Application of FLRT2 in preparation of medicine for intervening abnormal retinal neovascularization

Abstract

The invention relates to an application of leucine-rich transmembrane fibronectin 2 (FLRT2) in preparation of a medicine for intervening abnormal retinal neovascularization. The research finds that the leucine-rich transmembrane fibronectin 2 can promote the generation of retinal neovascularization, and the generation of pathological abnormal retinal neovascularization can be inhibited by inhibiting the expression of the leucine-rich transmembrane fibronectin 2; the leucine-rich transmembrane fibronectin 2 can be used as a new target for preventing and / or intervening eye retinal neovascularization related diseases, and provides a new direction and strategy for intervention of retinal abnormal neovascularization related diseases. The leucine-rich transmembrane fibronectin 2 inhibitor is any one or any combination of small interfering ribonucleic acid selected from small interfering ribonucleic acid 1, small interfering ribonucleic acid 2, small interfering ribonucleic acid 3, small interfering ribonucleic acid 4, small interfering ribonucleic acid 5 and small interfering ribonucleic acid 6.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of FLRT2 in the treatment of retinal neovascularization. Background Technology

[0002] Retinal neovascularization (RNV) is a common pathological feature of ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vascular occlusion, and retinopathy of prematurity (ROP). RNV can lead to serious complications such as tractional retinal detachment, vitreous hemorrhage, and neovascular glaucoma, and even blindness, affecting more than 130 million people worldwide.

[0003] Currently, clinical treatment for renal vascular endothelial growth factor (RNV) primarily involves intravitreal injection of anti-VEGF drugs and retinal photocoagulation. Although anti-VEGF therapy can reduce or eliminate neovascularization in over 60% of patients with peripheral retinal degeneration (PDR), more than 30% of PDR patients do not respond or respond poorly to anti-VEGF therapy. Anti-VEGF therapy is effective in 80% of patients with renal pelvic inflammatory disease (ROP), but in ROP children, peripheral retinal avascular areas persist after anti-VEGF injection, and long-term anti-VEGF therapy may affect systemic VEGF levels, suggesting that classic anti-VEGF therapy still has limitations. However, previous studies have shown that conditional knockout of Vegfa in adult mouse retinal pigment epithelial cells can lead to vision loss and choroidal capillary absence. Furthermore, isomorphic mouse embryos targeting VEGF receptor mutations die in utero during the mesodermal stage due to vascular developmental defects. Although retinal photocoagulation reduces VEGF secretion by destroying ischemic areas, it can induce decreased color vision, reduced contrast sensitivity, and even visual field defects. Therefore, exploring the pathogenesis of RNV and providing new therapeutic targets for RNV treatment is of great clinical significance.

[0004] The FLRT family (FLRT1, 2, 3) is a class of multi-domain proteins with functions in cell adhesion and signal regulation. Their core structure includes 10 leucine-rich repeats, a type III fibronectin region, and a short cytoplasmic tail. FLRT family members can influence vertebrate embryogenesis, including craniofacial development, cartilage formation, nervous system development, and heart development, by participating in cell sorting, adhesion, aggregation, inducing fibroblast growth factor (FGF) signaling, and acting in conjunction with FGF receptors. Studies have shown that FLRT2 is highly expressed in endothelial cells of tumor-associated aberrant angiogenesis, promoting and maintaining aberrant angiogenesis by mediating non-classical interendothelial adhesion. Endothelial-specific knockout of FLRT2 selectively prunes abnormal tumor vessels and increases the number of mature vessels. Furthermore, FLRT2 significantly enhances the proliferation of human umbilical vein endothelial cells (HUVECs), nasal septal cartilage (NSCs) and mandibular condyle cartilages (MCCs) in SD rats, promotes NSC and MCC migration, promotes HUVEC tube formation, and participates in the aggregation of anterior chondrocytes during cranial neural crest cell migration and craniofacial bone formation, suggesting a broad regulatory role in angiogenesis. Currently, there are few reports on the role of FLRT2 in rheumatoid arthritis (RNVs).

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides an application of FLRT2 in the treatment of retinal neovascularization.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0008] The first aspect of this invention provides the use of an FLRT2 inhibitor in the preparation of a medicament for treating or diagnosing retinal diseases, particularly for treating retinal neovascularization.

[0009] A second aspect of the present invention provides the use of FLRT2 as a target in screening drugs for the prevention and / or treatment of ocular retinal neovascularization-related diseases.

[0010] According to a preferred embodiment, the drug inhibits the expression of FLRT2.

[0011] A third aspect of this invention provides the use of FLRT2 inhibitors in the preparation of medicaments for the prevention and / or treatment of ocular retinal neovascularization-related diseases.

[0012] According to a preferred embodiment, the FLRT2 inhibitor includes nucleic acid molecules, proteins, and compounds.

[0013] According to a preferred embodiment, the FLRT2 inhibitor is a nucleic acid molecule, which includes siRNA or shRNA that silences or downregulates FLRT2 expression.

[0014] According to a preferred embodiment, the FLRT2 inhibitor is a siRNA, selected from any one or any combination of siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, and siRNA6, preferably a combination of siRNA1, siRNA2, and siRNA3 or a combination of siRNA4, siRNA5, and siRNA6; the siRNA sequence is as follows:

[0015] The positive strand sequence of siRNA1 is: CGUCAGGGAAUUAAAUAUGAATT;

[0016] The antisense strand sequence of siRNA1 is: UUCAUAUUUAAUUCCCUGACGTT;

[0017] The positive strand sequence of siRNA2 is: CCUCCACAACAACCAAAUUAATT;

[0018] The antisense strand sequence of siRNA2 is: UUAAUUUGGUUGUUGUGGAGGTT;

[0019] The positive strand sequence of siRNA3 is: GUCUCCUUAAAUAACGAUCAATT;

[0020] The antisense strand sequence of siRNA3 is: UUGAUCGUUAUUUAAGGAGACTT;

[0021] The positive strand sequence of siRNA4 is: GGAGGCUAUUAGCCUCAAA;

[0022] The antisense strand sequence of siRNA4 is: UUUGAGGCUAAUAGCCUCC;

[0023] The positive strand sequence of siRNA5 is: GGAUGAAAAUCGAAUUGCU;

[0024] The antisense strand sequence of siRNA5 is: AGCAAUUCGAUUUUCAUCC;

[0025] The positive strand sequence of siRNA6 is: GGCUGGAUAUAUCCAACAA;

[0026] The antisense strand sequence of siRNA6 is: UUGUUGGAUAUAUCCAGCC.

[0027] According to a preferred embodiment, the retinal neovascularization-related disease is one or more of the following: diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, uveitis, etc.

[0028] According to a preferred embodiment, the diabetic retinopathy is proliferative diabetic retinopathy (PDR), characterized by secondary retinal neovascularization.

[0029] According to a preferred embodiment, the drug treats retinal neovascularization-related diseases by at least one of the following methods:

[0030] (1) Inhibits the formation of pathological angiogenesis;

[0031] (2) Inhibits the proliferation of retinal vascular endothelial cells;

[0032] (3) Inhibits the migration ability of retinal vascular endothelial cells;

[0033] (4) Inhibits the tubular formation ability of retinal vascular endothelial cells;

[0034] (5) Reduce the non-perfusion area of ​​the retina;

[0035] (6) Reduce the area of ​​retinal vascular leakage.

[0036] The beneficial effects of this invention are:

[0037] The research team of this invention found that FLRT2 is highly expressed in the fibrovascular proliferative membrane and vitreous fluid of patients with proliferative diabetic retinopathy (PDR), in the serum and retina of high glucose-induced human retinal microvascular endothelial cells (HRMEC), and in the retina of oxygen-induced retinopathy (OIR) mice. FLRT2 is specifically expressed in abnormal retinal neovascularization (RNV) in diabetic retinopathy (DR) patients and OIR mouse models, but is not expressed in mature vessels. Downregulating FLRT2 expression in the retina of OIR mice can reduce abnormal RNV and non-perfusion areas.

[0038] Further research in this invention has revealed that FLRT2 can promote the formation of retinal neovascularization, while inhibiting the expression of FLRT2 can inhibit retinal neovascularization. Therefore, FLRT2 can serve as a new target for the prevention and / or treatment of ocular retinal neovascularization-related diseases, providing new directions and strategies for the treatment of retinal neovascularization-related diseases. Attached Figure Description

[0039] Figure 1 The following are single-cell transcriptome maps of the retina of mice in the control and OIR groups: A: UMAP visualization of single-cell transcriptome sequencing data, with different colors representing different cell types; BC: FLRT2 expression in HRMECs of the control and OIR groups; DE: FLRT2 gene expression in different cell types of the control and OIR groups; F: FLRT2 expression levels in HRMECs of the control and OIR groups at P14 and P17.

[0040] Figure 2 The successful construction of the OE-FLRT2 stable cell line is shown: A: Western blotting analysis of FLRT2 expression level in HRMEC; B: Gray value analysis and bar chart results of FLRT2 Western blotting results; *P<0.05; Data are expressed as mean ± standard deviation.

[0041] Figure 3The results show that FLRT2 promotes HRMEC proliferation, migration, and tube formation: A: CCK8 assay showed that the OE-FLRT2 group had enhanced proliferation ability compared to the OE-vector group; B: Cell migration assay showed that the OE-FLRT2 group had enhanced migration ability compared to the OE-vector group; Scale bar: 200 μm; C: Tube formation assay showed that the OE-FLRT2 group had enhanced tube formation ability compared to the OE-vector group; Scale bar: 200 μm; *P<0.05, **P<0.01; Data are expressed as mean ± standard deviation.

[0042] Figure 4 Immunofluorescence assays show that FLRT2 is specifically expressed in mouse RNVs and promotes RNV development and increases the area of ​​the non-perfused zone: AF: Immunofluorescence of mouse retinal slices shows that FLRT2 is specifically expressed in abnormal mouse RNVs. Scale bar: 1 mm (white), 20 μm (yellow); G, H: NAC downregulates FLRT2 expression in the retina of OIR mouse models, reducing RNVs and the area of ​​the non-perfused zone; *P<0.05, **P<0.01; Data are expressed as mean ± standard deviation.

[0043] Figure 5 A schematic diagram of the CreERT2 / loxP system construction is shown.

[0044] Figure 6 The results of gene identification of the transgenic mice and their littermates (WT mice) are shown.

[0045] Figure 7 Immunofluorescence results of mouse retinal retinal slices: Immunofluorescence showed the effect of Cdh5-Cre ERT2 Flrt2 flox / flox 4-OHT can specifically knock out Flrt2 in endothelial cells of mice (white arrow), and 4-OHT-induced conditional Flrt2 gene knockout mice have been successfully constructed: Scale bar: 20 μm.

[0046] Figure 8 The images show that FITC-dextran retroorbital injection and IB4 staining showed that knocking out Flrt2 in mouse endothelial cells inhibited RNV production and reduced the area of ​​retinal nonperfusion and retinal vascular leakage: AR: FITC-dextran retroorbital injection and IB4 staining showed abnormal RNV, retinal nonperfusion and retinal vascular leakage in mice, scale bar: 1 mm; SU: knocking out Flrt2 in mouse endothelial cells reduced the area of ​​RNV, retinal nonperfusion and retinal vascular leakage; *P<0.05, **P<0.01; data are expressed as mean ± standard deviation.

[0047] Figure 9The following diagram illustrates the downregulation of FLRT2 expression by FLRT2 siRNA: A: Western blotting analysis of FLRT2 expression levels in HRMECs transfected with si-FLRT2-1; B: Statistical analysis of grayscale values ​​and bar chart results of FLRT2 Western blotting; C: Western blotting analysis of FLRT2 expression levels in HRMECs transfected with si-FLRT2-2; D: Statistical analysis of grayscale values ​​and bar chart results of FLRT2 Western blotting; In the figure, si-FLRT2-1 is a mixture of siRNA1, siRNA2, and siRNA3, and si-FLRT2-2 is a mixture of siRNA4, siRNA5, and siRNA6; *P<0.05, **P<0.01; Data are expressed as mean ± standard deviation. Detailed Implementation

[0048] The following is a detailed explanation with reference to the accompanying drawings.

[0049] The following embodiments are for further explanation of the present invention only and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can understand the specific meaning of the terms used in the present invention based on the specific circumstances. Unless otherwise specified, the experimental procedures described in the following embodiments are conventional procedures. Unless otherwise specified, the reagents and other materials used in the following embodiments are commercially available.

[0050] Example 1

[0051] We analyzed the Gene Expression Omnibus (GEO) dataset GSE150703, which contains single-cell sequencing data of retinal cells from mice in the P14 and P17 oxygen-induced retinopathy (OIR) groups and their control groups. Analysis of this dataset revealed that in the OIR model, the expression of the fibronectin-rich transmembrane protein 2 (Flrt2) gene in retinal vascular endothelial cells was significantly upregulated compared to the control group. The Flrt2 mRNA levels in endothelial cells of the P14 and P17 OIR groups were 2.1-fold (P<0.05) and 1.52-fold (P>0.05) higher than those in the control group, respectively, suggesting that Flrt2 may play an important role in the development and progression of RNV. Figure 1 ).

[0052] Example 2: Cell and Mouse Experiments

[0053] 1. Main experimental materials and reagents

[0054] 1.1 Experimental cell lines

[0055] The human retinal microvascular endothelial cells (HRMEC) used in this experiment were purchased from Shanghai Yansheng Industrial Co., Ltd., China.

[0056] The human embryonic kidney cell line 293T was generously donated by Professor Ma Haiqing of the Department of Oncology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), affiliated with Southern Medical University.

[0057] 1.2 Bacteria

[0058] In this experiment, chemically transformed competent cells prepared from Escherichia coli DH5α strain were purchased from Nanjing Novizan Biotechnology Co., Ltd. in China and frozen at -80℃.

[0059] 1.3 Plasmids

[0060] The FLRT2 overexpression plasmid and empty vector plasmid were purchased from Guangzhou Hanyi Biotechnology Co., Ltd., China. The lentiviral packaging plasmids psPAX2 and pMD2.G were generously donated by Professor Ma Haiqing of the Department of Oncology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), affiliated with Southern Medical University.

[0061] The NCBI GenBank accession number for the human FLRT2 gene is: NM_001346143.2.

[0062] 1.4 Laboratory Animals

[0063] We constructed 4-hydroxytamoxifen (4-OHT)-induced endothelial cell-specific Flrt2 gene knockout mice:

[0064] Genotype Cdh5-Cre was constructed using male and female C57BL / 6J mice. ERT2 Flrt2 flox / flox and Cdh5-Cre ERT2 Flrt2 flox / flox Genetically engineered mice were used. Mice were housed at Guangdong Kaisheng Biotechnology Co., Ltd. before P6 and at the Guangdong Provincial Medical Laboratory Animal Center after P6. The environment was maintained at 22±4℃, humidity 40%–70%, with good ventilation and a 12-hour light / dark cycle. Free access to food and water was provided. This study strictly followed the Declaration of Helsinki and was approved by the hospital's ethics committee (ethics number KY2020-194-01-01). C57BL / 6J mice were purchased from Guangdong Kaisheng Biotechnology Co., Ltd. (Guangzhou, China).

[0065] The NCBI GenBank accession number for the mouse Flrt2 gene is: NM_201518.4.

[0066] 1.5 Main experimental consumables and reagents

[0067]

[0068] 1.6 PCR Primers and siRNA Sequences The PCR primer sequences are as follows:

[0069]

[0070]

[0071] The FLRT2 siRNA sequence is as follows:

[0072]

[0073] 1.7 Preparation of main reagents

[0074] (1) Complete Culture Medium (HRMEC): Pour 50 ml from 500 ml DMEM low-glucose medium into 50 ml centrifuge tubes as serum-free medium. Add 50 ml FBS to the remaining 450 ml DMEM low-glucose medium, vortex to mix, and prepare 10% FBS DMEM low-glucose medium. If penicillin-streptomycin is needed, add 1% penicillin-streptomycin (100×) solution to the medium and vortex to mix. If mycoplasma scavenging agent is needed, add 1‰ mycoplasma scavenging agent (1000×) solution to the medium in the dark and vortex to mix. Store the culture medium at 4℃.

[0075] (2) Complete Culture Medium (293T Cells): Pour 50 ml from 500 ml DMEM HG medium into a 50 ml centrifuge tube as serum-free medium. Add 50 ml FBS to the remaining 450 ml DMEM HG medium, vortex to mix, and prepare 10% FBS DMEM HG medium. If penicillin / streptomycin is needed, add 1% penicillin-streptomycin (100×) solution to the medium and vortex to mix. Store the culture at 4°C.

[0076] (3) Escherichia coli solid culture medium: Weigh 2.5g yeast powder, 5g NaCl, 7.5g agar powder, and 5g peptone using an electronic balance. Add 500ml of pure water. Heat in a microwave oven until boiling for a few seconds and clear, then cool to approximately 50℃. Add 35μl of ampicillin / 250mL. Shake well, add approximately 15ml of culture medium to each petri dish, irradiate with UV light, and wait for solidification. It can be stored at 4℃ for six months.

[0077] (4) Escherichia coli liquid culture medium: Weigh 2.5g yeast powder, 5g NaCl, 7.5g agar powder, and 5g peptone using an electronic balance. Add 500ml of pure water. Heat in a microwave oven until boiling for a few seconds and clear, then cool to approximately 50℃. Add 35μl of ampicillin / 250mL. Shake well and pour into 50ml centrifuge tubes, approximately 15ml per tube.

[0078] (5) Buffer P1: Before the first use, add all of the RNase A solution to Buffer P1, mix well, and store at 2-8℃ for 6 months. Before use, it needs to be placed at room temperature for a period of time and then used after returning to room temperature.

[0079] (6) Buffer PW: Before first use, add anhydrous ethanol to Buffer PW according to the instructions on the reagent bottle label.

[0080] (7) Protein lysis buffer: For each well of a six-well plate, take 50 μl of RIPA lysis buffer, add 100× protease inhibitor and 100× phosphatase inhibitor to 1× respectively, mix thoroughly, and use immediately.

[0081] (8) Use BCA protein quantification kit gradient standard solution: Use BSA to prepare gradient standard solution.

[0082] 2 methods

[0083] 2.1 Resuscitation, passage culture, cryopreservation, and counting of HREMC and 293T cells

[0084] Perform the procedure according to standard methods.

[0085] 2.2 Amplification of FLRT2 overexpression lentiviral plasmid

[0086] (1) Escherichia coli transformation: Take 10 μl of competent Escherichia coli into an EP tube, add 1 μl of plasmid, mix by swirl of the EP tube, do not vortex, place on ice for 20 min, heat shock at 42 degrees for 90 s, and place on ice for 1 min.

[0087] (2) Stirring: Use 20 μl of solid culture medium per plate and stir with a 200 μl pipette tip at an angle of about 30°. Incubate upside down in a 37°C incubator for 14–16 h, and store upside down at 4°C for up to 3 months.

[0088] (3) Picking bacteria: Use a 10ul pipette tip to pick single colonies from the solid culture medium and transfer them to the liquid culture medium. Incubate at 37℃ for 14-16 hours. Store at 4℃ for a maximum of 1 day.

[0089] 2.3 Plasmid Extraction

[0090] Take 5–15 ml of overnight culture and extract plasmids using standard methods. Detect the plasmid concentration using an ultra-micro spectrophotometer. Store the plasmids at -20°C.

[0091] 2.4 Lentiviral Vector Packaging

[0092] In this study, lentiviral vector packaging was performed in 293T cells, and the steps were as follows:

[0093] (1) Culture 293T cells in a six-well plate and start the experiment when the cell density reaches 60% to 70%.

[0094] (2) Prepare the following two tubes, A and B, for each well of the six-well plate:

[0095] Tube A: Take 100 μl of Opti-MEM into tube A, add 2000 ng of plasmid, 1500 ng of psPAX2 and 500 ng of pMD2.G, and gently pipette to mix.

[0096] Tube B: Take 100 μl of Opti-MEM into tube B, add 5 μl of Lipofectamine 2000, and gently mix by pipetting.

[0097] Let each liquid in the tube stand for 15 minutes. Then mix the two liquids together, gently blow and stir until well combined, and let stand for another 15 minutes.

[0098] (3) Aspirate the culture medium from the six-well plate, wash each well with PBS once, add 2 ml of serum-free DMEM and 200 μl of the mixed solution in (2), gently shake well using the cross method, and place in a humidified incubator containing 5% CO2 at 37°C for continued culture.

[0099] (4) After 4 to 6 hours, replace the culture medium with 2 ml of complete culture medium and continue culturing.

[0100] (5) After 24h and 48h respectively, the supernatant was aspirated and filtered with a 0.22μm filter to obtain the virus solution, which can be stored at -80℃ for 3 to 4 years. 2ml of complete culture medium was added to each well and cultured for a longer period.

[0101] (6) After another 24 hours, the supernatant was aspirated and filtered through a 0.22μm filter to obtain the virus solution, which can be stored at -80℃ for 3 to 4 years.

[0102] 2.5 Lentiviral infection of HRMEC and screening

[0103] (1) HRMECs were cultured in T25 culture flasks. The experiment was started when the cell density reached 60%.

[0104] (2) For each T25 culture flask, add 1 μl of mycoplasma scavenger, 0.8 μl of 10 mg / mL polybrene and 1 mL of virus solution to an EP tube, gently pipette to mix, and prepare a virus solution containing 1‰ mycoplasma scavenger and 8 μg / mL polybrene. Replace the culture medium with 1 mL of the above virus solution.

[0105] (3) After 6 hours, add 1 ml of complete culture medium containing 1‰ mycoplasma scavenger.

[0106] (4) After adding the virus solution for 24 hours, replace the virus-containing culture medium with 5 mL of normal complete culture medium containing 1‰ mycoplasma scavenging agent.

[0107] (5) 72 hours after adding the virus solution, when HRMECs show green fluorescence, cell screening begins. Add 1‰ of 10 mg / ml puromycin to the complete culture medium containing 1‰ mycoplasma scavenger in the centrifuge tube, shake to mix, and prepare a 10 μg / ml puromycin solution (preliminary experiments were conducted to determine the minimum drug concentration required to completely kill puromycin-free HRMECs within 24 hours). Replace the culture medium in the T25 culture flask with 5 mL of the above-mentioned culture medium.

[0108] (6) After screening for 24 hours, replace the culture medium in the T25 culture flask with 5 mL of normal complete culture medium containing 1‰ mycoplasma scavenging agent.

[0109] (7) After culturing the cells in a medium containing mycoplasma scavenger for 5 to 7 days, replace the medium with a complete medium containing 1% penicillin-streptomycin.

[0110] (8) One month later, gene expression was verified by Western blotting.

[0111] 2.6 Western blotting

[0112] (1) Protein extraction

[0113] (2) Protein concentration determination

[0114] Protein concentration was determined using the BCA protein quantification kit.

[0115] (3) Making adhesive

[0116] (4) Electrophoresis

[0117] (5) Electro-rotation

[0118] ①Prepare the electroporation solution: Total volume 1L, containing: Tris 3.03g, glycine 14.4g, methanol 200ml, water 800ml.

[0119] ② Soak the PVDF membrane in formaldehyde to ensure full wetting. Soak black and white cotton and filter paper in the transfer solution, and prepare the sandwich clamp in the following order: black clamp → sponge → filter paper → polyacrylamide gel → PVDF membrane → filter paper → sponge → white clamp. Remove air bubbles, clamp tightly, and place in the transfer tank, filling it with transfer solution. Transfer parameters: 250mA, duration 90min. Place the transfer tank at 4℃ to ensure the transfer process is carried out under low-temperature conditions.

[0120] (6) Closed

[0121] (7) Incubate antibodies

[0122] ① After sealing, place the PVDF membrane in a sealed box containing TBST solution and wash it three times on a shaker at 120r / min for 5min each time.

[0123] ② Dilute the primary antibody (FLRT2 1:1000, β-actin 1:1000); cut the PVDF membrane according to the molecular weight of the protein to be measured, place it in an incubation box containing the corresponding primary antibody, and incubate overnight at 4°C on a shaker at 50 r / min.

[0124] ③ Wash with TBST 3 times on a shaker, 5 minutes each time.

[0125] ④ Dilute the secondary antibody (1:20,000) and incubate at room temperature on a shaker at 50 r / min for 1 h.

[0126] ⑤ Wash three times on a shaker at 120r / min, for 5 minutes each time.

[0127] (8) Exposure, development and data processing.

[0128] 2.7 Cell Grouping

[0129] (1) Lentiviral empty vector (OE-vector) group: 5.5 mmol / L glucose medium + OE-vector;

[0130] (2) FLRT2 overexpression lentiviral vector (OE-FLRT2) group: 5.5 mmol / L glucose medium + OE-FLRT2.

[0131] 2.8 Cell Counting Kit Method

[0132] 2.9 Cell migration assay: standard method.

[0133] 2.10 Tube Formation Test

[0134] (1) The day before the experiment, the 48-well plate, matrix adhesive, and pipette tip were pre-cooled or dissolved at 4°C.

[0135] (2) Add 100 μl of matrix gel to each well of the 48-well plate using a pre-cooled pipette tip, operate on ice, place the 48-well plate at room temperature for 1 h, and then place it in an incubator for 30 min to gel.

[0136] (3) Digest the two groups of cells during the gelation process to prepare cell suspension.

[0137] (4) Add cell suspension to 48-well plates, 200 μl of culture medium per well, approximately 50,000 cells.

[0138] (5) Continue culturing in an incubator, and observe the cell tube formation under a microscope after 12 hours.

[0139] (6) ImageJ software was used to analyze the number of tube branches.

[0140] 2.11 Establishment, grouping, and administration methods of NAC-interventional WT mouse model

[0141] A mouse model of oxygen-induced retinopathy (OIR) was established, and the mice were divided into the following 3 groups:

[0142] (1) Control+PBS group: mice were fed in a normal oxygen environment (21% oxygen) for 17 days (P17), and intraperitoneal injection of solvent was given to control mice from P12 to P16 (PBS: the injection dose per mouse was 7.5 ml / (kg·d));

[0143] (2) OIR group: 7-day-old mice (P7) were placed in an oxygen chamber with their mothers and exposed to 75%–80% oxygen for 5 days. At 12 days old (P12), they were returned to a normal oxygen environment (21% oxygen) and continued to be fed for 5 days (P17). From P12 to P16, mice were given an intraperitoneal injection of an equal volume of solvent control (PBS: 7.5 ml / (kg·d) per mouse).

[0144] (3) OIR+NAC group: 7-day-old mice (P7) were placed in an oxygen chamber with their mothers and exposed to 75% to 80% oxygen for 5 days. At 12 days old (P12), they were returned to a normal oxygen environment (21% oxygen) and continued to be fed for 5 days (P17). From P12 to P16, they were injected intraperitoneally with 0.5 g / (kg·d) NAC dissolved in an equal volume of solvent (PBS).

[0145] 2.12 Establishment, grouping, and administration methods of transgenic mice and WT mouse models from the same batch

[0146] Mice at p7 age were numbered by clipping their toes, and these toes were used to extract genomic DNA for gene identification. The method is as follows:

[0147] (1) Extraction of mouse genomic DNA using the alkaline cleavage method:

[0148] (2) Using PCR to amplify DNA:

[0149] The PCR reaction system for gene identification is as follows: a 10 μl system contains 0.5 μl each of upstream and downstream primers, 1 μl of sample DNA, 5 μl of Taqmix, and 3 μl of ddH2O.

[0150] The PCR reaction conditions for gene identification are as follows:

[0151]

[0152]

[0153] (3) DNA electrophoresis:

[0154] Electrophoresis followed by exposure: The mice were exposed using a chemiluminescence analyzer to identify their genes.

[0155] Based on the mouse genotype, the genotypes WT and Cdh5-Cre were selected. ERT2 Flrt2 flox / flox and Cdh5-Cre ERT2 Flrt2 flox / flox The mice were divided into the following groups to verify whether the 4-OHT-induced conditional Flrt2 gene knockout mice were successfully constructed:

[0156] ①WT group: P12 to P17 were given intraperitoneal injection of 60mg / (kg·d)H2O2.

[0157] ②Cdh5-Cre ERT2 Groups: P12 to P17 were given intraperitoneal injection of 60 mg / (kg·d) H2O2.

[0158] ③Cdh5-Cre ERT2 +20μl 4-OHT group: P12 to P17 were given intraperitoneal injections of 60mg / (kg·d) H2O2 and 20μl / d of 4-OHT at a concentration of 1mg / ml.

[0159] ④Cdh5-Cre ERT2 +30μl 4-OHT group: P12 to P17 were given intraperitoneal injection of 60mg / (kg·d) H2O2 and 30μl / d of 4-OHT at a concentration of 1mg / ml.

[0160] ⑤Flrt2 flox / flox Groups: P12 to P17 were given intraperitoneal injection of 60 mg / (kg·d) H2O2.

[0161] ⑥Cdh5-Cre ERT2Flrt2 flox / flox Groups: P12 to P17 were given intraperitoneal injection of 60 mg / (kg·d) H2O2.

[0162] ⑦Cdh5-Cre ERT2 Flrt2 flox / flox +20μl 4-OHT group: P12 to P17 were given intraperitoneal injections of 60mg / (kg·d) H2O2 and 20μl / d of 4-OHT at a concentration of 1mg / ml.

[0163] ⑧Cdh5-Cre ERT2 Flrt2 flox / flox +30μl 4-OHT group: P12 to P17 were given intraperitoneal injection of 60mg / (kg·d) H2O2 and 30μl / d of 4-OHT at a concentration of 1mg / ml.

[0164] Based on the mouse genotype, the genotype is Flrt2. flox / flox and Cdh5-Cre ERT2 Flrt2 flox / flox In mice, the following groups were set up to analyze the function of FLRT2 in RNV:

[0165] ①Flrt2 flox / flox +Control group: fed in a normal oxygen environment (21% oxygen) for 17 days (P17). From P12 to P17, 20 μl of 1 mg / ml 4-OHT was injected intraperitoneally daily.

[0166] ②Flrt2 flox / flox +OIR group: 7-day-old mice (P7) were placed in an oxygen chamber with their mothers and exposed to 75%–80% oxygen for 5 days. At 12 days old (P12), they were returned to a normal oxygen environment (20% oxygen) and continued to be fed for 5 days (P17). From P12 to P17, 20 μl of 1 mg / ml 4-OHT was injected intraperitoneally daily.

[0167] ③Cdh5-Cre ERT2 Flrt2 flox / flox +OIR group: 7-day-old mice (P7) were placed in an oxygen chamber with their mothers and exposed to 75%–80% oxygen for 5 days. At 12 days old (P12), they were returned to a normal oxygen environment (20% oxygen) and continued to be fed for 5 days (P17). From P12 to P17, 20 μl of 1 mg / ml 4-OHT was injected intraperitoneally daily.

[0168] 2.13 Retinal Slide Immunofluorescence

[0169] (1) Mice were euthanized by cervical dislocation, the eyeballs were removed and placed in PBS, and a hole was made in the cornea with a syringe under a microscope and fixed with 4% PFA for 45-60 min.

[0170] (2) Separate the retina under a stereomicroscope.

[0171] (3) Fix the retina in 4% PFA at room temperature for 30 min.

[0172] (4) Wash the retina with PBS for 5 minutes each time, 3 times.

[0173] (5) Blocking and permeation: The retina was placed in 10% goat serum and 0.5% Triton X-100 (diluted with PBS) and blocked and permeated at room temperature for 1 hour.

[0174] (6) Primary antibody incubation: Dilute the primary antibody (FLRT2:1:100, CD31:1:200) with immunostaining primary antibody dilution buffer containing 0.2% Triton X-100. Place the retina in the diluted primary antibody and incubate overnight at 4°C and 40r / min to 60r / min on a shaker.

[0175] (7) Wash with PBS three times at room temperature and on a shaker at 40 r / min to 60 r / min for 15 min each time.

[0176] (8) Secondary antibody incubation: Dilute the secondary antibody with 5% goat serum and 0.2% Triton X-100 (1:400), place the retina in the diluted secondary antibody, and incubate at 4℃ and 40r / min to 60r / min for 4h.

[0177] (9) Wash with PBS 5 times at room temperature and on a shaker at 40r / min to 60r / min for 10 min each time.

[0178] (10) Mounting: Cut the retina into a four-leaf clover shape, place it on a glass slide, add anti-fluorescence attenuation mounting medium (containing DAPI), and cover with a coverslip.

[0179] (11) Result interpretation: Observe and acquire images under a laser confocal microscope.

[0180] 2.14 Retroorbital injection of fluorescein thiocyanate-dextran and retinal smear staining with isolectin

[0181] (1) Mice were anesthetized with isoflurane and injected with 0.1 ml of fluorescein isothiocyanate (FITC)-dextran into the orbit. They were sacrificed 5 min later.

[0182] (2) Remove the eyeball and place it in PBS. Under a microscope, use a syringe to make a hole in the cornea and place it in 4% PFA for 45-60 minutes.

[0183] (3) Separate the retina under a stereomicroscope.

[0184] (4) Fix the retina in 4% PFA at room temperature for 30 min.

[0185] (5) Wash the retina with PBS for 5 minutes each time, for a total of 3 times.

[0186] (6) Blocking and permeation: The retina was placed in 10% goat serum and 0.5% Triton X-100 (diluted with PBS) and blocked and permeated at room temperature for 1 h.

[0187] (7) Primary antibody incubation: Isolectin B4 (IB4) was diluted with immunostaining primary antibody dilution buffer containing 0.2% Triton X-100. The retina was placed in the diluted IB4 and incubated overnight at 4°C and 40-60 rpm on a shaker.

[0188] (8) Wash with PBS three times at room temperature and on a shaker at 40 r / min to 60 r / min for 15 min each time.

[0189] (9) Mounting: Cut the retina into a four-leaf clover shape, place it on a glass slide, add an anti-fluorescence attenuation mounting medium (containing DAPI), and cover with a coverslip.

[0190] (10) Result interpretation: Observe and acquire images under a laser confocal microscope.

[0191] 2.15 Statistical Methods

[0192] Statistical analysis was performed using R 4.4.1 and GraphPad Prism 10.2.1 software (GraphPad software, San Diego, CA, USA). Measurement data are expressed as mean ± standard deviation. Two-way ANOVA was used to assess differences in the OD values ​​of CCK8. Two-way independent samples t-tests were used to detect differences between the other two groups of measurement data. A p-value < 0.05 was considered statistically significant.

[0193] 3 Results

[0194] 3.1 FLRT2 promotes the proliferation, migration, and tubule formation of retinal vascular endothelial cells in vitro.

[0195] To investigate the function of FLRT2, we first constructed a stable OE-FLRT2 cell line and used Western blotting to verify FLRT2 expression in HRMECs. The results showed that FLRT2 expression in the OE-FLRT2 group was significantly higher than that in the OE-vector group, indicating that the stable OE-FLRT2 cell line was successfully constructed. Figure 2 ).

[0196] Given that the proliferation, migration, and tube formation of retinal vascular endothelial cells are key steps in RNV generation, we investigated the effect of FLRT2 on the proliferation, migration, and tube formation abilities of HRMECs in vitro.

[0197] (1) Proliferation capacity

[0198] The activity of HRMEC was detected using CCK8 assay. The results showed that after 24h, 48h, and 72h of culture, the activity of the OE-FLRT2 group was significantly higher than that of the OE-vector group (P<0.05). Figure 3 A) That is, FLRT2 can enhance the proliferation ability of HRMEC.

[0199] (2) Transferability

[0200] The migration ability of HRMECs was detected by cell migration assay. The results showed that after 24 h of culture, the number of cells that penetrated the filter membrane pores in the OE-FLRT2 group was higher than that in the OE-vector group, and the difference was statistically significant (P<0.05). Figure 3 B) demonstrates that FLRT2 can enhance the migration ability of HRMEC.

[0201] (3) Pipe forming capacity

[0202] The tube formation assay was used to detect the tube-forming ability of HRMECs. The results showed that after 12 h of culture, the number of branches formed by cells in the OE-FLRT2 group was higher than that in the OE-vector group, and the difference was statistically significant (P<0.05). Figure 3 C) indicates that FLRT2 enhances the tube-forming ability of HRMECs. In summary, FLRT2 enhances the proliferation, migration, and tube-forming abilities of HRMECs, playing a driving role in key stages of RNV generation. In conclusion, in vitro experiments confirm that FLRT2 can enhance the proliferation, migration, and tube-forming abilities of HRMECs, playing a driving role in key stages of RNV generation.

[0203] 3.2 Downregulation of FLRT2 in the OIR mouse model inhibits retinal neovascularization.

[0204] To investigate whether FLRT2 promotes RNV generation in an OIR mouse model, we isolated the retina of P17 mice and performed retinal patch immunofluorescence staining. The results showed that in the Control+PBS group, vascular endothelial cells (CD31) + The cells (radially distributed from the optic disc to the periphery of the retina) exhibited a consistent morphology and natural course, with a clear and regular vascular network. No RNVs or non-perfusion areas were observed, and no FLRT2 expression was seen. However, in the OIR+PBS group, vascular endothelial cells formed clusters of RNVs and large areas of non-CD31-stained non-perfusion areas. The endothelial cells of the RNVs expressed FLRT2. The FLRT2 expression level of the endothelial cells of the RNVs in the OIR+NAC group was lower than that in the OIR+PBS group. The area of ​​RNVs and the area of ​​non-perfusion areas in the OIR+NAC group were both smaller than those in the OIR+PBS group, and the differences were statistically significant (P<0.05). Figure 4 ).

[0205] In summary, when the expression level of FLRT2 increases, the RNV and the area of ​​the unperfused region increase; while when the expression level of FLRT2 decreases, the RNV and the area of ​​the unperfused region decrease, suggesting that inhibiting the expression of FLRT2 may suppress the generation of RNV.

[0206] 3.3 Construction, gene identification and validation of 4-hydroxytamoxifen-induced endothelial cell-specific Flrt2 gene knockout mice

[0207] To more accurately identify the function of FLRT2 in endothelial cells, we constructed 4-OHT-induced endothelial cell-specific Flrt2 gene knockout mice. As shown in the figure, LoxP sites were inserted on both sides of the Flrt2 gene, and the Cdh5-Cre gene was knocked out. ERT2 Specifically, the gene between two LoxPs in endothelial cells is knocked out under induction. Figure 5 To identify the genes in transgenic mice, we extracted mouse genomic DNA using the alkaline cleavage method, amplified the DNA using PCR, and then performed DNA electrophoresis. An example of the gene identification results is shown below. Figure 6 As shown: Genetic identification results indicate that the genotypes of the five mice are Flrt2. flox / flox Flrt2 flox / + Cdh5-Cre ERT2 WT, Flrt2 flox / + Select genotypes of WT, Cdh5-Cre. ERT2 Flrt2 flox / flox and Cdh5-Cre ERT2 Flrt2 flox / flox The mice were then used for subsequent experiments.

[0208] To determine whether the 4-OHT-induced endothelial cell-specific Flrt2 gene knockout mouse model was successfully constructed, we isolated the retinas of P17 mice and performed retinal patch immunofluorescence staining. Immunofluorescence staining of P17 mouse retinal patch showed that the WT group and Cdh5-Cre... ERT2 Group, Cdh5-Cre ERT2 +20μl 4-OHT group, Cdh5-Cre ERT2 +30μl 4-OHT group, Flrt2 flox / flox Group and Cdh5-Cre ERT2 Flrt2 flox / flox The retinal vascular endothelial cells in all groups expressed FLRT2. Cdh5-Cre ERT2 Flrt2 flox / flox +20 μl 4-OHT group and Cdh5-Cre ERT2 Flrt2 flox / flox Retinal vascular endothelial cells in the +30μl 4-OHT group did not express FLRT2 ( Figure 7 Therefore, for Cdh5-Cre ERT2 Flrt2 flox / flox In mice, 4-OHT can specifically knock out Flrt2 in endothelial cells and block H2O2-induced FLRT2 expression.

[0209] In summary, 4-OHT-induced endothelial cell-specific Flrt2 gene knockout mice were successfully constructed.

[0210] 3.4 In in vivo experiments, knocking out Flrt2 in mouse endothelial cells inhibited mouse RNV production.

[0211] To investigate whether FLRT2 promotes RNV generation in an OIR mouse model, we performed retroorbital injection of FITC-dextran and IB4-stained retinal retinal slices. Retinal slices from P17 mice showed that FLRT2... flox / flox In the +Control group, vessels radiated from the optic disc towards the periphery of the retina, exhibiting a consistent morphology and natural course. The vascular network was clear and regular, with no RNVs or non-perfusion areas observed. However, in Flrt2... flox / flox +OIR group showed endothelial cell clusters of RNV, non-perfusion areas without IB4 staining, and leaking FITC-dextran. Cdh5-Cre ERT2 Flrt2 flox / flox The RNV area, retinal non-perfusion area, and retinal vascular leakage area in the +OIR group were compared with those in Flrt2. flox / flox The OIR level decreased in the +OIR group, and the difference was statistically significant (P<0.05). Figure 8 ).

[0212] In summary, knocking out Flrt2 in mouse endothelial cells in in vivo experiments can inhibit the generation of RNV in mice, reduce the area of ​​retinal non-perfusion and retinal vascular leakage, and further confirm the key role of FLRT2 in pathological retinal neovascularization.

[0213] 3.5 FLRT2 siRNA downregulates FLRT2 expression in HRMECs

[0214] To investigate whether FLRT2 siRNA can downregulate FLRT2, we intervened in HRMEC with FLRT2 siRNA and used Western blotting to verify FLRT2 expression in HRMEC. The results showed that FLRT2 expression in the HG+si-FLRT2 group was significantly lower than that in the HG+si-NC group. Figure 9 ).

[0215] 4 Analysis

[0216] In vitro experiments demonstrated that FLRT2 enhances the proliferation, migration, and tube formation capabilities of HRMECs. In in vivo experiments, increased FLRT2 expression levels led to an increase in RNVs and the area of ​​unperfused regions; conversely, decreased FLRT2 expression levels resulted in a decrease in both RNVs and the area of ​​unperfused regions. These in vitro and in vivo results suggest that FLRT2 may promote RNV generation.

[0217] In vivo experiments showed that downregulating FLRT2 reduced the inhibition of RNV production in mice, and decreased the area of ​​retinal non-perfusion and retinal vascular leakage. Therefore, FLRT2 is a potential target for targeted therapy of RNV.

[0218] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. The use of an FLRT2 inhibitor in the preparation of a drug for the treatment or diagnosis of retinal diseases, especially for the treatment of retinal neovascularization.

2. Application of FLRT2 as a target in screening drugs for the prevention and / or treatment of ocular retinal neovascularization-related diseases.

3. The application according to claim 2, characterized in that, The drug inhibits the expression of FLRT2.

4. Application of FLRT2 inhibitors in the preparation of drugs for the prevention and / or treatment of ocular retinal neovascularization-related diseases.

5. The application according to claim 4, characterized in that, The FLRT2 inhibitors include nucleic acid molecules, proteins, and compounds.

6. The application according to claim 5, characterized in that, The FLRT2 inhibitor is a nucleic acid molecule, which includes siRNA or shRNA that silences or downregulates FLRT2 expression.

7. The application according to claim 6, characterized in that, The FLRT2 inhibitor is a siRNA, selected from any one or any combination of siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, and siRNA6; the siRNA sequence is as follows: The positive strand sequence of siRNA1 is: CGUCAGGGAAUUAAAUAUGAATT; The antisense strand sequence of siRNA1 is: UUCAUAUUUAAUUCCCUGACGTT; The positive strand sequence of siRNA2 is: CCUCCACAACAACCAAAUUAATT; The antisense strand sequence of siRNA2 is: UUAAUUUGGUUGUUGUGGAGGTT; The positive strand sequence of siRNA3 is: GUCUCCUUAAAUAACGAUCAATT; The antisense strand sequence of siRNA3 is: UUGAUCGUUAUUUAAGGAGACTT; The positive strand sequence of siRNA4 is: GGAGGCUAUUAGCCUCAAA; The antisense strand sequence of siRNA4 is: UUUGAGGCUAAUAGCCUCC; The positive strand sequence of siRNA5 is: GGAUGAAAAUCGAAUUGCU; The antisense strand sequence of siRNA5 is: AGCAAUUCGAUUUUCAUCC; The positive strand sequence of siRNA6 is: GGCUGGAUAUAUCCAACAA; The antisense strand sequence of siRNA6 is: UUGUUGGAUAUAUCCAGCC.

8. The application according to any one of claims 1 to 7, characterized in that, The retinal neovascularization-related diseases mentioned are one or more of the following: diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, uveitis, etc.

9. The application according to claim 8, characterized in that, The diabetic retinopathy mentioned is proliferative diabetic retinopathy (PDR), characterized by secondary retinal neovascularization.

10. The application according to any one of claims 1 to 9, characterized in that: The drug treats retinal neovascularization-related diseases through at least one of the following methods: (1) Inhibits the formation of pathological angiogenesis; (2) Inhibits the proliferation of retinal vascular endothelial cells; (3) Inhibits the migration ability of retinal vascular endothelial cells; (4) Inhibits the tubular formation ability of retinal vascular endothelial cells; (5) Reduce the area of ​​retinal non-perfusion; (6) Reduce the area of ​​retinal vascular leakage.

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