Application of Mafb as target spot in promotion of diabetic corneal epithelium injury repair

Abstract

The invention provides application of Mafb as a target spot in promotion of diabetic corneal epithelium injury repair, and belongs to the technical field of biological medicine. According to the application, the Mafb gene is taken as a treatment target, the repair speed of corneal epithelium injury can be obviously accelerated by over-expressing the Mafb gene in the cornea of a diabetic patient, and the expression levels of stem cell markers and cell proliferation markers in corneal epithelium tissues are obviously up-regulated. The invention also provides a recombinant adeno-associated virus AAV9-Mafb for overexpressing the Mafb gene, and the AAV9-Mafb can significantly recover the function of the corneal limbal stem cells of the diabetic mouse and promote the proliferation of corneal epithelial cells, thereby accelerating the repair of corneal epithelium injury of the diabetic mouse.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to... Mafb The application of this target in promoting the repair of corneal epithelial damage in diabetic patients. Background Technology

[0002] Diabetes mellitus is a globally prevalent metabolic disease, and chronic hyperglycemia can lead to various ocular complications. Epidemiological surveys show that more than half of diabetic patients also have diabetic keratopathy, whose clinical manifestations mainly include corneal epithelial abnormalities, superficial punctate keratitis, delayed epithelial wound healing, and neurotrophic keratopathy. Currently, clinical treatment mainly involves topical lubricants, antibiotic eye ointments, bandage-type soft contact lenses, and blepharoplasty. However, these methods can only temporarily relieve symptoms and cannot effectively stop disease progression. Persistent corneal epithelial defects may eventually cause severe visual impairment. However, corneal transplantation, as a terminal treatment option, is a complex procedure requiring high levels of surgical skill. Postoperative risks include graft rejection and various complications, and the global shortage of corneal donors remains a serious problem. Summary of the Invention

[0003] This invention provides a method for... Mafb To demonstrate the use of the target in promoting the repair of corneal epithelial damage in diabetic patients, the corneal overexpression of the described [target] was investigated. Mafb It can significantly restore the function of limbal stem cells in diabetic patients and promote the proliferation of corneal epithelial cells, thereby accelerating the repair of corneal epithelial damage in diabetic patients.

[0004] This invention provides an overexpression Mafb The application of the reagent in the preparation of drugs for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage.

[0005] In one embodiment of the present invention, the overexpression Mafb Including corneal overexpression of the Mafb .

[0006] In one embodiment of the present invention, the overexpression Mafb This includes using adeno-associated virus to overexpress the aforementioned [virus] in the cornea. Mafb .

[0007] In one embodiment of the present invention, the adeno-associated virus type includes AAV9.

[0008] This invention also provides a method for corneal overexpression Mafb The method for constructing recombinant adeno-associated virus includes the following steps: Mafb The gene was inserted into the AAV vector plasmid, and the recombinant adeno-associated virus was produced by transfection of the AAV-293 cell line.

[0009] In one embodiment of the present invention, the AAV vector plasmid includes GV825.

[0010] This invention also provides a method for constructing corneal overexpression using the above-described method. Mafb Recombinant adeno-associated virus.

[0011] The present invention also provides a medicament for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage, wherein the active ingredient includes the above-mentioned recombinant adeno-associated virus.

[0012] In one embodiment of the present invention, the dosage form of the drug includes an injection.

[0013] In one embodiment of the present invention, the packaging titer of the recombinant adeno-associated virus in the injection is 2.16 × 10⁻⁶. 13 vg / ml.

[0014] Beneficial effects: This invention uses Mafb The gene was identified as a therapeutic target, and it was found to be overexpressed in the cornea of ​​diabetic mice. Mafb Genes can significantly accelerate the repair of corneal epithelial damage, and in its corneal epithelial tissue, stem cell markers ( Gpha2 , Krt15 ) and cell proliferation markers ( Mki67 The expression levels of AAV9 were significantly upregulated. In this embodiment of the invention, a type 1 diabetic mouse model was established using 6-8 week old male C57BL / 6J mice, and AAV9- was injected subconjunctivally into the diabetic mice. Mafb A mouse corneal epithelial injury model was established 3-4 weeks after the injection to investigate AAV9- Mafb Regarding the therapeutic effect on diabetic corneal epithelial damage, experimental results showed that, compared with the control group of diabetic mice injected with AAV9 blank vector, AAV9- Mafb The treatment group of diabetic mice showed significantly accelerated corneal epithelial repair, and the expression levels of stem cell markers and cell proliferation markers were significantly upregulated in the corneal epithelial tissue of the treatment group. This indicates that AAV9- Mafb It can significantly restore the function of limbal stem cells in diabetic mice and promote the proliferation of corneal epithelial cells, thereby accelerating the repair of corneal epithelial damage in diabetic mice.

[0015] This invention achieves treatment through subconjunctival injection, exhibiting strong targeting and clear efficacy. The AAV9 vector enables efficient delivery. Mafb Gene delivery to target tissues specifically restores the function of limbal stem cells, greatly promoting the repair of diabetic corneal epithelial damage and fundamentally improving the treatment effect of diabetic corneal epithelial damage. Utilizing the AAV9- described in this invention... MafbGene therapy can be used to treat diabetic corneal epithelial damage by injecting gene drugs. The procedure is simple and can prevent or intervene in the disease at an early stage. It can effectively block the progression of the lesion to deeper layers, thereby avoiding irreversible vision loss. It provides a new early gene therapy strategy for the clinical management of diabetic corneal damage and is expected to significantly reduce the proportion of the disease that progresses to the end stage and requires corneal transplantation. Attached Figure Description

[0016] Figure 1 The present invention is a recombinant adeno-associated virus AAV9- Mafb Structural diagram; Figure 2 Subconjunctival injection of AAV9- Mafb Figure showing the effect of the treatment on the corneal epithelial repair rate in diabetic mice. Figure 3 Subconjunctival injection of AAV9- Mafb Figure showing the effect of gene expression in the corneal epithelium of diabetic mice; Figure 4 Subconjunctival injection of AAV9- Mafb Immunofluorescence results of protein expression levels of Mafb, stem cell markers and cell proliferation markers in limbal epithelial cells of diabetic mice. Detailed Implementation

[0017] This invention provides an overexpression Mafb The application of the reagent in the preparation of drugs for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage.

[0018] This invention discovers overexpression at the cornea Mafb It can significantly promote the repair of corneal epithelial damage in diabetic patients. Mafb When the source is human, its NCBI accession number is NM_005461.5, and its ID is 9935; this example uses mice as an example, therefore mouse-derived samples were selected. Mafb The gene is described, with its NCBI accession number NM_010658.3 and gene ID 16658. However, the contents of the mouse model should not be taken as the entire scope of protection of this invention. The purpose of this invention is to provide a theoretical basis and treatment plan for the clinical treatment of diabetic corneal epithelial damage.

[0019] The present invention relates to the above. Mafb There are no particular limitations on the method of overexpression, and the method can utilize techniques in the art that can specifically overexpress the substance in the cornea. Mafb Any method is acceptable. In one embodiment of the present invention, the adeno-associated virus method is used to overexpress the [therapeutic agent]. Mafb And constructed recombinant adeno-associated virus AAV9- Mafb.

[0020] In this embodiment of the invention, subconjunctival injection was used to treat delayed corneal epithelial injury healing in diabetic mice, and the efficacy was compared with that of the control group. Compared with the control group of diabetic mice injected with AAV9 blank vector, AAV9- Mafb The treatment group of diabetic mice showed significantly accelerated corneal epithelial repair, and the expression levels of stem cell markers (Gpha2, Krt15) and cell proliferation markers (Ki-67) were significantly upregulated in the corneal epithelial tissue of the treatment group. This indicates that AAV9- Mafb It can significantly restore the function of limbal stem cells in diabetic mice and promote the proliferation of corneal epithelial cells, thereby accelerating the repair of corneal epithelial damage in diabetic mice.

[0021] This invention also provides a method for corneal overexpression Mafb The method for constructing recombinant adeno-associated virus includes the following steps: Mafb The gene was inserted into the AAV vector plasmid, and the recombinant adeno-associated virus was produced by transfection of the AAV-293 cell line.

[0022] This invention relates to the adeno-associated virus AAV9- Mafb There are no particular limitations on the construction method; conventional methods in the field can be used for construction and packaging. For example, in one embodiment of this invention, the construction was entrusted to Jikai Gene (Shanghai) based on an adeno-associated virus serotype 9 (AAV9) gene delivery system. The construction method mainly includes: using mice... Mafb The coding sequence of the gene was inserted into the multiple cloning site of the commercial AAV transfer plasmid GV825 (which contains the CMV promoter, 3×Flag tag, F2A autocleavage peptide, EGFP green fluorescent protein reporter gene, WPRE enhancer, and BGH polyA signal sequence) using molecular cloning technology to construct a recombinant transfer plasmid. Subsequently, transfection with the AAV-293 cell line was used to produce... Figure 1 The recombinant AAV9-CMV-Mafb-3Flag-FT2A-EGFP-WPRE-BGH polyA virus shown is abbreviated as AAV9- Mafb The AAV9- was determined using droplet digital PCR (ddPCR). Mafb The genome titer was 2.16 × 10⁻⁶. 13 vg / ml.

[0023] This invention also provides a method for constructing corneal overexpression using the above-described method. Mafb Recombinant adeno-associated virus.

[0024] The recombinant adeno-associated virus AAV9-Mafb described in this invention can be used for the repair of corneal epithelial damage in diabetic patients via subconjunctival injection.

[0025] The present invention also provides a medicament for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage, wherein the active ingredient includes the above-mentioned recombinant adeno-associated virus.

[0026] The dosage form of the drug described in this invention includes an injection, wherein the packaging titer of the recombinant adeno-associated virus in the injection is 2.16 × 10⁻⁶. 13 vg / ml.

[0027] To further illustrate the present invention, the following description, in conjunction with embodiments, demonstrates the invention's features. Mafb The use of the target in promoting the repair of corneal epithelial damage in diabetic patients is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0028] Unless otherwise specified, the methods and reagents used in the embodiments of this invention are derived from commercially available products and conventional methods. For example, the method for constructing a type 1 diabetic mouse model (T1DM) was constructed according to the method described in the article (Yang L, Di G, Qi X, Qu M, Wang Y, Duan H, et al. Substance P promotes diabetic corneal epithelial woundhealing through molecular mechanisms mediated via the neurokinin-1 receptor. Diabetes 2014;63:4262–74.); AAV9-Mafb was purchased from Jikai Gene (Shanghai), with a viral titer of 2.16 × 10⁻⁶. 13 vg / ml.

[0029] Example 1 Type 1 diabetes model was established by intraperitoneal injection of 50 mg / kg streptozotocin (STZ) into 6-8 week old C57BL / 6J mice for 5 consecutive days. Blood glucose was monitored. Qualified diabetic mice with blood glucose levels that were consistently higher than 16.7 mmol / L were injected subconjunctivally with adeno-associated virus.

[0030] During adeno-associated virus (AAV) injection, type 1 diabetic mice were randomly divided into a control group and an experimental group: Control group: Subconjunctival injection of blank control virus AAV9-NC 5 μL, titer: 2.16 × 10⁻⁶ 13 vg / ml; Mafb overexpression group: Subconjunctival injection of AAV9- Mafb 5 μL, titer: 2.16 × 10⁵ 13 vg / ml; A corneal epithelial injury model was established 3-4 weeks after subconjunctival injection. The corneal epithelial wound area was marked using a 2.5 mm trephine, and corneal epithelial tissue was scraped away using an Alger Brush II scraper. The wound was stained with sodium fluorescein at 0 and 24 hours after epithelial scraping, and the area of ​​corneal epithelial injury was observed using a slit-lamp microscope. Subsequently, tissue samples were collected at 36 hours, and qPCR was performed on the corneal epithelial tissue for observation. Mafb , Gpha2 and Mki67 The study investigated the changing trends of gene expression and observed the changes in the protein levels of Mafb, Ki-67, Gpha2, and Krt15 in corneal epithelial tissue through immunofluorescence staining of frozen sections and corneal smears.

[0031] Table 1 RT-qPCR primers

[0032] At 0 and 24 hours after corneal epithelial scraping, the control group and the overexpressing group were stained with sodium fluorescein to mark their respective groups. Mafb The area of ​​corneal epithelial defects in each group was observed and statistically analyzed using a slit-lamp microscope (n=8). The results are as follows: Figure 2 As shown, overexpression was observed 24 hours after corneal epithelial scraping. Mafb The corneal epithelial defect area in the diabetic mice group was significantly smaller than that in the control group.

[0033] Subconjunctival injection of AAV9- Mafb In the corneal epithelium of diabetic mice, Mafb Stem cell markers ( Gpha2 ) and cell proliferation markers ( Mki67 Gene expression levels such as Figure 3 As shown, the results were significantly higher than those in the control group (n=3).

[0034] Immunofluorescence results as follows Figure 4 As shown, subconjunctival injection of AAV9- Mafb Subsequently, the protein expression levels of Mafb, stem cell markers (Gpha2, Krt15), and cell proliferation markers (Ki-67) in the limbal epithelial cells of diabetic mice were significantly upregulated (n=3).

[0035] In summary, adeno-associated virus (AAV) vector-mediated... MafbOverexpression of the gene in limbal epithelial cells of mice significantly accelerates the repair rate of corneal epithelial damage in diabetic mice. Specifically, it promotes the expression of stem cell markers (such as Gpha2 and Krt15) and proliferation cell markers (Ki-67) in the limbal region of mice, effectively restores the function of limbal stem cells in diabetic mice, promotes the proliferation of corneal epithelial cells, and thus accelerates the repair of corneal epithelial damage.

[0036] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An overexpression Mafb The application of the reagent in the preparation of drugs for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage.

2. The application according to claim 1, characterized in that, The overexpression Mafb Including corneal overexpression of the Mafb .

3. The application according to claim 1 or 2, characterized in that, The overexpression Mafb This includes using adeno-associated virus to overexpress the aforementioned [virus] in the cornea. Mafb .

4. The application according to claim 3, characterized in that, The types of adeno-associated viruses mentioned include AAV9.

5. A substance overexpressing in the cornea Mafb The method for constructing recombinant adeno-associated virus is characterized by, Includes the following steps: Will Mafb The gene was inserted into the AAV vector plasmid, and the recombinant adeno-associated virus was produced by transfection of the AAV-293 cell line.

6. The construction method according to claim 5, characterized in that, The AAV vector plasmid includes GV825.

7. The corneal overexpression obtained by the construction method according to claim 5 or 6 Mafb Recombinant adeno-associated virus.

8. A drug for treating diabetic corneal epithelial damage and / or promoting the repair of diabetic corneal epithelial damage, characterized in that, The active ingredient includes the recombinant adeno-associated virus as described in claim 7.

9. The drug according to claim 8, characterized in that, The dosage form of the drug includes injections.

10. The drug according to claim 9, characterized in that, The packaging titer of the recombinant adeno-associated virus in the injection is 2.16 × 10⁻⁶. 13 vg / ml.

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