IGFBP3 protein as cataract marker and application thereof

Abstract

The invention discloses IGFBP3 protein as a cataract marker and application of the IGFBP3 protein, the cataract biomarker is IGFBP3 protein, and the expression level of the IGFBP3 protein in cataract patients is increased. According to the IGFBP3 protein as the cataract marker and the application of the IGFBP3 protein, the IGFBP3 protein is applied to clinic, early diagnosis of cataract can be achieved, and the IGFBP3 protein has important significance in preventing the blinding rate of patients. Through early recognition and intervention, visual impairment caused by cataract can be reduced, so that the life quality of a patient is improved. In addition, the detection of the IGFBP3 protein can help to evaluate disease progression and treatment effect, and provides important reference basis for clinical treatment. The introduction of the detection method not only can improve the diagnosis accuracy of cataract, but also can bring a more personalized treatment scheme for the patient, thereby improving the long-term prognosis and life quality of the patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, 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 according to their causes. Age-related cataracts are the most common type in adults, with the onset age between 45 and 50 years old. Lens opacity is the most prominent feature of cataracts. Lens epithelial cells are the most metabolically active cells in the lens, undergoing a series of reactions such as oxidation, insolubility and cross-linking, which ultimately lead to lens opacity.

[0003] At present, the exploration of basic and clinical research on cataracts is increasing, but its pathogenesis is still not very clear, and many patients are already in the middle and late stages when diagnosed. There is no significant progress in clinical treatment except surgery, and the current situation is still not optimistic. Therefore, further exploring the relevant mechanisms of cataract occurrence and development, and finding new markers and therapeutic targets have become urgent issues to be solved. 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 is 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: 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.

[0006] A reagent for detecting a biomarker, the reagent 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.

[0007] A reagent for detecting a biomarker is used in the preparation of a product for cataract diagnosis and / or prognosis assessment, wherein the biomarker is IGFBP3 protein.

[0008] Preferably, in the above technical solution, the reagent for detecting the biomarker is a reagent for detecting the expression level of the IGFBP3 protein.

[0009] 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.

[0010] Preferably, in the above technical solution, the product is a kit, a medicine or a device.

[0011] An inhibitor for inhibiting the expression or activity of IGFBP3 protein.

[0012] 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 amount of IGFBP3.

[0013] Use of an inhibitor in preparing a product for preventing and / or treating cataract.

[0014] A pharmaceutical composition comprising an inhibitor.

[0015] The above technical solution of the present invention has the following beneficial effects: The IGFBP3 protein provided in the present application is used as a cataract marker and its application. When applied to 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, providing an important reference 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0017] Figure 1 To detect the expression level of IGFBP3 protein in lens epithelial cells; Figure 2 To detect the expression level of IGFBP3 protein in rat lens; Figure 3 To detect the expression level of IGFBP3 protein in the aqueous humor of 100 individuals with different diseases. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that 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 unless otherwise specifically stated.

[0019] 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.

[0020] The animals, cells and clinical samples involved in the experiments of the present invention are as follows: (1) Experimental animals: SD rats (weight 200 g, 8 weeks old, male SD rats, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.); (2) Cells: SRA01 / 04 human lens epithelial cells (purchased from Shanghai Xinyu Biotechnology Co., Ltd.); (3) Clinical samples: aqueous humor of individuals with different diseases (transparent lens group, age-related cataract group, and diabetic cataract group).

[0021] Experimental methods: Cell experiment group: (1) Control group: DMSO was added; (2) High glucose group: DMSO and 50 mM D-glucose were added.

[0022] Animal experiment grouping: 30 mice were equally divided into two groups, with 15 mice in each group.

[0023] (1) Control group: Rats were fasted for 16 h and intraperitoneally injected with 0.1 mol / L citrate-phosphate buffer, pH 4.5.

[0024] (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.

[0025] Clinical sample grouping: (1) CN (transparent crystal group); (2) AC (age-related cataract group); (3) (diabetic cataract group).

[0026] The experimental process is described in detail below.

[0027] Example 1 Cell experiment Human lens epithelial cells in the logarithmic growth phase were seeded in six-well plates (seeding density was about 50%) and cultured overnight in a cell incubator at 37°C with 5% CO2. After 24 hours, different drugs were added for treatment according to the groups.

[0028] The specific groups are: (1) Control group: DMSO was added; (2) High glucose group: DMSO and 50 mM D-glucose were added.

[0029] After the cells of each treatment group were cultured for 24 hours at 37°C in an incubator containing 5% CO2, the culture medium was discarded, 1 ml of pre-cooled PBS was added for washing 3 times, and 100 μl of lysis solution containing PMSF was added to each well after the PBS was aspirated (10 μl of PMSF was added to each 1 ml of lysis solution). The cells were scraped on one side of the culture plate with a clean cell scraper, and then the cell fragments and lysis solution were transferred to a 1.5 ml centrifuge tube with a pipette, and placed on ice for lysis for 30 minutes (the vortex mixer was shaken for 30 seconds every 5 minutes to ensure complete cell lysis); after sufficient lysis, the cells were centrifuged at 12000g and 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.

[0030] The protein concentration was determined by the BCA method, and the corresponding volume of 20ug 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 ul protein sample), and the protein sample was obtained by incubating at 98℃ metal bath for 10min. 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.

[0031] The results are as follows Figure 1 As shown, Figure 1 The results of immunoblotting experiments of lens epithelial cells in different groups. The expression of IGFBP3 protein was detected by protein immunoblotting. Compared with the control group, the expression level of IGFBP3 in the high glucose group was significantly increased, while the expression level of β-actin did not change significantly. The above results show that under high glucose stimulation, the expression of IGFBP3 in lens epithelial cells is significantly increased.

[0032] Example 2 Animal Experiment SD rats were killed by overdose of anesthesia 12 weeks after the diabetic model was established. The eyeballs were removed and the lenses were taken out. 200ul of lysis solution containing PMSF was added (10μl of PMSF was added to every 1ml of lysis solution), and the rats were fully ground with a tissue grinder and placed on ice for lysis for 30min (vortex mixer was oscillated 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 cell protein product.

[0033] The protein concentration was determined by BCA method, and the corresponding volume of 20ug protein was calculated and taken out as the sample volume for western blot experiment. Then 5*SDS-PAGE protein loading buffer was added (1 μl protein loading buffer was added for every 4 ul protein sample) and the protein sample was obtained by metal bath at 98℃ for 10min. The samples of SD rats in the control group were obtained by the same method. The samples were used for western blot experiment to detect the protein expression level of IGFBP3 in the lens of rats in each group.

[0034] The results are as follows Figure 2 As shown, Figure 2 The results of the lens tissue immunoblotting experiment of SD rat proteins in different groups. The expression of IGFBP3 protein was detected by western blotting. Compared with the control group rats, the expression level of IGFBP3 in the diabetic group rats was significantly increased, while the expression level of β-actin did not change significantly. The above results show that the expression of IGFBP3 in the lens of SD rats is significantly increased under the condition of diabetes.

[0035] Example 3 Enzyme-linked immunosorbent assay Aqueous humor samples of 100 patients with different types of eye diseases (13 patients in the transparent lens group, 59 patients in the age-related cataract group, and 28 patients in the diabetic cataract group) were collected for follow-up testing.

[0036] Follow these steps: (1) Dilution of standard sample.

[0037] (2) Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, 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 well, and then add 10 μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0038] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0039] (4) Solution preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0040] (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. Repeat this 5 times and pat dry.

[0041] (6) Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well.

[0042] (7) Incubation: Same operation as (3).

[0043] (8) Washing: Same operation as (5).

[0044] (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.

[0045] (10) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0046] Determination: Use the blank well to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution. Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to draw a standard curve on the coordinate paper. According to the OD value of the sample, find the corresponding concentration from the standard curve; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample. Perform statistical analysis on the final results of different groups of samples.

[0047] The results are as follows Figure 3 As shown, Figure 3 The enzyme-linked immunosorbent assay (ELISA) experiment was performed on the aqueous humor of the eyes of patients with different groups of diseases. The content of IGFBP3 in the aqueous humor of the eyes was detected by ELISA. Compared with the transparent lens group, the content of IGFBP3 in the aqueous humor of the patients in the age-related cataract group was increased, while the content of IGFBP3 in the aqueous humor of the patients in the diabetic cataract group was higher than that in the other two groups. These results show that the expression of IGFBP3 in different patients is significantly different and can be used as a marker to distinguish different cataracts.

[0048] Although the present invention has been disclosed as above by the embodiments, it is 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 protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A cataract biomarker, characterized in that: The cataract biomarker is IGFBP3 protein, and the expression level of the IGFBP3 protein is increased in cataract patients.

2. A reagent for detecting a biomarker, characterized in that: The reagent is used to detect the expression level of IGFBP3 protein, and contains an antibody that can specifically bind to IGFBP3 protein, and the antibody is a monoclonal antibody or a polyclonal antibody.

3. 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.

4. The use according to claim 3, characterized in that: The reagent for detecting the biomarker is a reagent for detecting the expression level of the IGFBP3 protein.

5. The use according to claim 3, characterized in that: The cataract is diabetic cataract or age-related cataract, and the expression level of the IGFBP3 protein is increased in cataract patients.

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

7. An inhibitor, characterized in that The inhibitor is used to inhibit the expression or activity of IGFBP3 protein.

8. The inhibitor according to claim 7, characterized in that The inhibitor includes an IGFBP3 antibody, a small molecule compound or a natural drug that can reduce the expression amount of IGFBP3.

9. Use of the inhibitor according to claim 7 in the preparation of a product for preventing and / or treating cataracts.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the inhibitor according to any one of claims 7-8.

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