Oral cancer screening and prognosis kit based on detection of phosphorylated Cofilin protein expression level and application of oral cancer screening and prognosis kit

Abstract

The invention relates to the field of biomedicine, in particular to an oral cancer screening and prognosis kit based on detection of phosphorylated Cofilin protein expression level and application of the oral cancer screening and prognosis kit. The kit contains xylene, alcohol, a citrate buffer solution, hydrogen peroxide, a confining liquid, a primary antibody, a secondary antibody, a DAB developing solution, hematoxylin and neutral gum. According to the kit, the malignant process of the oral squamous cell carcinoma is judged by detecting the expression level of the phosphorylated Cofilin protein in the oral mucosa tissue of a human body, the kit is used for auxiliary diagnosis of clinical oral squamous cell carcinoma, an effective theoretical basis is provided for taking targeted blocking measures or decisions for patients, and the clinical application and transformation prospects are good.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an oral cancer screening and prognosis kit based on detecting the expression level of phosphorylated Cofilin protein and its application. Background Art

[0002] Oral cancer, primarily oral squamous cell carcinoma (OSCC), is the sixth most common cancer worldwide, with a five-year survival rate of approximately 50%. In recent years, its incidence and younger onset have been gradually increasing. This is primarily due to factors such as late detection, acquired drug resistance, and lymph node metastasis. Traditional treatments, including surgery combined with broad-spectrum radiotherapy and chemotherapy, are ineffective. Because the specific mechanisms underlying the malignant metastatic behavior of OSCC remain unclear, currently available clinically targeted tumor therapies are very limited, particularly the lack of individual-specific targeted tumor treatment strategies. Early screening and accurate diagnosis are crucial for improving the prognosis of oral cancer patients and can significantly improve their survival rate and quality of life. While some oral cancer detection kits are currently available on the market, their high technical barriers and high cost prevent widespread use. Developing detection indicators based on existing conventional detection methods that target high-risk and prognostic indicators for cancer progression would provide new options for oral cancer screening and survival monitoring.

[0003] Cofilin is a small actin-binding protein whose activity is primarily regulated by phosphorylation at the third site, serine (Ser3). Studies have shown that its activity is highly correlated with processes such as cell motility and migration, and tumor metastasis. It plays a particularly important role in neurological diseases, osteoarthritis, and cardiovascular and renal diseases. However, phosphorylated cofilin has not been reported as a marker for malignant progression and prognosis in oral squamous cell carcinoma (OSCC). Summary of the Invention

[0004] The purpose of the present invention is to provide an oral cancer screening and prognosis kit based on the detection of phosphorylated Cofilin protein expression levels and its application; the kit of the present invention can be used for screening early oral cancer diseases and predicting survival by the strength of phosphorylated Cofilin protein expression.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an oral cancer screening and prognosis kit based on detecting the expression level of phosphorylated cofilin protein. The kit contains xylene, alcohol, citrate buffer, hydrogen peroxide, blocking solution, primary antibody, secondary antibody, DAB color developing solution, hematoxylin and neutral gum; the primary antibody is rabbit-derived phosphorylated cofilin.

[0007] Preferably, the secondary antibody is HRP-labeled goat anti-rabbit IgG polymer.

[0008] The present invention also provides a method for using the kit, which is characterized by comprising the following steps:

[0009] (1) After baking the paraffin sections, dewax them in xylene;

[0010] (2) Dewaxing the sections by sequentially passing through graded alcohol dehydration steps and then rinsing with distilled water to obtain dehydrated sections;

[0011] (3) Soak the dehydrated sections in hydrogen peroxide to block endogenous peroxidase;

[0012] (4) The sections with blocked endogenous peroxidase were placed in citrate buffer, microwaved for 2 minutes, and then cooled naturally to repair the antigen;

[0013] (5) Add blocking solution to the sections after antigen retrieval and block at room temperature for 30 minutes to block nonspecific signals;

[0014] (6) After blocking nonspecific signals, rabbit phosphorylated cofilin was added to the slices, incubated overnight at 4°C, and washed twice with PBS;

[0015] (7) Add HRP-labeled goat anti-rabbit IgG polymer to the slice obtained in step (6), incubate at room temperature for 1 hour, and wash twice with PBS;

[0016] (8) DAB colorimetric solution was added dropwise to the slice obtained in step (7), and the reaction was terminated in PBS after the tissue showed positive results.

[0017] (9) adding hematoxylin to the slice obtained in step (8) for counterstaining, and then washing with water to return the blue color;

[0018] (10) After the blueing is completed, the slices are dehydrated through a gradient of alcohol and then placed in xylene for transparency;

[0019] (11) Seal the transparent sections with neutral gum and let them dry naturally.

[0020] Preferably, the baking time of the paraffin sections in step (1) is 0.5 to 1.5 hours, and the baking temperature is 55 to 70°C.

[0021] Preferably, the alcohol gradient in step (2) is set to 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, and each dehydration takes 0.5 to 2 minutes.

[0022] Preferably, the blocking solution in step (5) is composed of 10% NGS, 1% BSA, 0.3% Triton-100, and the rest is supplemented by PBS.

[0023] Preferably, the alcohol gradient in step (10) is set to 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol, and each dehydration is 0.5 to 2 minutes.

[0024] The present invention also provides a use of the oral cancer screening and prognosis kit based on detecting the expression level of phosphorylated Cofilin protein in preparing a detection reagent for oral cancer screening and prognosis.

[0025] Preferably, the detection reagent is a reagent for immunohistochemical staining test.

[0026] Preferably, the detection reagent is an enzyme-linked immunosorbent assay reagent.

[0027] Preferably, the detection reagent is a Western blot test reagent.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The kit of the present invention detects the expression level of phosphorylated cofilin protein in human oral mucosal tissue to determine the malignant progression of oral squamous cell carcinoma. If the expression level of phosphorylated cofilin protein is lower than that of healthy individuals, the risk of oral squamous cell carcinoma and the progression of malignant transformation are low; if the expression level of phosphorylated cofilin protein is high, the risk of oral squamous cell carcinoma is high and the malignant transformation is rapid. Furthermore, as the expression level of phosphorylated cofilin protein increases, the prognosis worsens, that is, the course of oral squamous cell carcinoma prolongs, the outcome is unsatisfactory, and the survival period is shortened. This kit can be used for auxiliary diagnosis of oral squamous cell carcinoma in clinical practice, providing an effective theoretical basis for patients to adopt targeted blocking measures or make decisions, and has good prospects for clinical application and translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0031] Figure 1 Schematic diagram of the scoring criteria for staining results;

[0032] Figure 2Cofilin phosphorylation protein immunohistochemical staining diagram and statistical results;

[0033] Figure 3 Figure 2 is a curve diagram showing the negative correlation between the expression of Cofilin phosphorylation protein and survival time. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0036] Example 1

[0037] The expression level of phosphorylated Cofilin protein was detected using pathological sections of normal oral mucosa tissue and oral squamous cell carcinoma (OSCC) patient tissue.

[0038] 1. Components of an oral cancer screening and prognosis kit based on detecting phosphorylated cofilin protein expression levels

[0039] Table 1 Kit components

[0040]

[0041]

[0042] 2. Instructions for using the kit:

[0043] 1. Collect tissue samples

[0044] Methods: Lesion tissues were collected from patients clinically diagnosed with oral squamous cell carcinoma (OSCC). Oral mucosal tissues from healthy adults served as normal controls. Surgical specimens were obtained from the Department of Mucosal and Head and Neck Surgery, West China Hospital of Stomatology, Sichuan University. Lesion tissues of approximately 0.5 cm in diameter were obtained from patients clinically diagnosed with OSCC during surgery. The tissues were immediately wrapped tightly in tin foil and placed in cryovials, which were then quickly frozen in liquid nitrogen for later use. There were 4 cases in the normal control group and 109 cases in the OSCC group.

[0045] 2. Preparation of paraffin sections (fixation - dehydration - clearing - wax immersion - sectioning)

[0046] (1) Rinse the obtained tissue sample with PBS to remove blood, dry it with paper, and promptly place it in fresh 4% paraformaldehyde (tissue to fixative volume ratio 1:10) and fix it at room temperature for 24 hours;

[0047] (2) The fixed tissue is appropriately trimmed, the tissue section is determined, the grouping is marked, and the tissue is placed in an embedding frame and rinsed with running water overnight.

[0048] (3) Place the embedding frame into an automatic dehydrator to complete dehydration and make it transparent;

[0049] (4) Remove the embedding frame and immerse it in paraffin wax I, paraffin wax II, and finally embed it in sectioning wax;

[0050] (5) After the paraffin wax is completely solidified, freeze it in a refrigerator at -20 degrees Celsius for 1 hour to facilitate slicing;

[0051] (6) Set the microtome to 4 μm slices, spread the slides in a 45-degree water bath, pick the slides, and mark the slides;

[0052] (7) Place in a 55 degree oven for 2 hours and store the slices at room temperature for later use.

[0053] 3. Immunohistochemical staining

[0054] (1) Place the paraffin sections in a 65°C oven for 1 h;

[0055] (2) Xylene dewaxing: dewax in xylene I for 10 min and in xylene II for 10 min respectively;

[0056] (3) Gradient alcohol hydration: 100%, 95%, 90%, 80% and 70% gradient alcohol, 5 min each time;

[0057] (4) Rinse with distilled water for 5 minutes;

[0058] (5) 3% hydrogen peroxide to block endogenous peroxidase: paraffin sections were immersed in H2O2 and kept in the dark at room temperature for 15 min;

[0059] (6) Rinse with PBS for 5 minutes;

[0060] (7) Antigen retrieval: The antigen retrieval box is filled with antigen retrieval solution, the slide is placed in it, heated to boiling in a microwave oven for 2 minutes, and cooled naturally at room temperature. The present invention uses 0.01 mol / L citrate buffer (pH 6.0);

[0061] (8) Blocking nonspecific signals: 10% NGS, 1% BSA, 0.3% Triton-100, PBS, blocking at room temperature for 30 minutes;

[0062] (9) Drain the blocking solution without washing, directly add the prepared primary antibody, and incubate overnight at 4°C;

[0063] (10) Wash twice with PBS, 5 minutes each time;

[0064] (11) enzyme-labeled goat anti-rabbit IgG polymer incubation at room temperature for 1 h;

[0065] (12) DAB color development, color development time is controlled under a microscope, and PBS is used to terminate color development;

[0066] (13) Hematoxylin counterstaining of nuclei for 5 seconds, and running water returns to blue;

[0067] (14) Dehydration with graded alcohol (80%, 90%, 95%, 100%), and clearing with xylene for 5 minutes;

[0068] (15) Seal the slides with neutral gum and let them dry naturally;

[0069] (16) Scan the slides under a microscope and perform statistical analysis of the results.

[0070] 4. Result scoring

[0071] Five unique fields of view on each section were observed under a high-power microscope. Phosphorylated cofilin was identified as positive by the appearance of brownish-yellow granules in the cytoplasm. The immunohistochemical staining score (IRS) was calculated by assessing the average staining intensity of the entire section and the percentage of positive cells relative to tumor cells in each field. All sections were blindly evaluated by an experienced researcher, who then calculated the average score for each section.

[0072] IHC scoring criteria are:

[0073] Staining result evaluation system: Qualifications of scorers: The evaluation team consists of three experts with experience in pathological diagnosis, including two main scorers and one arbitrator. Double-blind evaluation process: Initial evaluation stage: The two main scorers independently score, and the patient's clinical information and the other party's scoring results are shielded during the evaluation. Arbitration mechanism: When the scores of the two main scorers differ, the third arbitration expert will conduct a re-evaluation, and the opinion closest to the arbitration score will be used as the final result. Observation method: Five high-power fields are randomly selected for each slice, and no less than 150 tumor cells are counted in each field, excluding necrotic areas, hemorrhagic areas and interstitial areas. Scoring criteria: Semi-quantitative integration method is used for evaluation: 1 point: negative (basic no staining of cells), 2 points: weak positive (stained cells are light yellow), 3 points: moderately positive (brown-yellow), 4 points: strongly positive (stained cells>50%, dark brown). Quality control: Olympus microscopes are used for unified observation, with standard light source intensity. Instruments are calibrated before each batch of evaluation. The staining result standards are as follows: Figure 1 shown.

[0074] 5. Results Analysis

[0075] According to the IHC scoring standard, statistical analysis showed that the expression of phosphorylated Cofilin in the mucosal tissue of the normal group was low, while the expression of phosphorylated Cofilin in the lesion mucosal tissue of oral squamous cell carcinoma (OSCC) patients was very high. In addition, the higher the tumor grade of the OSCC group, the higher the expression of phosphorylated Cofilin. There was a statistically significant difference between the OSCC group and the normal mucosal group ( Figure 2 , P<0.01**), and the expression of phosphorylated Cofilin was negatively correlated with the prognosis of survival ( Figure 3 ), indicating that the expression level of phosphorylated Cofilin protein can be used for the purpose of screening and judging the prognosis of oral squamous cell carcinoma, and drugs that block the Cofilin phosphorylation pathway can be taken in advance to slow down the malignant progression and prolong survival.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An oral cancer screening and prognosis kit based on detecting the expression level of phosphorylated Cofilin protein, characterized in that: The kit contains xylene, alcohol, citrate buffer, hydrogen peroxide, blocking solution, primary antibody, secondary antibody, DAB color developing solution, hematoxylin and neutral gum; the primary antibody is rabbit-derived phosphorylated Cofilin.

2. The kit according to claim 1, wherein The secondary antibody was HRP-labeled goat anti-rabbit IgG polymer.

3. A method for using the kit according to claim 1 or 2, characterized in that: The following steps are involved: (1) After baking the paraffin sections, dewax them in xylene; (2) Dewaxing the sections by sequentially passing through graded alcohol dehydration steps and then rinsing with distilled water to obtain dehydrated sections; (3) Soak the dehydrated sections in hydrogen peroxide to block endogenous peroxidase; (4) The sections with blocked endogenous peroxidase were placed in citrate buffer, microwaved for 2 minutes, and then cooled naturally to repair the antigen; (5) Add blocking solution to the sections after antigen retrieval and block at room temperature for 30 minutes to block nonspecific signals; (6) After blocking nonspecific signals, rabbit phosphorylated cofilin was added to the slices, incubated overnight at 4°C, and washed twice with PBS; (7) Add HRP-labeled goat anti-rabbit IgG polymer to the slice obtained in step (6), incubate at room temperature for 1 hour, and wash twice with PBS; (8) DAB colorimetric solution was added dropwise to the slice obtained in step (7), and the reaction was terminated in PBS after the tissue showed positive results. (9) adding hematoxylin to the slice obtained in step (8) for counterstaining, and then washing with water to return the blue color; (10) After the blueing is completed, the slices are dehydrated through a gradient of alcohol and then placed in xylene for transparency; (11) Seal the transparent sections with neutral gum and let them dry naturally.

4. The method of use according to claim 3, characterized in that: The baking time of the paraffin sections in step (1) is 0.5 to 1.5 hours, and the baking temperature is 55 to 70°C.

5. The method of use according to claim 3, characterized in that: The alcohol gradient in step (2) is set to 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, and each dehydration is 0.5 to 2 minutes.

6. The method of use according to claim 3, characterized in that: The components of the blocking solution in step (5) are 10% NGS, 1% BSA, 0.3% Triton-100, and the rest is supplemented with PBS.

7. Use of the oral cancer screening and prognosis reagent based on detecting the expression level of phosphorylated Cofilin protein according to claim 1 or 2 in the preparation of a detection reagent for oral cancer screening and prognosis.

8. The use according to claim 7, characterized in that The detection reagent is a reagent for immunohistochemical staining test.

9. The use according to claim 7, characterized in that The detection reagent is an enzyme-linked immunosorbent assay reagent.

10. The use according to claim 7, characterized in that The detection reagent is a reagent for protein immunoblotting test.

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