A biomarker for diagnosing diabetic retinopathy, a detection kit and its application

Abstract

The present invention relates to the field of biomedicine, and in particular to a biomarker, a detection kit and an application thereof for diagnosing diabetic retinopathy (DR). The present invention proposes an innovative solution, using the biomarker TFF2 to diagnose and assist in the diagnosis of DR. The present invention verifies its effectiveness in diagnosing DR by detecting the expression level of TFF2 in the aqueous humor sample of the patient. Research data show that the ROC curve AUC value of TFF2 as a DR diagnostic indicator is as high as 0.9122, indicating that it has extremely high sensitivity, specificity and accuracy. This result significantly improves the reliability and accuracy of early diagnosis of DR. In addition, the present invention has found that TFF2 significantly promotes the formation of new blood vessels by promoting the proliferation, migration, tube formation, sprouting of retinal vascular endothelial cells and increasing the permeability of endothelial cells, thereby exacerbating the progression of DR. Therefore, the present invention not only proposes an efficient and accurate DR diagnostic method, but also provides important theoretical support and practical guidance for future DR treatment strategies.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a biomarker for diagnosing diabetic retinopathy, a detection kit and applications thereof. Background Art

[0002] Diabetic retinopathy (DR) is one of the most common and serious microvascular complications of diabetes and the main cause of vision loss in diabetic patients. With the increase in the incidence of diabetes, the incidence of DR is rising rapidly, and it is estimated that the number of diabetic patients worldwide will reach 700 million by 2045. Abnormal increase in blood sugar leads to retinal microvascular damage, resulting in a series of fundus manifestations such as retinal hemorrhage, exudation, macular edema, vitreous hemorrhage, and retinal detachment. At present, imaging methods (such as wide-angle retinal imaging, optical coherence tomography, etc.) are commonly used in clinical practice to screen and diagnose DR. However, due to the hidden nature of DR in the early stage of the disease, clinical symptoms are often difficult to detect on fundus images, resulting in many patients having reached the middle and late stages of the disease when they seek medical treatment, and their vision has been irreversibly damaged. At present, the treatment methods for diabetic retinopathy are mainly laser, drugs and surgery. This type of treatment is mainly aimed at advanced patients with existing visual impairment and fundus proliferative vascular lesions, and plays a role in delaying the progression of the disease. However, the effect on restoring the visual quality of DR patients is limited, and there are problems such as invasive operation, poor efficacy and high cost. Therefore, it is of great clinical significance to find sensitive and easy-to-use detection markers for the diagnosis and intervention of diabetic retinopathy.

[0003] Secretory proteins refer to proteins that are synthesized inside cells and then secreted outside the cells to function. They mainly include proteins with important physiological functions such as cytokines, growth factors, complement, degradation enzymes, antibodies, peptide hormones, and immunoglobulins. During the occurrence of various diseases, disease-related cells will secrete a variety of proteins such as cytokines and proteolytic enzymes, which participate in regulating the occurrence and development of the disease. Aqueous humor is an intraocular fluid that is in a dynamic circulation. Its composition is closely related to the local physiological and pathological environment of the eye. Aqueous humor can reflect changes in the intraocular microenvironment better than blood. Detecting the expression of secretory proteins in aqueous humor can reflect the state of the disease.

[0004] Trefoil Factor Family (TFF) is a class of small molecule polypeptides containing one or several trefoil factor domains, also known as trefoil peptides. TFF2 is a member of the TFF family and is a secreted protein that is acid-resistant, heat-resistant, and resistant to protease hydrolysis. Current reports show that the expression of TFF2 is significantly increased in patients with tumor infiltration and lymph node and hematogenous metastasis. Continuously secreted TFF2 affects the pathological processes of tumor cell proliferation, migration, and invasion by mediating changes in oncogenes and tumor suppressor genes. However, the role of TFF2 in diabetic retinopathy has not been widely explored. Summary of the invention

[0005] In view of this, the present invention proposes a biomarker TFF2 for diagnosing or assisting in the diagnosis of diabetic retinopathy. The expression level of the biomarker in the aqueous humor of patients with diabetic retinopathy is higher than that of patients without diabetic retinopathy, and the difference is statistically significant. When the expression level of TFF2 is used to detect diabetic retinopathy, the AUC value of its ROC curve is 0.9122, and the diagnostic value is 864.534pg / mL. It can be seen that TFF2, as a diagnostic marker for DR, has high diagnostic sensitivity, specificity and accuracy. Detecting TFF2 in samples can detect DR early and take corresponding treatment measures, which can effectively delay the progression of the disease and reduce the occurrence of complications.

[0006] In in vitro cell experiments, the present invention found that TFF2 can promote the proliferation, migration, tube formation and sprouting of retinal vascular endothelial cells, thereby accelerating the formation of new blood vessels; TFF2 also affects the permeability of endothelial cells, increases the permeability of retinal new blood vessels, and aggravates the progression of the disease. Therefore, TFF2 may become a potential target for DR treatment. By intervening in the expression of TFF2, it is expected to antagonize the angiogenesis-related phenotype of endothelial cells, thereby delaying the occurrence and development of DR.

[0007] The technical solution of the present invention is achieved in this way:

[0008] In a first aspect, the present invention provides a use of a reagent for detecting a biomarker in a sample in the preparation of a product for diagnosing or assisting in the diagnosis of diabetic retinopathy, wherein the biomarker is TFF2.

[0009] In some preferred embodiments, the product includes reagents for detecting the level of the biomarker by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, and mass spectrometry technology.

[0010] In some preferred embodiments, the reagents include one or more of the following: (1) a probe that specifically recognizes the biomarker; (2) a primer that specifically amplifies the biomarker; (3) a binder that specifically binds to the protein encoded by the biomarker.

[0011] Further, in some preferred embodiments, the sample is aqueous humor.

[0012] Aqueous humor is a colorless, transparent liquid in the anterior chamber that provides nutrients and oxygen to intraocular tissues, discharges metabolites, and maintains intraocular pressure. Because aqueous humor is in direct contact with intraocular tissues, it can directly reflect changes in the intraocular microenvironment and better reveal the condition of eye lesions. In addition, under normal circumstances, the composition of aqueous humor is relatively stable and is not easily disturbed by external factors, so the test results are highly accurate.

[0013] In some preferred embodiments, the kit comprises one or more of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, and an electrochemiluminescence detection kit.

[0014] In some preferred embodiments, the kit is selected from an ELISA kit, and the components include: a detection antibody labeled with horseradish peroxidase (HRP), a standard, a color developing solution, and a stop solution.

[0015] Furthermore, in some preferred embodiments, the diabetic retinopathy is proliferative diabetic retinopathy, and the pathological feature is the concomitant retinal neovascularization.

[0016] In some preferred embodiments, the steps of diagnosing or assisting in the diagnosis of diabetic retinopathy include: S1 collecting an aqueous humor sample from the patient to be tested; S2 detecting the protein expression level of the biomarker in the aqueous humor sample. If the protein expression level is higher than 864.534 pg / mL, the patient to be tested has diabetic retinopathy.

[0017] In a second aspect, the present invention provides the use of a reagent for detecting a biomarker in a sample in the preparation of a product for prognostic evaluation of diabetic retinopathy, wherein the biomarker is TFF2, and the prognosis of diabetic retinopathy is evaluated based on changes in the protein expression level of the biomarker, and the sample is aqueous humor.

[0018] In a third aspect, the present invention also provides the use of a TFF2 inhibitor in the preparation of a drug for the treatment and prevention of diabetic retinopathy, characterized in that the drug for the treatment and prevention of diabetic retinopathy is used to inhibit one or more pathological behaviors of endothelial cell proliferation, migration, tube formation, sprouting, and increased permeability, and the diabetic retinopathy is proliferative diabetic retinopathy, and the pathological feature is the accompanying retinal neovascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The results of ELISA test in aqueous humor of patients with diabetic retinopathy and patients without diabetic retinopathy; the horizontal axis represents the aqueous humor samples of different patients, and the vertical axis represents the relative expression level of TFF2;

[0021] Figure 2 This is the ROC analysis result of TFF2 in the diagnosis of diabetic retinopathy;

[0022] Figure 3 The results of ELISA test in aqueous humor of patients with diabetic retinopathy before and after treatment; the horizontal axis represents the aqueous humor samples of different patients, and the vertical axis represents the relative expression level of TFF2;

[0023] Figure 4 The EdU proliferation assay was used to detect the effect of TFF2 on the proliferation of retinal endothelial cells;

[0024] Figure 5 Transwell cell migration assay was used to detect the effect of TFF2 on the migration ability of retinal vascular endothelial cells;

[0025] Figure 6 The effect of TFF2 on the tube-forming ability of retinal endothelial cells was tested in the Matrigel tube-forming assay.

[0026] Figure 7 The 3D endothelial cell spheroid sprouting assay was used to detect the effect of TFF2 on the sprouting ability of retinal vascular endothelial cells;

[0027] Figure 8 EB-Transwell experiment was used to detect the effect of TFF2 on the permeability of retinal vascular endothelial cells. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] Example 1 Application of TFF2 as a biomarker for the diagnosis of diabetic retinopathy

[0031] In this example, aqueous humor samples were collected from 30 patients with diabetic retinopathy and 30 patients without diabetic retinopathy, and the expression level of TFF2 in the aqueous humor samples was detected using an ELISA kit. Then, the expression difference of TFF2 in the aqueous humor of patients with diabetic retinopathy and patients without diabetic retinopathy was statistically analyzed. In addition, the present invention also draws a single ROC curve using GraphPad Prism 9.0 to analyze the specificity, accuracy and sensitivity of TFF2 protein for the diagnosis of diabetic retinopathy. The details are as follows:

[0032] 1.1 Experimental Materials

[0033] The 96-well ELISA plate (8 rows × 12 columns) of TFF2 protein was purchased from Jiangsu ELISA Industrial Co., Ltd.; the TFF2 ELISA kit was purchased from Jiangsu ELISA Industrial Co., Ltd. (Cat No: MM-61899H1), and its reagent components are shown in the following table:

[0034]

[0035] 1.2 Experimental procedures

[0036] (1) Collecting aqueous humor samples

[0037] Aqueous humor was collected from 30 patients with diabetic retinopathy, and aqueous humor was collected from 30 patients without diabetic retinopathy (control group). Among them, the aqueous humor of 30 patients with diabetic retinopathy came from patients diagnosed by imaging; the aqueous humor of 30 patients without diabetic retinopathy came from patients without any fundus lesions. The collected aqueous humor was placed in a 1.5mL EP tube, and the sample number was marked on the top and side of the EP tube. It was frozen in a -80℃ refrigerator and the collection date and storage location were recorded. Before use, the aqueous humor was taken out and thawed in a 4℃ refrigerator and aliquoted to avoid repeated freezing and thawing.

[0038] (2) ELISA detection of TFF2 protein content

[0039] A) Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well.

[0040] B) First add 10 μL of the sample to be tested into the sample well, and then add 40 μL of sample diluent.

[0041] C) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well and seal the wells with a sealing film. D) Incubate the wells at 37°C in a water bath or incubator for 60 min.

[0042] E) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0043] F) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0044] G) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0045] (3) Statistical analysis

[0046] Draw a standard curve: In an Excel worksheet, use the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve of the standard, and calculate the concentration value of TFF2 in each sample according to the curve equation. The t-test method was used to analyze whether there was a significant difference in the TFF2 protein level between the diabetic retinopathy group and the non-diabetic retinopathy group. The test results are as follows Figure 1 shown.

[0047] (4) ROC curve analysis

[0048] GraphPad Prism 9.0 was used to draw ROC curves to analyze the value of TFF2 protein in the diagnosis of diabetic retinopathy. Based on the expression level of TFF2 protein in aqueous humor samples of 30 patients with diabetic retinopathy and 30 patients without diabetic retinopathy, ROC curves were drawn to evaluate the ability of TFF2 to distinguish between patients with diabetic retinopathy and those without diabetic retinopathy.

[0049] The ROC curve for distinguishing patients with diabetic retinopathy from those without diabetic retinopathy based on TFF2 protein diagnosis was used as the cutoff value (i.e., diagnostic value) with the maximum OD value of Youden index. The corresponding AUC and 95% confidence interval, sensitivity, and specificity were calculated at the same time. Figure 2 shown.

[0050] 1.3 Results Analysis

[0051] (1) Figure 1 As shown, aqueous humor of patients with diabetic retinopathy and non-diabetic retinopathy was collected, and ELISA was used to detect the difference in TFF2 protein levels. The results showed that compared with patients without diabetic retinopathy, the TFF2 protein level in the aqueous humor of patients with diabetic retinopathy was significantly increased, indicating that the expression level of TFF2 is closely linked to diabetic retinopathy and can be used as a biomarker for the diagnosis of diabetic retinopathy.

[0052] (2) Figure 2 As shown, when TFF2 is used to diagnose diabetic retinopathy, the diagnostic value is 864.534 pg / mL, and the AUC value of the ROC curve is 0.9122. The results show that the expression level of TFF2 in human aqueous humor has very high specificity, accuracy and sensitivity in the diagnosis of diabetic retinopathy.

[0053] Example 2 Application of TFF2 as a biomarker for prognostic assessment of diabetic retinopathy

[0054] In this example, aqueous humor of 20 patients with diabetic retinopathy was collected before and after treatment, and the expression level of TFF2 in the aqueous humor samples was detected using an ELISA kit. Then, the expression difference of TFF2 in the aqueous humor of patients with diabetic retinopathy before and after treatment was statistically analyzed.

[0055] 2.1 Experimental Materials

[0056] The experimental materials of this embodiment are the same as the experimental materials 1.1 in embodiment 1.

[0057] 2.2 Experimental procedures

[0058] (1) Collecting aqueous humor samples

[0059] Aqueous humor was collected from 20 patients with diabetic retinopathy before and after treatment. The collected aqueous humor was placed in a 1.5 mL EP tube, the sample number was marked on the top and side of the EP tube, and it was frozen in a -80°C refrigerator. The collection date and storage location were recorded. Before use, the aqueous humor was taken out and thawed in a 4°C refrigerator and aliquoted to avoid repeated freezing and thawing.

[0060] (2) ELISA detection of TFF2 protein content

[0061] A) Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well.

[0062] B) First add 10 μL of the sample to be tested into the sample well, and then add 40 μL of sample diluent.

[0063] C) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well and seal the wells with a sealing film. D) Incubate the wells at 37°C in a water bath or incubator for 60 min.

[0064] D) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0065] E) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0066] F) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0067] (3) Statistical analysis

[0068] Draw a standard curve: In an Excel worksheet, use the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve of the standard, and calculate the concentration value of TFF2 in each sample according to the curve equation. The t-test method was used to analyze whether there was a significant difference in the TFF2 protein level in the aqueous humor of patients with diabetic retinopathy before and after treatment. The test results are as follows Figure 3 shown.

[0069] 2.3 Results Analysis

[0070] Compared with before treatment, Figure 3 As shown in the figure, the level of TFF2 protein in the aqueous humor of patients with diabetic retinopathy decreased after treatment, indicating that TFF2 protein is expected to become a biomarker for evaluating the prognosis of patients with diabetic retinopathy.

[0071] Example 3 Study on the mechanism of action of TFF2 in diabetic retinopathy

[0072] Experimental Materials

[0073] Retinal vascular endothelial cells (HRVECs) were purchased from ATCC Cell Bank, USA; DMEM medium (Cat No: C11995) was purchased from Gibco Biotechnology, USA; fetal bovine serum (Cat No: FBS500-s) was purchased from AusGeneX, Australia; recombinant TFF2 protein (Cat No: HY-P71929A) was purchased from MCE, USA; trypsin-EDTA (Cat No: 25200114) was purchased from Thermo Fisher Scientific, USA; BeyoClick TM EdU-488 cell proliferation detection kit (Cat No: C0075S) was purchased from China Biotech Co., Ltd.; crystal violet (Cat No: C805211) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; Matrigel Matrix Basement Membrance (Cat No: 356234) was purchased from Corning Incorporated; carboxymethyl cellulose (Cat No: 419273) was purchased from Sigma Biotechnology Co., Ltd. Type I rat tail collagen (Cat No: 354236) was purchased from Corning Incorporated; Evans Blue (Cat No: K29136) was purchased from Beijing Keerhui.

[0074] (I) EdU cell proliferation assay to detect the effect of TFF2 on the proliferation of retinal vascular endothelial cells

[0075] (1) Experimental procedures

[0076] A) HRVECs were seeded in 24-well plates. When the cell confluence reached about 60%, the treatment group was stimulated with TFF2 recombinant protein solution for 48 hours, while the control group was not treated with any measures;

[0077] B) discard the old culture medium, wash twice with PBS buffer, add 5-ethynyl-2′deoxyuridine (EdU) working solution (EdU stock solution: complete culture medium = 1:1000) into the 24-well plate at a volume of 1 mL / well, and incubate for 2-4 hours;

[0078] C) After the incubation, discard the EdU working solution, add 1 mL of 4% paraformaldehyde to each well, and fix at room temperature for 15 minutes;

[0079] D) Discard the paraformaldehyde, add 500 μL of washing solution (PBS containing 3% BSA) to each well, and wash three times, each time for 3-5 minutes;

[0080] E) Discard the washing solution, add 500 μL of permeabilization solution (PBS containing 0.3% Triton X-100) to each well, and incubate at room temperature for 10-15 minutes;

[0081] F) Discard the permeabilization solution and add 500 μL of washing solution to each well and wash 1-2 times, each time for 3-5 minutes;

[0082] G) Discard the washing solution, prepare the Click reaction solution according to the manufacturer's instructions, add 200 μL Click reaction solution to each well, and incubate at room temperature in the dark for 30 minutes;

[0083] H) Discard the reaction solution and add 500 μL of washing solution to each well and wash 1-2 times, each time for 3-5 minutes;

[0084] I) discard the washing solution, add 200 μL DAPI to each well, and incubate at room temperature in the dark for 10 min;

[0085] J) Discard the DAPI staining solution and add 500 mL PBST to each well and wash three times, each time for 3-5 minutes.

[0086] K) Observation and recording using a fluorescence microscope.

[0087] (2) Experimental results

[0088] like Figure 4 As shown in the figure, after TFF2 treatment, the cell proliferation rate in the treatment group was higher than that in the control group. TFF2 has the effect of promoting the proliferation of HRVECs.

[0089] (II) Transwell cell migration assay to detect the effect of TFF2 on retinal vascular endothelial cell migration

[0090] (1) Experimental procedures

[0091] A) HRVECs were seeded in 24-well plates. When the cell confluence reached about 60%, the treatment group was stimulated with TFF2 recombinant protein solution for 48 hours, while the control group was not treated with any measures;

[0092] B) Preparation of 0.2% crystal violet solution: weigh 0.08 g of crystal violet powder and dissolve it in 40 mL of PBS, shake overnight, filter twice with filter paper and then use for staining;

[0093] C) The treated HRVECs were collected and resuspended in DMEM without fetal bovine serum (FBS) to adjust the cell density to 1×10 5 Pieces / mL;

[0094] D) Add 500 μL of DMEM complete medium containing 10% FBS to the lower chamber of the 24-well plate, select a chamber with a pore size of 8.0 μm and place it in the 24-well plate, remove the bubbles between the chamber and the liquid, and add 200 μL of cell suspension to the upper chamber, taking care to avoid the generation of bubbles;

[0095] E) Continue culturing in a CO2 incubator for 24 h, and gently wipe off the unmigrated cells in the upper chamber with a cotton swab;

[0096] F) Add 500 μL of 4% paraformaldehyde to a clean well of a 24-well plate, place the chamber in it and fix for 15 min, take out the chamber and invert it on the table, and air dry for 20 min;

[0097] G) Discard the fixative solution, add 500 μL of 0.2% crystal violet solution, and stain for 5 min;

[0098] H) Discard the crystal violet, add 500 μL PBS, and wash three times, 2 min each time;

[0099] I) Gently wipe the upper chamber with a cotton swab to remove the crystal violet that has not passed through the upper chamber, and observe and record using an inverted microscope.

[0100] (2) Experimental results

[0101] like Figure 5 As shown in the figure, after TFF2 treatment, the number of cells migrating in the treatment group was higher than that in the control group. TFF2 has the effect of promoting the migration of HRVECs.

[0102] (III) Matrigel tube formation assay to detect the effect of TFF2 on retinal vascular endothelial cell tube formation

[0103] (1) Experimental procedures

[0104] A) HRVECs were seeded in 24-well plates. When the cell confluence reached about 60%, the treatment group was stimulated with TFF2 recombinant protein solution for 48 hours, while the control group was not treated with any measures;

[0105] B) Take out the aliquoted Matrigel Matrix from -20℃, put it into 4℃ crushed ice to melt, and pre-cool the 24-well plate and pipette tip at -20℃;

[0106] C) Take out the matrix gel, 24-well plate and pipette tip from the 4°C refrigerator and place them on crushed ice in the clean bench. Rinse the 24-well plate with cold PBS, add 50 μL of matrix gel Matrigel Matrix to each well, avoid bubbles, and incubate at 37°C for 30 min.

[0107] D) Collect the treated HRVECs and adjust the cell density to 8×10 4 Pieces / mL;

[0108] E) Add 1 mL of cell suspension to a 24-well plate pre-coated with matrix gel, incubate at 37°C for 4 h, and observe and record under an inverted microscope.

[0109] (2) Experimental results

[0110] like Figure 6 As shown in the figure, after TFF2 treatment, the ability of cell tube formation in the treated group was stronger than that in the control group. TFF2 has the effect of promoting HRVECs tube formation.

[0111] (IV) 3D endothelial cell spheroid sprouting assay to detect the effect of TFF2 on retinal vascular endothelial cell sprouting

[0112] (1) Experimental procedures

[0113] A) HRVECs were seeded in 24-well plates. When the cell confluence reached about 60%, the treatment group was stimulated with TFF2 recombinant protein solution for 48 hours, while the control group was not treated with any measures;

[0114] B) Preparation of methylcellulose stock solution: 3 g of methylcellulose was weighed and added to a 500 mL culture flask, and a clean magnetic stirring rotor was added. The flask was sterilized by autoclaving at 121°C for 20 min, followed by the addition of 250 mL of DMEM preheated to 60°C, stirred at room temperature for 20 min, shaken at 4°C overnight, and centrifuged at 5000 rpm for 30 min. The upper layer was collected for cell sphere culture;

[0115] C) Collect the treated HRVECs and adjust the cell density to 2×10 4 / mL, mix the cell suspension with methylcellulose stock solution (mixing ratio 4:1), and inoculate 25μL / drop on the cover of the culture dish, taking care to avoid bubbles;

[0116] D) Incubate inverted in a CO2 incubator for 24 h, use a pipette to draw 10 mL of PBS to gently wash away the suspended spheres, transfer the liquid to a 15 mL centrifuge tube, centrifuge at 800 rpm for 3 min to precipitate the cell spheres, discard the upper layer, add 2 mL of methylcellulose stock solution containing 20% ​​FBS and 2 mL of collagen stock solution, and mix carefully to avoid generating bubbles;

[0117] E) The cell spheroid mixture was added into a 24-well plate at a volume of 1 mL / well and incubated in a CO2 incubator for 30 min to promote collagen polymerization.

[0118] F) After the collagen is polymerized, add 200 μL of DMEM to each well and continue incubation for 24 h.

[0119] (2) Experimental results

[0120] like Figure 7 As shown in the figure, after TFF2 treatment, the sprouting ability of cells in the treated group was stronger than that in the control group. TFF2 has the effect of promoting the sprouting of HRVECs.

[0121] (V) Evans Blue-Transwell assay to detect the effect of TFF2 on the permeability of retinal endothelial cells

[0122] (1) Experimental procedures

[0123] A) Prepare 4% bovine serum albumin (BSA) solution and 2% Evans blue (EB) solution in advance.

[0124] B) HRVECs were inoculated into 24-well plates. When the cell confluence reached about 60%, the treated group was stimulated with TFF2 recombinant protein solution for 48 hours, while the control group was not treated with any measures;

[0125] C) Collect the treated HRVECs, place a Transwell chamber with a pore size of 0.4 μm on a 24-well plate, add 100 μL of cell suspension to the upper chamber for culture, and add 600 μL of 10% FBS to the lower chamber;

[0126] D) After 8 h, the liquid in the chamber was removed, 100 μL of EBA solution was added to the upper chamber, and 600 μL of 4% BSA solution was added to the lower chamber;

[0127] E) After incubation in the dark for 1 hour, the chamber was removed and the OD value of the solution in the lower chamber was measured using an ELISA reader at a wavelength of 595 nm.

[0128] (2) Experimental results

[0129] like Figure 8 As shown in the figure, after TFF2 treatment, the cell permeability of the treated group was higher than that of the control group. TFF2 has the effect of promoting the permeability of HRVECs.

[0130] In summary, in in vitro cell experiments, TFF2 promotes the formation of new blood vessels by regulating the ability to promote the proliferation, migration, tube formation and sprouting of retinal vascular endothelial cells; TFF2 also increases the permeability of endothelial cells, exacerbating the progression of retinal lesions. Therefore, TFF2 may become a potential target for the treatment of DR. By intervening in the expression of TFF2, it is expected to antagonize the angiogenesis-related phenotype of endothelial cells, thereby delaying the occurrence and development of DR.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting biomarkers in a sample in the preparation of a product for diagnosing or assisting in the diagnosis of diabetic retinopathy, characterized in that: The biomarker is TFF2.

2. The use according to claim 1, characterized in that: The product includes reagents for detecting the level of the biomarker through sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, and mass spectrometry technology.

3. The use according to claim 2, characterized in that: The reagents include one or more of the following: (1) a probe that specifically recognizes the biomarker; (2) a primer that specifically amplifies the biomarker; (3) a binding agent that specifically binds to a protein encoded by the biomarker.

4. The use according to any one of claims 1 to 3, characterized in that: The sample was collected from the aqueous humor.

5. The use according to claim 1, characterized in that: The product for diagnosing or assisting in the diagnosis of diabetic retinopathy is a kit, which includes one or more of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit and an electrochemiluminescence detection kit.

6. The use according to any one of claims 1 to 3 and 5, characterized in that: The diabetic retinopathy is proliferative diabetic retinopathy, and the pathological feature is the accompanying retinal neovascularization.

Images