammatory factors, flow cytometry was used to verify whether ANG recombinant protein treatment could reduce the level of islet cell apoptosis caused by inflammation. The following are included:

[0124] 3.1 Evaluation Methodology

[0125] (1) The method of Example 1 was used to isolate Ang knockout mice (Ang - / - ) of the pancreatic islets.

[0126] (2) The pancreatic islets / islet cells were pretreated with ANG recombinant protein, and a mixture of inflammatory factors was added to induce islet cell apoptosis. Finally, the apoptosis level of islet cells was analyzed by flow cytometry and immunofluorescence experiments.

[0127] 3.2 Results

[0128] 3.2.1 Flow cytometry evaluation of the effect of ANG on islet cell apoptosis

[0129] (1) Islet processing: - / - ANG recombinant protein (final concentration of 1 μg / mL) was added to the culture medium of islets, and an equal volume of PBS buffer was added to the culture medium of islets in the control group. The cells were incubated in a cell culture incubator for 12 h, and then an inflammatory factor mixture (final concentrations: 10 ng / mL TNF-α, 10 ng / mL IL-1β, and 100 ng / mL IFN-γ) was added to the culture medium for 48 h.

[0130] (2) Single cell preparation: Digest the islets treated in step (1) with 0.25% trypsin solution at 37°C for 3-5 min. Use a pipette to gently disperse the islets into single cells. Add 2 times the volume of trypsin solution in RPMI 1640 complete medium (Gibco #31800) containing 10% FBS to terminate the digestion reaction. Centrifuge at 500 × g for 3 min, discard the supernatant, resuspend in PBS and centrifuge again.

[0131] (3) Staining: Resuspend the islet cells (about 1×10 40 cells / mL) obtained in step (2) in 100 μL of binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, 5% BSA, pH 7.4). 5 cells), add 2.5 μL Annexin-V-FITC and 5 μL PI solution to each sample and mix gently, and stain at room temperature in the dark; after 10-15 minutes, add 200 μL binding buffer, mix well and place on ice.

[0132] (4) Flow cytometric analysis: The stained samples in step (3) were transferred to flow tubes and analyzed using a DxFLEX flow cytometer. FSC-A and SSC-A scatter plots were drawn and gated for intact cells (P1). FSC-H and SSC-A scatter plots were drawn and gated for non-adherent cells (P2). The maximum excitation wavelength of FITC is 488 nm, and the maximum emission wavelength is 525 nm. The green fluorescence of FITC is detected in the FL1 channel; the maximum excitation wavelength of the PI-DNA complex is 535 nm, and the maximum emission wavelength is 615 nm. The red fluorescence of PI is detected in the FL2 or FL3 channel. A scatter plot was drawn with PI as the horizontal axis and FITC as the vertical axis. Fluorescence channel compensation was adjusted using negative control samples, PI single-labeled samples, and FITC single-labeled samples. Finally, the apoptosis level of the experimental samples was detected.

[0133] Results Figure 6 In the flow chart, Annexin-V + Single positive and Annexin-V + PI + The double-positive cell population is apoptotic pancreatic islet cells.

[0134] from Figure 6 It can be seen that ANG incubation reduced the Annexin-V positive and Annexin-V + PI + The proportion of double-positive islet cells indicated that ANG treatment could inhibit islet cell apoptosis induced by inflammatory factors.

[0135] 3.2.2 Immunofluorescence evaluation of the effect of ANG on islet cell apoptosis

[0136] (1) Preparation of pancreatic islet cells: Separate the Ang cells with 0.25% pancreatic enzyme solution. - / - After the pancreatic islets were digested into single cells, they were seeded into a glass-bottom 8-well chamber cell culture dish (about 70,000 islet cells per well) and cultured statically for 24 hours to allow the islet cells to adhere to the wall.

[0137] (2) Islet cell treatment: Ang - / - ANG recombinant protein (final concentration 1 μg / mL) was added to the culture medium of pancreatic islet cells, and an equal volume of PBS was added to Ang - / - Islet cells were used as a control and incubated for 12 h, and then treated with a mixture of inflammatory factors (final concentrations: 10 ng / mL TNF-α, 10 ng / mL IL-1β, and 100 ng / mL IFN-γ) for 24 h.

[0138] (3) Fixation and blocking: Fix with 300 μL 4% paraformaldehyde for 15 min, wash three times with PBS, and then add sheep serum to block for 1 h at room temperature.

[0139] (4) Primary antibody incubation: dilute the antibody with PBS at a certain ratio (INS, 1:500; CC3, 1:100), add 50 μL to each section, and incubate in a humidified chamber at 4°C overnight.

[0140] (5) Rinse: Wash three times with PBS, 5 min each time.

[0141] (6) Secondary antibody incubation: Add AF488 / AF555 fluorescently labeled anti-mouse / rabbit IgG antibody (1:500 dilution) working solution to each section and incubate at room temperature in the dark for 1 h.

[0142] (7) Rinse: Wash three times with PBS, 5 min each time.

[0143] (8) Nuclear staining: Use DAPI solution to stain the cell nucleus.

[0144] (9) Sealing and observation: Add 20 μL of anti-fluorescence quencher onto each slide and cover with a coverslip; use an Olympus OSR laser confocal microscope to image and record.

[0145] Results Figure 7 , blue fluorescence marks the cell nucleus, green fluorescence marks insulin-positive islet cells, and red fluorescence marks CC3-positive apoptotic islet cells. The ratio of apoptotic cells to all cells in the field of view is taken as the apoptotic cell ratio.

[0146] from Figure 7 It can be seen that after incubation with ANG recombinant protein, Ang - / -The apoptosis rate of islet cells was lower; the statistical results showed that compared with the control group, the apoptosis level of islet cells in the ANG-treated group was significantly reduced, which further proved the conclusion in step 3.2.1, indicating that ANG can inhibit the apoptosis of islet cells.

[0147] The above description only represents the specific implementation mode of the present application, which helps those skilled in the art to understand or implement the contents of the present application. Any modifications, changes and improvements made within the spirit and principles of the claims of the present invention are within the protection scope of the claims of the present invention.

Claims

1. Use of ANG as a target in screening drugs for the prevention, treatment or diagnosis of diabetes.

2. Use of ANG or ANG promoter in the preparation of drugs for preventing or treating diabetes.

3. The use according to claim 1 or 2, characterized in that The drug has at least one of the following effects: (1) Inhibit pancreatic islet cell apoptosis; (2) Increase pancreatic islet area; (3) Reduce blood sugar concentration.

4. The use according to claim 2, characterized in that: The ANG promoter refers to a substance that can promote the transcription or translation of the ANG gene, or can promote the expression or activity of the ANG protein; And / or, the ANG or the ANG enhancer is the only active ingredient or one of the active ingredients of the drug.

5. The use according to claim 4, characterized in that The promoter comprises biological materials related to the ANG, and the biological materials are selected from one or both of a recombinant expression vector and a bioengineering bacterium.

6. Use of a reagent for specifically detecting ANG expression levels in the preparation of a diabetes detection product.

7. The use according to claim 6, characterized in that The product is selected from one or more of a kit, a chip, a probe and a membrane strip.

8. A method for screening drugs for treating diabetes, characterized in that: The method comprises the following steps: screening drugs with the ANG gene as a target, and taking drugs that can increase the expression level of the ANG gene as candidate drugs.

9. The use according to any one of claims 1 to 7 or the method according to claim 8, characterized in that The diabetes is selected from pancreatic diabetes.

10. The use or method according to claim 9, characterized in that The diabetes mellitus is selected from diabetes mellitus secondary to pancreatitis.

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