Application of hepatocyte nuclear factor 1 alpha in promoting beta cell regeneration and preventing and treating diabetes

Abstract

The invention relates to the field of biological medicines, in particular to application of a hepatocyte nuclear factor 1 alpha (HNF1 alpha) in promoting beta cell regeneration and preventing and treating diabetes. Specifically, by regulating and controlling the expression of an HNF1alpha coding gene and the activity of HNF1alpha, pancreas islet alpha cells can be promoted to be transdifferentiated into beta cells to achieve beta cell regeneration, the functions of pancreas islet beta cells are reserved, the occurrence of diabetes mellitus is delayed, and a new medical approach is provided for prevention and treatment of diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of hepatocyte nuclear factor 1α in promoting β-cell regeneration and preventing and treating diabetes. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. Diabetic complications severely impact patients' health and quality of life, and can even be life-threatening. Pancreatic β-cell dysfunction is a central link in the development and progression of diabetes, and the continuous loss of total functional β-cells is the core mechanism leading to insulin secretion defects. Therefore, exploring effective β-cell regeneration strategies to restore the total number of functional pancreatic β-cells is the most effective means of treating diabetes.

[0003] To date, pancreas transplantation or islet transplantation is the most direct method for restoring the total number of β cells. However, pancreas transplantation surgery is highly invasive and has many postoperative complications, making it difficult to use as a routine treatment. Allogeneic islet transplantation has shown good efficacy in treating diabetes, but its availability is limited by a shortage of donors. Promoting islet proliferation can increase the number of β cells in situ, but the potential risks of pancreatic exocrine (especially ductal) cell proliferation and tumorigenesis remain uncontrollable. The directed differentiation of stem cells into mature β cells still faces several unresolved technical challenges and is some distance from clinical application.

[0004] In recent years, a significant shift in academic perspectives has emerged, with the view that β cells in the later stages of diabetes do not undergo complete apoptosis but rather "dedifferentiation." Therefore, inducing cell reprogramming to "redifferentiate" or transform other cells into β cells has become a highly promising new therapeutic direction.

[0005] From a developmental biology perspective, alpha cells are an ideal source for the regeneration and functional remodeling of pancreatic β cells. Alpha cells appear first during pancreatic islet development, second only to β cells in number. They are anatomically adjacent, share similar genetic backgrounds and transcription factor expression, and have similar mechanisms for sensing blood glucose and hormone secretion, exhibiting close mutual regulation. Furthermore, animal and human studies have shown that under various β cell damage conditions, the number of alpha cells remains unchanged or even increases compensatorily. Simultaneously, since the conversion of alpha cells to β cells can increase the number of β cells and decrease the number of alpha cells, restoring the balance between insulin and glucagon, and thus remodeling pancreatic islet function and maintaining blood glucose homeostasis, reprogramming pancreatic alpha cells into β cells is a very promising new strategy for diabetes treatment and has rapidly become a research hotspot in the field of islet regeneration.

[0006] However, translating this scientific discovery into practical therapies hinges on identifying core molecules capable of efficiently and specifically initiating the transdifferentiation of α cells into β cells. While several transcription factors have been shown to participate in this process, an ideal target possessing both potent driving force and clear clinical relevance remains lacking. Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor playing a crucial role in pancreatic development and function; mutations in its gene are a major cause of monogenic diabetes (MODY3), suggesting its indispensable role in maintaining normal β cell function. Current research has not fully elucidated the decisive role of HNF1α in regulating α cell fate, particularly its significant potential as an overexpression agent in inducing α cell transdifferentiation and preventing diabetes. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the object of the present invention is to provide the use of hepatocyte nuclear factor 1α (HNF1α) in promoting β-cell regeneration and preventing and treating diabetes, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention first provides the use of HNF1α or an promoter thereof in the preparation of a medicament for treating diabetes.

[0009] The present invention also provides a method for treating diabetes, wherein the method comprises administering HNF1α or a promoter thereof to a subject.

[0010] The present invention also provides a composition for treating diabetes, wherein the active ingredient contains HNF1α or an promoter thereof and a pharmaceutically acceptable carrier or excipient.

[0011] As described above, the use of hepatocyte nuclear factor 1α in promoting β-cell regeneration and preventing diabetes of the present invention has the following beneficial effects:

[0012] 1) Promote β-cell regeneration: Achieve in situ β-cell regeneration by inducing pancreatic α-cells to transdifferentiate into β-cells;

[0013] 2) Promotes the transdifferentiation of pancreatic α cells into β cells: Overexpression of HNF1α in α cells can significantly downregulate α cell markers (such as Glucagon, Arx, Dpp4) and upregulate β cell markers (such as Insulin, Nkx6.1, Glut2), resulting in "triple-positive cells" that co-express insulin, glucagon, and HNF1α, confirming that cell identity has been reprogrammed;

[0014] 3) Delaying the onset of diabetes: In the STZ-induced diabetic mouse model, transgenic mice that specifically overexpress HNF1α in α cells showed slower blood glucose elevation, reduced incidence of diabetes, improved survival rate, and better weight maintenance. Attached Figure Description

[0015] Figure 1 The diagram illustrates the verification of β-cell regeneration and HNF1α expression upregulation in α-cells under extreme β-cell destruction conditions in Example 1 of this invention. Figure a shows the immunofluorescence detection at different time points after STZ treatment; Figure b shows the change in the number of single pancreatic β-cells (* p < 0.01); Figure c shows the change in the expression of insulin genes (Insulin1 and Insulin2) (* p < 0.01); Figure d shows the expression of HNF1α protein in α-cells.

[0016] Figure 2 The PCR reaction procedure shown is from Example 1 of the present invention.

[0017] Figure 3 The diagram shows the transdifferentiation of α cells overexpressing HNF1α into β cells in Example 2 of the present invention. Figure a shows the α cell-specific molecular changes in α cells; Figure b shows the β cell-specific molecular changes in α cells; Figure c is an immunofluorescence detection diagram.

[0018] Figure 4 The figures show the changes in glucagon and insulin expression in the α cell line induced by HNF1α overexpression in Example 3 of the present invention. Figure a shows the expression of glucagon and insulin in α cells after HNF1α overexpression, as detected by Western blotting. Figure b shows the immunofluorescence of glucagon and insulin in α cells after HNF1α overexpression. Figure c shows the protein expression of glucagon and insulin in α cells after HNF1α overexpression, as detected by ELISA.

[0019] Figure 5 This is shown in Example 4 of the present invention, where α cells overexpressing HNF1α secrete insulin in response to glucose stimulation.

[0020] Figure 6 The figures shown are graphs illustrating the remission of diabetes in different groups of mice in Example 5 of the present invention. Figure A shows the survival analysis; Figure B shows the incidence rate; Figure C shows the weight change; and Figure D shows the random blood glucose level change. Detailed Implementation

[0021] The present invention first provides the use of hepatocyte nuclear factor 1α (HNF1α) or an promoter thereof in the preparation of a medicament for treating diabetes.

[0022] In some embodiments of the present invention, the diabetes is type 1 diabetes or type 2 diabetes.

[0023] In this invention, the term "treatment" refers to a series of positive effects exerted after a disease has already begun to develop. Specifically, it can slow the progression of the disease, controlling its rapid progression; it can interrupt the continuous deterioration of the disease, preventing it from evolving further into a more severe state; it can effectively control the severity of the disease, preventing it from exceeding the body's tolerance; it can stop the adverse development of the disease, preventing it from continuing to worsen; it can alleviate various uncomfortable symptoms caused by the disease, relieving the patient's suffering; and it can even, to some extent, reverse a specific sign, symptom, disorder, condition, or the direction or severity of the disease's progression. However, it should be clarified that such treatment does not necessarily mean the complete elimination of all disease-related signs, symptoms, conditions, or disorders, but rather improving the state of the disease and alleviating disease-related signs, symptoms, conditions, or disorders.

[0024] In some embodiments of the present invention, the drug has any one or more of the following effects:

[0025] 1) Promotes β-cell regeneration;

[0026] 2) Promotes the transdifferentiation of pancreatic α cells into β cells;

[0027] 3) Delay the onset of diabetes.

[0028] In some embodiments of the present invention, the promotion of β-cell regeneration and the ability of α-cells overexpressing HNF1α to secrete insulin upon glucose stimulation indicate that they have acquired the core β-cell function of glucose-responsive insulin secretion. In animal models of STZ-induced diabetes, transgenic mice with α-cell-specific overexpression of HNF1α (TgGCG-HNF1α) exhibited higher survival rates, lower diabetes incidence, and better weight maintenance, demonstrating an effective replenishment of functional β-cells in vivo.

[0029] In some embodiments of the present invention, the process of promoting the transdifferentiation of pancreatic α cells into β cells involves overexpression of HNF1α in the α cell line (alpha TC1 clone 6). This results in a significant decrease in the mRNA and protein expression levels of α cell-specific molecular markers (including glucagon, transcription factor Arx, and dipeptidyl peptidase Dpp4), while the expression of β cell-specific molecular markers (including insulin, transcription factor Nkx6.1, and glucose transporter Glut2) significantly increases. The presence of "triple-positive cells" co-labeled with insulin, glucagon, and HNF1α indicates that the α cells have undergone identity reprogramming, transitioning to a β cell phenotype.

[0030] In some embodiments of the present invention, the delay in the onset of diabetes is demonstrated by the fact that, at the animal level, diabetic mice overexpressing HNF1α in α cells have significantly lower random blood glucose levels than wild-type mice and maintain a lower level throughout the observation period, proving that HNF1α can effectively improve hyperglycemia in a diabetic model.

[0031] Specifically, the blood glucose reduction refers to the situation where, after administering the drug to the subject, the subject's blood glucose level is close to or reaches the blood glucose level of a healthy individual compared to before administration. For example, based on the blood glucose level of a healthy individual, the drug can restore the subject's blood glucose level to at least 70%, 80%, 90%, or 100% of that of a healthy individual.

[0032] In some embodiments of the present invention, the HNF1α promoter is selected from HNF1α functional activators or HNF1α overexpression reagents.

[0033] In some embodiments of the present invention, the HNF1α functional activator is selected from small molecule compounds, peptides, or antibodies.

[0034] In some embodiments of the present invention, the HNF1α overexpression reagent is a nucleic acid construct containing the HNF1α coding gene or a virus whose genome integrates the HNF1α coding gene.

[0035] In some embodiments of the present invention, the nucleic acid construct containing the HNF1α coding gene refers to a nucleic acid molecule containing the coding sequence of HNF1α and capable of driving the transcription and translation of the gene within a host cell. This construct can be obtained using conventional molecular cloning techniques in the art, for example, by cloning the HNF1α coding gene fragment into various vectors known in the art.

[0036] The nucleic acid construct is in genomic form of a viral vector. In this form, the nucleic acid construct contains cis-acting elements necessary for viral replication and packaging, and can be packaged into infectious recombinant viral particles in packaging cells that provide trans-acting factors. Viruses obtained in this manner can efficiently deliver the HNF1α-encoding gene into target cells. In this invention, the type of viral vector is not limited, such as one or more of adenovirus vectors, lentiviral vectors, retroviral vectors, and adeno-associated virus vectors.

[0037] In some embodiments of the present invention, the virus whose genome integrates the HNF1α coding gene is selected from one or more of lentiviruses, adenoviruses, adeno-associated viruses, or retroviruses.

[0038] The drug for treating diabetes is a molecule that can specifically promote the transcription or translation of the HNF1α-encoding gene, or can specifically promote the expression of the HNF1α-encoding gene or the activity of HNF1α, thereby increasing the level of HNF1α in the patient's body.

[0039] The diabetes treatment drugs prepared by HNF1α or its promoters include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, peptides, proteins, adenoviruses, adeno-associated viruses, or lentiviruses.

[0040] The dosage of the diabetes treatment drug is sufficient to upregulate the transcription or translation of the HNF1α-encoding gene, or sufficient to upregulate HNF1α expression or activity, such that the expression of the HNF1α-encoding gene is upregulated by at least 50%, 80%, 90%, 95%, or 99%.

[0041] HNF1α promoters refer to substances that have an upregulating effect on HNF1α. Those with an upregulating effect on HNF1α include, but are not limited to:

[0042] 1) Promotes the expression of the HNF1α-encoding gene or the activity of HNF1α;

[0043] 2) Overexpression was used to increase the expression level of the HNF1α encoding gene;

[0044] 3) Directly increase the level of the HNF1α encoding gene within α cells.

[0045] Promoting HNF1α activity means increasing the activity of HNF1α. Preferably, compared with before upregulation, the activity of HNF1α protein is increased by at least 10%, more preferably by at least 30%, even better by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0046] Promoting the expression of the HNF1α coding gene can specifically mean promoting the transcription or translation of the HNF1α coding gene. Specifically, it can mean increasing the transcription or expression of the HNF1α coding gene by at least 10%, preferably by at least 30%, even better by at least 50%, better by 70%, and best by at least 90%.

[0047] The aforementioned medication for treating diabetes must include HNF1α or its promoters, and use HNF1α or its promoters as the active ingredient for the aforementioned effects.

[0048] In the aforementioned medication for treating diabetes, the active ingredient performing the aforementioned function may be only HNF1α or its promoter, or it may contain other molecules that can perform similar functions. HNF1α or its promoter may be the sole active ingredient or one of the active ingredients in the aforementioned medication for treating diabetes.

[0049] The diabetes treatment drug can be a single-component substance or a multi-component substance. There are no special restrictions on the form of the diabetes treatment drug; it can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0050] The drug is primarily targeted at mammals. Preferred mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Preferred primates include monkeys, apes, or humans.

[0051] This invention also provides a method for treating diabetes, wherein the method involves administering HNF1α or a promoter thereof to a subject. The subject may be a mammal. The mammal is preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, primate, etc. The primate is preferably a monkey, ape, or human. The subject may be a patient suffering from diabetes or an individual seeking to prevent or alleviate diabetes. Alternatively, the subject may be an ex vivo tissue or cell of a diabetic patient or an individual seeking to treat diabetes.

[0052] HNF1α or its promoters can be administered to subjects before, during, or after diabetes treatment.

[0053] The application of the aforementioned medication for treating diabetes provides a method for treating diabetes, specifically a method for treating diabetes in a subject, comprising administering an effective dose of the medication to the subject. The subject of this method can be a human being.

[0054] The present invention also provides a composition for treating diabetes, wherein the active ingredient contains HNF1α or an promoter thereof and a pharmaceutically acceptable carrier or excipient.

[0055] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0056] A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, meaning it can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include 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 gum 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, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0057] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0059] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0060] Example 1: β cell regeneration and upregulation of HNF1α expression in α cells under extreme β cell destruction conditions

[0061] 1.1 Establishment of a mouse model of extreme β-cell destruction

[0062] Eight C57 mice aged 8-10 weeks and weighing 25-30 g were selected. All mice were weighed after fasting for 12 hours, and the dosage of streptozotocin (STZ) (160 mg / kg) was calculated based on body weight. Each mouse served as a self-control before and after administration.

[0063] STZ was prepared fresh in citrate buffer (pH 4.5) under light-protected conditions and administered intraperitoneally within 15 minutes of dissolution. Mouse islet tissue was collected at 8, 16, 24, and 48 hours post-STZ injection for later use. Islet tissue for immunofluorescence assay was fixed in paraformaldehyde, while islet tissue for flow cytometry analysis was directly placed in PBS buffer and digested with collagenase.

[0064] 1.2 Immunofluorescence detection of pancreatic β-cell damage and regeneration in mice

[0065] Mouse pancreatic tissue was collected, embedded in paraffin, sectioned, dewaxed, dehydrated with xylene-methanol, and repaired with citric acid before immunofluorescence staining. The specific steps were as follows: incubation with primary antibody insulin at 4°C overnight, followed by washing three times with PBS; then incubation with anti-mouse fluorescent secondary antibody at 37°C for 1 hour, followed by washing three times with PBS again; finally, staining with DAPI for 8-10 minutes, mounting, and imaging under a fluorescence microscope.

[0066] The results show that ( Figure 1 (a, 1b) After 16 hours of STZ treatment, β cells in the mouse pancreas almost completely disappeared; by 48 hours, significant in situ regeneration of β cells could be observed.

[0067] 1.3 Isolation and purification of mouse pancreatic islet cells

[0068] Mice were anesthetized with 3% sodium pentobarbital, their limbs were fixed in a supine position, and the whole body was disinfected with alcohol. The chest cavity was disinfected with iodine and alcohol. A few incisions were made in the abdomen, and the abdominal cavity was accessed layer by layer. The intestines were retracted to expose the pancreas and common bile duct. The mice were euthanized by exsanguination. 8-10 ml of pre-cooled 0.5 mg / ml collagenase IV solution was injected retrogradely through a cannula inserted into the common bile duct to inflate the pancreas. The entire pancreas was quickly removed and transferred to a digestion bottle pre-filled with 6 ml of Hank's solution. The digested pancreatic tissue was allowed to digest at 38°C for 10 minutes. The digested pancreatic tissue was removed and placed in an ice bath. It was then shredded with ophthalmic forceps and washed with 10 ml of 4°C serum and 4°C Hank's solution. The cells were filtered through a 600 μm stainless steel mesh. The cell suspension was centrifuged in 50 ml centrifuge tubes at 1000 rpm and 4°C for 2 minutes. The supernatant was discarded and the precipitate was collected. The precipitate was washed with Hank's solution at 4°C, and centrifuged and washed in the same way. After resuspending in cold Hank's solution, the precipitate was divided into two 15 ml centrifuge tubes and centrifuged at 1000 rpm and 4°C for 2 minutes. The supernatant was discarded and the precipitate was collected.

[0069] Add 4 ml of 25% Ficoll solution to the precipitate and mix well. Then, add 2 ml each of 23%, 20%, and 11% Ficoll solution and Hank's solution sequentially. Centrifuge at 3000 rpm and 4°C for 20 minutes. Aspirate the islets at the 23%–20% and 20%–11% interfaces, wash twice with Hank's solution at 4°C in a 50 ml centrifuge tube, and finally resuspend the islet cells in DMEM medium for culture.

[0070] 1.4 Real-time PCR detection of insulin mRNA expression

[0071] The collected pancreatic islet tissue was placed in Trizol and allowed to stand for 5-10 minutes. Chloroform was then added, and the sample was centrifuged at 12,000 rpm for 5 minutes, separating the sample into an aqueous layer, an intermediate layer, and an organic layer. RNA was present in the aqueous layer. After collecting the aqueous layer, two volumes of isopropanol were added to precipitate the RNA. After standing for 10 minutes, the sample was centrifuged at 12,000 rpm for 15 minutes. The solvent was discarded, and the sample was washed once with 75% ethanol and centrifuged at 12,000 rpm for 8 minutes. The solvent was then discarded. After air-drying, 50 μl of RNAase-free water was added to dissolve the RNA. The RNA concentration was then measured using a NanoDrop spectrophotometer and stored at -80°C for later use.

[0072] 2 μg of total RNA was isolated and reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega) in a 25 μl reaction volume. The real-time PCR reaction system included 0.5 μl of cDNA and a 0.2 μM primer set. The reaction procedure was as follows: Figure 2 As shown.

[0073] PCR products were detected using Evagreen fluorescent dye (Molecular Probes). Experiments were performed on an RG-3000 real-time thermal cycler (Corbett Research, Sydney, Australia).

[0074] Using β-actin as an internal reference gene, the relative expression levels of insulin genes (Insulin1 and Insulin2) mRNA were calculated using the 2-ΔΔCT method.

[0075] The results show that ( Figure 1 c) After 48 hours of STZ treatment, the expression of insulin genes (Insulin1 and Insulin2) increased significantly, consistent with the trend of β-cell regeneration observed by immunofluorescence.

[0076] 1.5 Western blot analysis of HNF1α expression

[0077] Mouse islet cells were isolated and purified at 0, 16, 24, and 48 hours after STZ injection. Cells positive for α-cell-specific surface markers were sorted by flow cytometry. Sorted α-cells were collected and lysed with 150 μl of RAPI lysis buffer (Beyotime) containing a protease inhibitor. The mixture was pipetted, homogenized, and incubated on ice for 30 minutes. The supernatant was collected after centrifugation at 14,000 rpm for 20 minutes at 4°C. Protein concentration was estimated using Bradford reagent (Sangon, Shanghai, China).

[0078] Equal amounts of protein (120 μg) were subjected to 10% SDS-PAGE electrophoresis and then transferred to a transmold. The mixture was incubated overnight with a specific anti-HNF1α rabbit polyclonal antibody (Cell signal, USA). The results were detected using an ECL Plus Western blot detection system (Amersham).

[0079] The results show that ( Figure 1 d) During the in situ regeneration of β cells, the expression of HNF1α protein in α cells increased significantly, suggesting that HNF1α may be related to the regeneration process of β cells.

[0080] Example 2: Overexpression of HNF1α induces α cells to transdifferentiate into β cell phenotype.

[0081] 2.1 Preparation and amplification of HNF1α-overexpressing adenovirus

[0082] a) Virus construction: The HNF1α overexpressing adenovirus was constructed by Hanheng Biotechnology (Shanghai) Co., Ltd. The overexpressing adenovirus vector, pCDH-CMV-MCS-EF1-copGFP, was purchased from System Biosciences. The cloned gene sequence was the full-length sequence of the HNF1α gene, with Gene ID 6927.

[0083] b) Virus amplification and titration: in a 100 mm culture dish or 75 cm² culture medium. 2 Inoculate 5×10 in culture flasks 6 293T cells were cultured in 10 ml of DMEM medium. 0.5 μl of virus preservation solution was added to DMEM medium and diluted to 1 ml, then mixed thoroughly (MOI value approximately 5). The original cell culture medium was removed, and the virus dilution was carefully added, taking care not to disrupt the cell monolayer. The cells were gently shaken in a cross-shaped motion three times and incubated at 37°C in a CO2 incubator for 90 minutes. Subsequently, 9 ml of DMEM medium was added, and the cells were cultured for another 72 hours. At this point, approximately 5 × 10⁶ cells / mL of the solution were present in 10 ml of DMEM medium. 9 ~5×10 10 For each virus particle, an MOI (Mean Interval) determination is performed to estimate the viral particle size. If the viral load is sufficient for the experiment, the virus is released by three freeze-thaw cycles at -20°C and 37°C. Then, the virus is transferred to a 15 ml sterile centrifuge tube and centrifuged for 10 minutes. The supernatant is collected and stored at -20°C or -80°C for later use.

[0084] 2.2 Infection of α cells with HNF1α-overexpressing adenovirus

[0085] Mouse pancreatic α cell line (alphaTC1 clone 6) was digested and collected, and seeded at an appropriate density in 6-wells. After cell adhesion, the medium was replaced with adenovirus containing HNF1α overexpression for infection, and cultured for 7 days to maintain stable HNF1α overexpression. An empty vector adenovirus infection group was set as a negative control. After infection, cells were collected for RNA extraction and immunofluorescence assay.

[0086] 2.3 Real-time PCR detection of the expression of α-cell and β-cell specific molecules

[0087] AlphaTC1 clone6 cells treated with HNF1α-overexpressing adenovirus were extracted using the Trizol method, and the specific experimental steps were the same as in 1.4 of Example 1.

[0088] Two μg of RNA was reverse transcribed to synthesize cDNA. Using specific primers, the mRNA expression levels of α-cell-specific molecules (Glucogen, Arx, Dpp4) and β-cell-specific molecules (Insulin2, NKx6.1, Glut2) were quantitatively detected by real-time PCR. β-actin was used as an internal reference gene, and the relative expression levels of each gene were calculated using the 2–ΔΔCT method.

[0089] The results show that ( Figure 3 (a, 3b) Compared with the control group, the expression of β cell-specific molecules (Insulin, Nkx6.1, Glut2) gradually appeared and was significantly upregulated in α cells overexpressing HNF1α, while the expression of α cell-specific molecules (Glucagon, Arx, Dpp4) was significantly decreased.

[0090] 2.4 Detection of the expression of α-cell and β-cell specific molecules using cellular immunofluorescence techniques.

[0091] Collect treated alphaTC1 clone 6 cells, fix with 10% paraformaldehyde for 10 minutes, and wash three times with PBS. Then, incubate cells with 5% BSA for 1 hour; subsequently, treat cells with 0.1% Triton X-100 for 10 minutes to enhance cell membrane permeability. Add primary antibodies against the specific molecules mentioned above and incubate overnight at 4°C. The next day, remove cells, wash three times with PBS, add the corresponding immunofluorescent secondary antibody, incubate for 1 hour, stain with DAPI for 8-10 minutes, mount, and image under a fluorescence microscope.

[0092] Cell immunofluorescence results ( Figure 3 c) Further confirmation showed that in α cells overexpressing HNF1α, the expression of β cell-specific proteins increased, while the expression of α cell-specific proteins decreased, consistent with the results of real-time quantitative PCR.

[0093] Example 3: Overexpression of HNF1α induces downregulation of glucagon expression and upregulation of insulin expression in α cell lines.

[0094] 3.1 Western blot analysis of Glucagon and Insulin expression

[0095] Alpha TC1 clone 6α cells treated with HNF1α-overexpressing adenovirus were collected and lysed in 150 μl of RAPI lysis buffer (Beyotime) containing a protease inhibitor. The mixture was pipetted, homogenized, and incubated on ice for 30 minutes. Then, the cells were centrifuged at 14,000 rpm for 20 minutes at 4°C, and the supernatant was collected. Protein concentration was estimated using Bradford reagent (Sangon, Shanghai, China).

[0096] Equal amounts of protein (120 μg) were loaded onto a 10% SDS-PAGE gel for electrophoresis, followed by transfer to a PVDF membrane. After transfer, the membrane was incubated overnight with specific anti-Glucagon rabbit polyclonal antibody (Cell Signal, USA) and anti-Insulin mouse monoclonal antibody (HuaAn Biotechnology, China). Finally, the results were detected using an ECL Plus Western blot detection system (Amersham).

[0097] The results show that ( Figure 4 a) Compared with the control group, insulin protein expression was significantly increased in alpha TC1 clone 6 cells overexpressing HNF1α, while glucagon protein expression was significantly decreased.

[0098] 3.2 Detection of Glucagon and Insulin expression using immunofluorescence techniques

[0099] Collect the treated cells, fix them with 10% paraformaldehyde for 10 minutes, and wash them three times with PBS. Then, incubate the cells with 5% BSA for 1 hour, followed by treatment with 0.1% Triton X-100 for 10 minutes to enhance cell membrane permeability. Next, add primary antibodies against Gluconium and Insulin, and incubate overnight at 4°C. The next day, remove the cells, wash them three times with PBS, add the corresponding immunofluorescent secondary antibodies, incubate for 1 hour, stain with DAPI for 8-10 minutes, mount the slides, and image them under a fluorescence microscope.

[0100] Immunofluorescence results ( Figure 4 b) It was confirmed that in alpha TC1 clone 6 cells overexpressing HNF1α, triple-positive cells expressing insulin, glucagon and HNF1α could be observed, with enhanced insulin fluorescence signal and weakened glucagon fluorescence signal.

[0101] 3.3 ELISA detection of Glucagon and Insulin protein expression

[0102] Alpha TC1 clone 6 cells overexpressing HNF1α were stimulated with glucose, and the cell supernatant was collected. Insulin concentration was measured using an insulin ELISA kit, and glucagon concentration was measured using a glucagon ELISA kit.

[0103] ELISA results ( Figure 4c) Consistent with the protein level changes detected by WB, it was shown that after overexpression of HNF1α, the expression of Insulin protein secreted into the cell supernatant was significantly upregulated, while the expression of Glucagon protein was correspondingly downregulated.

[0104] Experimental Example 4: α cells overexpressing HNF1α secrete insulin in response to glucose stimulation

[0105] First, the alpha TC1 clone 6 cell line was cultured and infected with HNF1α-overexpressing adenovirus (Ad-HNF1α) and control adenovirus (Con), respectively. After infection, the cells were stimulated with glucose solutions of different concentrations for 1 hour, and the cell supernatant was collected. Then, the insulin concentration in the supernatant was measured using an insulin ELISA kit to assess the effect of HNF1α overexpression on the insulin secretion capacity of alpha cells.

[0106] By comparing insulin secretion levels in the control group (Con) and the experimental group (Ad-HNF1α) at ​​different glucose concentrations and performing statistical analysis (such as t-test), the differences between the experimental and control groups were determined to be statistically significant. This process, functionally speaking, suggests that HNF1α has the potential to transdifferentiate α cells into β cells. Figure 5 ).

[0107] Example 5: The effect of pancreatic α-cell-specific overexpression of HNF1α in delaying the onset of diabetic mice

[0108] 5.1 Preparation of α-cell-specific knockout HNF1α mice

[0109] HNF1α conditional knockout mice (HNF1α) fl / fl The glucagon promoter-driven Cre recombinase transgenic mice (GluCre tool mice, JacksonLab number 030663) have been kindly provided by Professor Xie Weifen's research group in the Department of Gastroenterology, Changzheng Hospital, Second Military Medical University. These mice have been purchased from Southern Model Biotechnology Co., Ltd. The two types of mice were mated to obtain α-cell-specific knockout HNF1α gene mice (HNF1α...). αKO ).

[0110] 5.2 Preparation of transgenic mice expressing HNF1α specifically in α cells

[0111] The transgenic mouse that specifically expresses HNF1α in α cells is a mouse overexpressing the human HNF1α gene driven by the GCG promoter (named HNF1α). α-cell / OE Mice). HNF1α α-cell / OE The mice were created by conditionally overexpressing the CAG-LSL-HNF1α-IRES-tdTomato gene and Gcg. iCreThe knock-in mice were obtained through mating. The CAG-LSL-HNF1α-IRES-tdTomato gene conditionally overexpressing mouse was obtained using CRISPR / Cas9 technology via homologous recombination, by inserting the CAG-LSL-HNF1α-IRES-tdTomato-WPRE-pA (SEQ ID NO.17) expression cassette into the Rosa26 gene locus. This mouse model has been successfully constructed by Shanghai Southern Model Biotechnology Co., Ltd. iCre Knock-in mice (Strain #: 030663) were purchased from Jackson Laboratory. Gcg iCre Knock-in mice express a codon-optimized Cre recombinase gene in pancreatic α cells under the control of the glucagon promoter (i.e., the GCG promoter).

[0112] 5.3 A single high-dose STZ injection induces a diabetes model

[0113] Male C57BL / 6J background mice, aged 8-10 weeks, were selected and divided into the following 3 groups:

[0114] 1) Wild-type group (WT): 6 wild-type mice;

[0115] 2) Knockout group (αKO): α-cell-specific knockout HNF1α gene mice (HNF1α... αKO ), 14;

[0116] 3) Overexpression group (OE): α-cell-specific overexpression of the HNF1α gene in mice (HNF1α... α-cell / OE (7 mice).

[0117] All mice were fasted the evening before the experiment, and a high dose of STZ (160 mg / kg) was injected into them on an empty stomach at 8:00 a.m. the next day to induce diabetes. After STZ injection, the following indicators were observed and recorded daily: 1) weight change; 2) random blood glucose level; 3) whether typical symptoms of diabetes such as polyuria and polydipsia appeared; and 4) mouse mortality.

[0118] Survival analysis results showed that ( Figure 6 A), the overexpression group had the highest survival rate, with no deaths occurring during the experimental period, significantly superior to the wild-type and knockout groups; the survival rate of the knockout group decreased sharply, indicating that the absence of HNF1α in α cells significantly increased the risk of death after STZ treatment. The results regarding the incidence of diabetes showed ( Figure 6B), the overexpression group showed a significantly delayed and reduced incidence of diabetes, demonstrating a strong anti-diabetic effect; the knockout group rapidly reached 100% incidence, and the onset time was earlier than that of the wild type, indicating that HNF1α plays a crucial role in maintaining glucose homeostasis and resisting diabetes in α cells. Weight change results showed ( Figure 6 C), the overexpression group maintained the best weight and experienced the smallest weight loss, reflecting better overall metabolic health; the knockout group suffered the most severe weight loss, consistent with its high mortality and morbidity, indicating metabolic disorder and deteriorating health. The weight change trend corroborated the survival rate and blood glucose results. Random blood glucose levels showed ( Figure 6 D), the overexpression group had a relatively low blood glucose level, which was significantly better than the wild type, demonstrating its effective blood glucose lowering ability; the knockout group showed a severe diabetic state.

[0119] In summary, both gain-of-function (overexpression) and loss-of-function (knockout) models strongly demonstrate that HNF1α is a key protective factor against STZ-induced diabetes in pancreatic α cells. Overexpression of HNF1α not only effectively controls blood glucose and delays the onset of diabetes, but also significantly improves the survival rate and overall health of mice; conversely, knockout of this gene leads to rapid deterioration of diabetes and increased mortality.

[0120] The specific primer sequences involved in this application are as follows:

[0121] SEQ ID NO.1

[0122] Forward-mInsulin1:5'-AAGCTGGTGGGCATCCAGTAAC-3'

[0123] SEQ ID NO.2

[0124] Reverse-mInsulin1:5'-TTCACGGCGGGACTTGGGTGTG-3'

[0125] SEQ ID NO.3

[0126] Forward-mInsulin2: 5'-GTTTCAACATGGCCCTGTGGATGC-3'

[0127] SEQ ID NO.4

[0128] Reverse-mInsulin2:5'-CCACCTCCAGTGCCAAGGTCTG-3'

[0129] SEQ ID NO.5

[0130] Forward-mGlucagon:5'-CTTCCCAGACAGAAGCGCATGAG-3'

[0131] SEQ ID NO.6

[0132] Reverse-mGlucagon:5'-CTGCCTGGCCCTCCAAGTAAG-3'

[0133] SEQ ID NO.7

[0134] Forward-mArx:5'-CCAGAAGACGCACTACCCTGAC-3'

[0135] SEQ ID NO.8

[0136] Reverse-mArx:5'-CGGTCCAGGCCGAATCAAGC-3'

[0137] SEQ ID NO.9

[0138] Forward-mDpp4:5'-CCGCCCGACCATGAAGACAC-3'

[0139] SEQ ID NO.10

[0140] Reverse-mDpp4:5'-CCATGTTCAGCATTGAGCAGCAAG-3'

[0141] SEQ ID NO.11

[0142] Forward-mNKx6.1:5'-CCCCCCATCAAGGATCCATTTTG-3'

[0143] SEQ ID NO.12

[0144] Reverse-mNKx6.1:5'-CTTCTTCTTGGCCGTGGCCATC-3'

[0145] SEQ ID NO.13

[0146] Forward-mGlut2:5'-CATCGGTGTGATCAATGCACCTC-3'

[0147] SEQ ID NO.14

[0148] Reverse-mGlut2:5'-CCCACTGCAAAGCTGGACAC-3'

[0149] SEQ ID NO.15

[0150] Forward-mβ-actin:5'-GTGACGTTGACATCCGTAAAGA-3'

[0151] SEQ ID NO.16

[0152] Reverse-mβ-actin:5'-GCCGGACTCATCGTACTCC-3'

[0153] SEQ ID NO.17

[0154]

[0155] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of HNF1α or its promoters in the preparation of drugs for treating diabetes.

2. The use according to claim 1, characterized in that, The diabetes referred to is either type 1 diabetes or type 2 diabetes.

3. The use according to claim 1, characterized in that, The drug has one or more of the following effects: 1) Promotes β-cell regeneration; 2) Promotes the transdifferentiation of pancreatic α cells into β cells; 3) Delay the onset of diabetes.

4. The use according to claim 3, characterized in that, The transdifferentiation of pancreatic α cells into β cells includes one or more of the following characteristics: a) β-cell-specific molecules are expressed in α cells; b) Downregulation of α-cell-specific molecules in α-cells.

5. The use according to claim 4, characterized in that, The β-cell-specific molecule is selected from insulin, transcription factor Nkx6.1, or glucose transporter Glut2; And / or, the α-cell-specific molecule is selected from glucagon, transcription factor Arx, or dipeptidyl peptidase Dpp4.

6. The use according to claim 1, characterized in that, The HNF1α promoter is selected from HNF1α functional activators or HNF1α overexpression reagents; preferably, the HNF1α functional activator is selected from small molecule compounds, peptides or antibodies; preferably, the HNF1α overexpression reagent is a nucleic acid construct containing the HNF1α coding gene or a virus whose genome integrates the HNF1α coding gene.

7. The use according to claim 6, characterized in that, The nucleic acid construct containing the HNF1α encoding gene is in the genomic form of a viral vector; preferably, the viral vector is selected from one or more of adenovirus vectors, lentivirus vectors, retrovirus vectors, or adeno-associated virus vectors.

8. The use according to claim 6, characterized in that, The virus whose genome integrates the HNF1α coding gene is selected from one or more of lentiviruses, adenoviruses, adeno-associated viruses, or retroviruses.

9. The use according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

10. The use according to claim 1, characterized in that, The form of the drug is selected from one or more of solid, liquid, gel, semi-liquid or aerosol.

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