IGFBP3 protein as cataract marker and its application

Abstract

The present invention discloses an IGFBP3 protein as a cataract marker and its application. The cataract biomarker is IGFBP3 protein, and the expression level of IGFBP3 protein is increased in cataract patients. The IGFBP3 protein provided in this application is used as a cataract marker and its application. When applied in clinical practice, it can achieve early diagnosis of cataracts, which is of great significance for preventing blindness in patients. Through early identification and intervention, visual impairment caused by cataracts can be reduced, thereby improving the quality of life of patients. In addition, the detection of IGFBP3 protein can also help evaluate the progression of the disease and the effect of treatment, and provide an important reference basis for clinical treatment. The introduction of this detection method can not only improve the diagnostic accuracy of cataracts, but also provide patients with more personalized treatment plans, thereby improving the long-term prognosis and quality of life of patients.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an IGFBP3 protein as a cataract marker and application thereof. Background Art

[0002] Cataracts can be divided into age-related cataracts, pediatric cataracts, and secondary cataracts based on their etiology. Age-related cataracts are the most common type in adults, with onset occurring between the ages of 45 and 50. Lens opacity is the primary characteristic of cataracts. Lens epithelial cells are the most metabolically active cells in the lens, undergoing a series of reactions including oxidation, insolubilization, and cross-linking, ultimately leading to lens opacity.

[0003] While basic and clinical research on cataracts continues to grow, its pathogenesis remains unclear, and many patients are diagnosed in the advanced stages. Clinical treatments other than surgery have seen limited progress, and the current situation remains grim. Therefore, further exploration of the mechanisms underlying cataract development and progression, as well as the identification of novel biomarkers and therapeutic targets, are pressing challenges. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an IGFBP3 protein as a cataract marker and its application, which can be applied in clinical practice to achieve early diagnosis of cataracts, reduce the blindness rate of patients and improve the quality of life.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A cataract biomarker is provided, wherein the cataract biomarker is IGFBP3 protein, and the expression level of the IGFBP3 protein is increased in cataract patients.

[0007] A reagent for detecting a biomarker, which is used to detect the expression level of an IGFBP3 protein. The reagent contains an antibody that can specifically bind to the IGFBP3 protein, and the antibody is a monoclonal antibody or a polyclonal antibody.

[0008] A use of a reagent for detecting a biomarker in the preparation of a product for cataract diagnosis and / or prognosis assessment, wherein the biomarker is IGFBP3 protein.

[0009] Preferably, in the above technical solution, the reagent for detecting the biomarker is a reagent for detecting the expression level of the IGFBP3 protein.

[0010] Preferably, in the above technical solution, the cataract is diabetic cataract or age-related cataract, and the expression level of the IGFBP3 protein is increased in cataract patients.

[0011] Preferably, in the above technical solution, the product is a kit, a drug or a device.

[0012] An inhibitor for inhibiting the expression or activity of IGFBP3 protein.

[0013] Preferably, in the above technical solution, the inhibitor includes an IGFBP3 antibody, a small molecule compound or a natural medicine that can reduce the expression level of IGFBP3.

[0014] Use of an inhibitor in preparing a product for preventing and / or treating cataracts.

[0015] A pharmaceutical composition comprising an inhibitor.

[0016] The above technical solution of the present invention has the following beneficial effects:

[0017] The IGFBP3 protein provided in this application is used as a cataract marker and its application in clinical practice, which can achieve early diagnosis of cataracts, which is of great significance for preventing blindness in patients. Through early identification and intervention, visual impairment caused by cataracts can be reduced, thereby improving the quality of life of patients. In addition, the detection of IGFBP3 protein can also help assess disease progression and treatment effects, providing an important reference basis for clinical treatment. The introduction of this detection method can not only improve the diagnostic accuracy of cataracts, but also provide patients with more personalized treatment plans, thereby improving their long-term prognosis and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0019] Figure 1 To detect the expression level of IGFBP3 protein in lens epithelial cells;

[0020] Figure 2 To detect the expression level of IGFBP3 protein in rat lens;

[0021] Figure 3 To detect the expression level of IGFBP3 protein in the aqueous humor of 100 individuals with different diseases. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention.

[0023] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.

[0024] The animals, cells, and clinical samples involved in the experiments of this invention are as follows:

[0025] (1) Experimental animals: SD rats (200 g, 8-week-old, male SD rats, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.);

[0026] (2) Cells: SRA01 / 04 human lens epithelial cells (purchased from Shanghai Xinyu Biotechnology Co., Ltd.);

[0027] (3) Clinical samples: aqueous humor of individuals with different diseases (clear lens group, age-related cataract group, diabetic cataract group).

[0028] Experimental methods:

[0029] Cell experiment group:

[0030] (1) Control group: DMSO was added;

[0031] (2) High glucose group: DMSO and 50 mM D-glucose were added.

[0032] Animal experimental groups: 30 mice were divided into two groups, with 15 mice in each group.

[0033] (1) Control group: Rats were fasted for 16 h and intraperitoneally injected with 0.1 mol / L citrate-phosphate buffer, pH 4.5.

[0034] (2) Diabetic group: Rats were fasted for 16 h, intraperitoneally injected with freshly prepared streptozotocin (STZ, Sigma Chemical Co, St Louis, MO, USA) (65 mg / kg), and then gavaged with normal saline for 12 weeks.

[0035] Clinical sample grouping:

[0036] (1) CN (transparent crystal group);

[0037] (2) AC (age-related cataract group);

[0038] (3) (diabetic cataract group).

[0039] The following is a detailed description of the experimental process.

[0040] Example 1 Cell experiment

[0041] Human lens epithelial cells in logarithmic growth phase were seeded into six-well plates (seeding density approximately 50%) and cultured overnight in a 37°C incubator with 5% CO₂. After 24 hours, different drugs were added according to group.

[0042] The specific groups are:

[0043] (1) Control group: DMSO was added;

[0044] (2) High glucose group: DMSO and 50 mM D-glucose were added.

[0045] After incubating cells in each treatment group for 24 hours at 37°C in a 5% CO2 incubator, the culture medium was discarded and the cells were washed three times with 1 ml of pre-chilled PBS. After aspirating the PBS, 100 μl of lysis buffer containing PMSF was added to each well (10 μl of PMSF per 1 ml of lysis buffer). Using a clean cell scraper, the cells were scraped to the side of the culture plate. Cell debris and lysis buffer were then pipetted into a 1.5 ml centrifuge tube and lysed on ice for 30 minutes (vortexing the tube for 30 seconds every 5 minutes to ensure complete cell lysis). After complete lysis, the cells were centrifuged at 12,000 g at 4°C for 15 minutes, and the supernatant was transferred to a new 1.5 ml centrifuge tube to obtain the total cell protein product.

[0046] The protein concentration was determined by the BCA method, and the corresponding volume of 20 μg of protein was calculated and taken out as the sample volume for protein immunoblotting experiment. Then, 5*SDS-PAGE protein loading buffer was added (1 μl protein loading buffer was added for every 4 μl protein sample) and the protein sample was obtained by incubating at 98°C in a metal bath for 10 min. The protein sample was then used for protein immunoblotting experiment to detect the protein expression level of IGFBP3 in human lens epithelial cells in each group.

[0047] The results are as follows Figure 1 As shown, Figure 1 The following are the results of immunoblotting experiments on lens epithelial cells from different groups. IGFBP3 protein expression was detected by Western blotting. Compared with the control group, IGFBP3 expression levels were significantly increased in the high glucose group, while β-actin expression levels remained unchanged. These results indicate that high glucose stimulation significantly increases IGFBP3 expression in lens epithelial cells.

[0048] Example 2 Animal Experiment

[0049] Twelve weeks after the SD rats were induced to have diabetes, they were killed by overdose of anesthesia, their eyes were removed and the lenses were taken out. 200ul of lysis buffer containing PMSF (10μl of PMSF was added to every 1ml of lysis buffer) was added and the rats were fully ground with a tissue grinder and lysed on ice for 30min (vortex mixer was shaken for 30s every 5min). After full lysis, the rats were centrifuged at 12000g and 4℃ for 15min, and the supernatant was transferred to a new 1.5ml centrifuge tube to obtain the total cellular protein product.

[0050] Protein concentration was determined using the BCA assay. A volume corresponding to 20 μg of protein was calculated and used as the sample for western blotting. 5×SDS-PAGE protein loading buffer was then added (1 μl of protein loading buffer per 4 μl of protein sample) and incubated at 98°C for 10 minutes. Samples were obtained from the SD rats in the control group using the same method. These samples were then used for western blotting to determine IGFBP3 protein expression levels in the lenses of the rats in each group.

[0051] The results are as follows Figure 2 As shown, Figure 2 Results of a protein immunoblotting experiment on lens tissue from different groups of SD rats. IGFBP3 protein expression was assessed by Western blotting. Compared with the control group, IGFBP3 expression levels were significantly elevated in the diabetic rats, while β-actin expression levels remained unchanged. These results demonstrate that IGFBP3 expression is significantly elevated in the lens of SD rats under diabetic conditions.

[0052] Example 3 Enzyme-linked immunosorbent assay

[0053] Aqueous humor samples from 100 patients with different types of eye conditions (13 patients in the transparent lens group, 59 patients in the age-related cataract group, and 28 patients in the diabetic cataract group) who visited the ophthalmology department were collected for follow-up testing.

[0054] Follow these steps:

[0055] (1) Dilution of the standard.

[0056] (2) Sample addition: Set up blank wells (blank control wells do not contain sample and enzyme-labeled reagent, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50 μl of the standard sample to the enzyme-labeled plate. First add 40 μl of sample diluent to the test sample wells, and then add 10 μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the plate well, trying not to touch the well wall, and gently shake to mix.

[0057] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0058] (4) Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0059] (5) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry.

[0060] (6) Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well.

[0061] (7) Incubation: The operation is the same as (3).

[0062] (8) Washing: The operation is the same as (5).

[0063] (9) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and incubate at 37°C in the dark for 10 minutes.

[0064] (10) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0065] Assay: Use a blank well to zero the sample and measure the absorbance (OD) of each well sequentially at 450 nm. Measurements should be performed within 15 minutes after adding the stop solution. Plot a standard curve on graph paper, with the standard concentration as the horizontal axis and the OD value as the vertical axis. Use the standard curve to determine the corresponding concentration based on the sample OD value; then multiply by the dilution factor. Alternatively, calculate the linear regression equation for the standard curve using the standard concentration and OD value. Substitute the sample OD value into the equation to calculate the sample concentration, which will then be multiplied by the dilution factor to determine the actual sample concentration. Statistically analyze the final results for different sample groups.

[0066] The results are as follows Figure 3 As shown, Figure 3 Enzyme-linked immunosorbent assay (ELISA) was performed on the aqueous humor of patients with different disease groups. IGFBP3 levels were measured by ELISA. Compared with the crystalline lens group, IGFBP3 levels were elevated in the age-related cataract group, while those with diabetic cataract had even higher levels compared to the other two groups. These results suggest that IGFBP3 expression varies significantly among patients and can serve as a biomarker for distinguishing different types of cataract.

[0067] Although the present invention has been disclosed above with reference to the embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. Use of a reagent for detecting a biomarker in the preparation of a product for cataract diagnosis and / or prognosis assessment, characterized in that: The biomarker is IGFBP3 protein, the expression level of the IGFBP3 protein is increased in cataract patients, and the cataract is age-related cataract.

2. The use according to claim 1, characterized in that The reagent for detecting the biomarker is a reagent for detecting the expression level of the IGFBP3 protein.

3. The use according to claim 1, characterized in that The product is a kit, a drug or a device.

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