Application of exosome MZF1 protein marker in liver cirrhosis diagnosis / treatment medicine or reagent, kit and detection method

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to application of an exosome MZF1 protein marker in a liver cirrhosis diagnosis / treatment medicine or reagent, a kit and a detection method. The invention provides an application of an exosome MZF1 protein marker in a liver cirrhosis diagnosis / treatment medicine or reagent, a kit for liver cirrhosis diagnosis / treatment and a detection method of a serum exosome MZF1 protein. The exosome MZF1 protein is used as a marker, a valuable reagent or drug application prospect is provided for early diagnosis and treatment of liver cirrhosis, and help is provided for analysis of more serum exosome proteins applied to liver cirrhosis markers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application, kits, and detection methods of an exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of liver cirrhosis. Background Technology

[0002] Liver cirrhosis is one of the most common chronic liver diseases, with high morbidity and mortality. Liver cirrhosis often presents asymptomatic in its early stages, leading to delayed diagnosis and poor prognosis. Currently, the diagnosis of liver cirrhosis mainly relies on magnetic resonance imaging (MRI) and liver biopsy, which are costly and difficult to implement widely. Therefore, further exploration of non-invasive biomarkers for the early diagnosis of liver cirrhosis is an urgent problem to be solved.

[0003] Exosomes are membrane-bound vesicles with diameters ranging from 50 to 200 nm, secreted by various cells. They carry biomolecules such as proteins and nucleic acids, are released from cells into the extracellular environment, play a crucial role in intercellular communication, and participate in various physiological and pathological processes. In recent years, exosomes and their protein components have shown great potential in disease diagnosis, especially in the field of non-invasive liquid biopsy, but reports in the field of liver cirrhosis are limited. Therefore, studying exosomal proteins in circulating body fluids is of great significance for the diagnosis of liver cirrhosis.

[0004] In related technologies, clinical diagnostic markers for cirrhosis include hyaluronic acid (HA), type III procollagen (PCⅢ), type IV collagen (Ⅳ-C), and laminin (LN). These are non-invasive tests for assessing the degree of liver fibrosis, but their specificity is not high and they cannot directly diagnose cirrhosis.

[0005] Therefore, there is an urgent need to develop an application, kit, and detection method for exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of cirrhosis, in order to solve the technical problem that non-invasive examinations for assessing the degree of liver fibrosis have low specificity and cannot directly diagnose cirrhosis.

[0006] It should be noted that the information disclosed in this background section is only for understanding the background technology of this application concept, and therefore may include information that does not constitute prior art. Summary of the Invention

[0007] This disclosure provides at least one application, kit, and detection method of exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of liver cirrhosis.

[0008] In a first aspect, embodiments of this disclosure provide the application of an exosomal MZF1 protein marker in diagnostic / therapeutic drugs or reagents for liver cirrhosis.

[0009] In one alternative implementation, the exosomes are serum exosomes.

[0010] Secondly, embodiments of this disclosure also provide a kit for the diagnosis / treatment of cirrhosis, comprising exosomal MZF1 protein as a diagnostic / therapeutic biomarker.

[0011] Thirdly, this disclosure also provides a method for detecting serum exosomal MZF1 protein, applied to the detection of exosomal MZF1 protein as described above, the method comprising the following steps: Step S1: Prepare reagents for extracting serum exosomes; Step S2: Collect and prepare serum; Step S3: Separate and extract serum exosomes from serum using a reagent for extracting serum exosomes; Step S4: Detect MZF1 protein in serum exosomes using ELISA.

[0012] In one optional embodiment, the serum is prepared as follows: Step S21: Take 5ml of blood sample and shake well. Step S22: After standing at room temperature for 30 minutes, centrifuge at 3500 rpm for 5 minutes using a horizontal centrifuge to obtain the upper serum layer; Step S23: Use a 1ml pipette to transfer the serum into a sterile EP tube and store at -80℃ for later use.

[0013] In one alternative implementation, the step of separating serum exosomes from serum is as follows: Step S31: Bring the serum from -80℃ to room temperature, and use a pipette to draw 200 μl into a sterile EP tube, add 50 μl of the reagent for extracting serum exosomes, and gently pipette to mix. Step S32: Let stand overnight at 4°C, and centrifuge at 4°C and 1500g for 30 minutes the next day. Step S33: Discard the supernatant, add an appropriate amount of PBS, and centrifuge again at 4°C and 1500g for 30 min to wash the precipitate. Step S34: Discard the supernatant and add 50 to 100 μl of sterile 1xPBS to resuspend the exosome precipitate at the bottom of the tube; Step S35: Let stand at 4°C for several hours, then gently mix by blowing to obtain an exosome suspension.

[0014] In one optional implementation, the steps for detecting MZF1 protein in serum exosomes are as follows: The steps for detecting MZF1 protein in serum exosomes using ELISA are as follows: Step S41: Set up standard wells, sample wells, and blank wells on the microplate. Add 50 μL of standard and 50 μL of sample to the corresponding positions on the microplate, respectively. Do not add any sample to the blank wells. Gently shake the microplate. Step S42, incubation: After sealing the ELISA plate with sealing film, incubate at 37°C for 30 min; Step S43: Wash, remove the sealing film from the microplate, discard the liquid, shake dry, fill the blank wells, standard wells and sample wells with washing buffer, let stand for 30 seconds and then discard, repeat this process 5 times, and pat dry. Step S44: Add enzyme, add 50 μl of enzyme-labeled reagent to the standard well and the sample well to be tested respectively; Step S45, incubation: After sealing the ELISA plate with sealing film, incubate at 37°C for 30 min; Step S46: Wash, remove the sealing film, discard the liquid, spin dry, fill the blank well, standard well and sample well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry. Step S47: Add 50 μL of colorimetric reagent A to the blank well, standard well, and sample well, and then add 50 μL of colorimetric reagent B to each well. Gently shake to mix, and develop color at 37°C in the dark for 10 min. Step S48: Add 50 μL of stop solution to each blank well, standard well, and sample well to terminate the reaction; Step S49: Zero the aperture with a blank aperture and measure the absorbance of each aperture sequentially at a wavelength of 450nm.

[0015] The beneficial effects of this invention are that it provides an application of exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of cirrhosis. Using exosomal MZF1 protein as a marker provides a valuable reagent or drug application prospect for the early diagnosis and treatment of cirrhosis, and also helps to analyze more serum exosomal proteins for use as markers for cirrhosis.

[0016] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained in accordance with the structures particularly pointed out in the description, claims and drawings.

[0017] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The identification of serum exosomes provided in the embodiments of this disclosure, Figure 1 A shows the morphology of serum exosomes as detected by transmission electron microscopy. Figure 1 B represents the particle size number of serum exosomes detected by nanoparticle tracer analysis. Figure 1 C represents a marker protein for Western blot detection of serum exosomes; Figure 2 The ELISA method provided in this embodiment of the present disclosure is used to detect the content of serum exosomal MZF1 protein; Figure 3 The diagnostic efficacy of serum exosome MZF1 was analyzed using ROC curve analysis, as provided in the embodiments of this disclosure. Figure 3 A. Diagnostic efficacy of serum exosome MZF1 for MASH; Figure 3 B. Diagnostic efficacy of serum exosome MZF1 for cirrhosis; Figure 4 The correlation between serum exosome MZF1 and liver function is provided in the embodiments of this disclosure. Figure 4 A. Correlation analysis of serum exosome MZF1 and ALT; Figure 4 B. Correlation analysis of serum exosome MZF1 and AST; Figure 5 The immunohistochemical detection of MZF1 expression in liver tissues of cirrhotic patients and healthy individuals is provided in the embodiments of this disclosure. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Research has found that among the relevant technologies, clinically used diagnostic markers for cirrhosis include hyaluronic acid (HA), type III procollagen (PCⅢ), type IV collagen (Ⅳ-C), and laminin (LN). These are non-invasive tests for assessing the degree of liver fibrosis, but their specificity is not high and they cannot directly diagnose cirrhosis.

[0022] Therefore, there is an urgent need to develop an application, kit, and detection method for exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of cirrhosis, in order to solve the technical problem that non-invasive examinations for assessing the degree of liver fibrosis have low specificity and cannot directly diagnose cirrhosis.

[0023] The shortcomings of the above solutions are the result of the inventor's practical experience and careful research. Therefore, the discovery process of the above problems and the solutions proposed in this disclosure below should be considered as the inventor's contribution to this disclosure.

[0024] Based on the above research, this disclosure provides a method for detecting serum exosome proteins, including reagents for extracting serum exosomes; The reagent used to extract serum exosomes is ExoQuickexosomeprecipitation solution; The present invention does not specifically limit the source of the reagents and immunohistochemical kits used for extracting serum exosomes; any commercially available reagents in the art can be used. For example, in this embodiment, the ExoQuick exosome precipitation solution, purchased from SBI (catalog number EXOQ20A-1), was used for extracting serum exosomes; the immunohistochemical kit was purchased from Bosterone (catalog number SA1020).

[0025] The separation and extraction of serum exosomes according to the present invention preferably includes: mixing serum with ExoQuickexosomeprecipitation solution in a ratio of 4:1, letting stand overnight, centrifuging at 1500g for 30 min at 4°C, discarding the supernatant, and the precipitate is serum exosomes; This invention describes the detection of MZF1 protein in exosomes after resuspending serum exosomes in sterile 1xPBS. The preferred method includes: adding 40 μL of sample diluent to an enzyme-labeled plate, followed by 10 μL of the sample to be tested. The plate is sealed with sealing film and incubated at 37°C for 30 min. Washing is repeated 5 times, and the plate is patted dry. 50 μL of enzyme-labeled reagent is added, and the plate is incubated at 37°C for 30 min, followed by washing. 50 μL of chromogenic reagent A is added to each well, followed by 50 μL of chromogenic reagent B. The mixture is gently vortexed and incubated at 37°C in the dark for 10 min. 50 μL of stop solution is added to each well to terminate the reaction. The absorbance (OD value) of each well is measured sequentially at 450 nm.

[0026] The present invention will be further described in detail below with reference to specific embodiments.

[0027] At least one embodiment provides a method for extracting serum exosomes, comprising the following steps: I. Collection and Preparation of Serum 1. Collect 5ml of blood from the median cubital vein using a blood collection tube that promotes coagulation. Immediately after blood collection, gently invert the tube three times to mix thoroughly. 2. After standing at room temperature for 30 minutes, centrifuge at 3500 rpm for 5 minutes using a horizontal centrifuge to obtain the supernatant serum; 3. Use a 1ml pipette to transfer the serum into a sterile EP tube and store at -80℃ for later use or at 4℃ for immediate use.

[0028] II. Isolation and Extraction of Serum Exosomes 1. Remove the serum from -80℃ or 4℃ and bring it to room temperature. Use a pipette to draw 200μl into a new sterile EP tube, then add 50μl of the serum exosome extraction reagent (ExoQuick exosome precipitation solution) and gently pipette to mix. 2. Let stand overnight at 4℃, then centrifuge at 4℃ and 1500g for 30 minutes the next day; 3) Discard the supernatant, add an appropriate amount of PBS, and centrifuge again at 4℃ and 1500g for 30 min to wash the precipitate; 4) Discard the supernatant, add 50-100 μl of sterile 1xPBS to resuspend the exosome precipitate at the bottom of the tube, incubate at 4°C for several hours, then gently pipette to mix and prepare an exosome suspension for further use.

[0029] Transmission electron microscopy results showed that serum exosomes are biconcave disc-shaped structures, such as... Figure 1 As shown in Figure A; nanoparticle tracing analysis revealed that the particle size of serum exosomes was approximately 110 nm, as... Figure 1 As shown in B; Western blot was used to detect serum exosome expression protein markers Alix, TSG101, CD63, and CD9, as shown in Figure B. Figure 1 As shown in Figure C. The above results indicate that the extracted serum exosomes conform to the general characteristics of exosomes.

[0030] At least one embodiment provides a method for detecting exosomal MZF1 protein, namely enzyme-linked immunosorbent assay (ELISA), comprising the following steps: 1. Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme labeling reagent, all other steps are the same), standard wells, and sample wells on the ELISA plate. Add 50 μL of standard and 50 μL of sample to be tested to the corresponding positions on the ELISA plate, respectively. Do not add any sample to the blank wells. Gently shake the ELISA plate. 2. Incubation: After sealing the ELISA plate with sealing film, incubate it at 37°C for 30 minutes; 3. Washing: Remove the sealing film from the microplate, discard the liquid, shake dry, fill the blank wells, standard wells and sample wells with washing buffer, let stand for 30 seconds and then discard. Repeat this process 5 times, then pat dry. 4. Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank wells; 5. Incubation: After sealing the ELISA plate with sealing film, incubate it at 37°C for 30 minutes; 6. Washing: Remove the sealing film, discard the liquid, shake dry, fill the blank well, standard well, and sample well with washing solution, let stand for 30 seconds and then discard. Repeat this process 5 times, then pat dry. 7. Color development: Add 50 μL of color developer A to the blank well, standard well, and sample well, and then add 50 μL of color developer B to each well. Gently shake to mix, and develop color at 37°C in the dark for 10 min. 8. Termination: Add 50 μL of stop solution to each blank well, standard well, and sample well to terminate the reaction; 9. Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm.

[0031] At least one embodiment provides a method for detecting MZF1 protein in liver tissue, comprising the following steps: 1. Place paraffin sections of liver tissue in a 60℃ oven for 6-8 hours and dewax to remove water using standard procedures; 2. Add 3% H2O2 dropwise to inactivate endogenous peroxidase; 3. Boil liver tissue in citrate buffer (pH 6.0) for 45 minutes to repair antigens; 4. Use 5% BSA to seal the slide at room temperature for 1 hour to remove non-specific staining; 5. Incubate liver tissue with MZF1 primary antibody overnight at 4°C; 6. On the second day, wash three times with PBS for 5 minutes each time to remove excess primary antibody from the slide; 7. Incubate liver tissue with biotin-anti-mouse / rabbit IgG antibody and SABC at 37°C for 30 min, respectively; 8. Develop the color using the DAB horseradish peroxidase colorimetric kit, counterstain with hematoxylin for 2 min, and then invert the blue color with running water for 15 min.

[0032] 9. After routine dehydration, the slides are mounted with neutral resin and examined under a microscope.

[0033] The value of serum exosome MZF1 in liver cirrhosis as a diagnostic biomarker provided in at least one embodiment is as follows: This invention included 30 patients with cirrhosis who visited Wujin People's Hospital and Changzhou Third People's Hospital in Changzhou between July 2024 and June 2025. Basic information such as age, sex, ALT, and AST was collected, and serum exosome MZF1 levels were collected and detected. Simultaneously, 30 healthy individuals undergoing physical examinations and 30 MASH patients were included, matched by age and sex. All three groups of samples were collected, sampled, aliquoted, and stored under identical conditions for serum exosome MZF1 content detection and analysis. Immunohistochemical detection of MZF1 was performed on paraffin sections of liver tissue from 3 healthy individuals and 3 patients with cirrhosis.

[0034] Data Analysis: The data from this experiment were analyzed using GraphpadPrism 8.0.0. The differences between different groups were compared using the researcher test. ROC curves were plotted to analyze the diagnostic sensitivity and specificity of exosome MZF1 in hepatitis and cirrhosis. Correlation analysis was used to observe the correlation between exosome MZF1 and liver injury markers ALT and AST.

[0035] result: Serum exosomes and liver tissue MZF1 expression levels in patients with cirrhosis, hepatitis, and healthy individuals undergoing physical examinations like Figure 2 As shown, exosomal MZF1 is highly expressed in serum exosomals of patients with cirrhosis (Cirrhosis-ex), and the expression level increases sequentially in healthy individuals (Healthy-ex), hepatitis patients (MASH-ex), and Cirrhosis-ex. The diagnostic efficacy of serum exosomal MZF1 for hepatitis and cirrhosis is as follows: Figure 3 As shown, the areas under the AUC curves are 0.7711 (A) and 0.8700 (B), respectively. Figure 4 As shown, exosome MZF1 showed a significant positive correlation with commonly used clinical liver injury markers ALT and AST, with correlation coefficients of 0.5033 (A) and 0.5466 (B), respectively. Furthermore, as... Figure 5 As shown, the expression of MZF1 in the liver tissue of patients with cirrhosis was significantly higher than that in the liver tissue of healthy individuals. These results indicate that exosome MZF1 has promising clinical application potential in the diagnosis and differential diagnosis of cirrhosis.

[0036] In conclusion, serum exosome MZF1 can provide a novel biomarker with potential value for the early diagnosis and differential diagnosis of cirrhosis. The detection method for serum exosome MZF1 provided can offer reference and assistance for exploring more serum exosomal protein biomarkers for disease diagnosis.

[0037] Based on the above research and verification, the exosomal MZF1 protein provided in this application, as a biomarker, can be applied to scenarios or fields including but not limited to the following: 1. Application of exosomal MZF1 protein markers in drugs or reagents for the diagnosis / treatment of liver cirrhosis.

[0038] 2. Kits for the diagnosis / treatment of cirrhosis, including exosomal MZF1 protein as a diagnostic / therapeutic biomarker.

[0039] 3. Application of exosomal MZF1 protein markers in products that differentiate between individuals with cirrhosis and healthy individuals.

[0040] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. The apparatus embodiments described above are merely illustrative. For example, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. Furthermore, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Based on the above-described ideal embodiments of the present invention, and through the above description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the disclosed embodiments. The technical scope of the embodiments of this disclosure is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. The application of an exosomal MZF1 protein marker in drugs or reagents for the diagnosis / treatment of liver cirrhosis.

2. The application of the exosomal protein marker as described in claim 1 in diagnostic / therapeutic drugs or reagents for liver cirrhosis, characterized in that, The exosomes are serum exosomes.

3. A reagent kit for the diagnosis / treatment of liver cirrhosis, characterized in that, Including the exosomal MZF1 protein, as a diagnostic / therapeutic biomarker.

4. A method for detecting serum exosome MZF1 protein, characterized in that, The method for detecting serum exosomal MZF1 protein as described in any one of claims 1-2 includes the following steps: Step S1: Prepare reagents for extracting serum exosomes; Step S2: Collect and prepare serum; Step S3: Separate and extract serum exosomes from serum using a reagent for extracting serum exosomes; Step S4: Detect MZF1 protein in serum exosomes using ELISA.

5. The method for detecting serum exosome MZF1 protein as described in claim 4, characterized in that, The method for preparing serum is as follows: Step S21: Take 5ml of blood sample and shake well. Step S22: After standing at room temperature for 30 minutes, centrifuge at 3500 rpm for 5 minutes using a horizontal centrifuge to obtain the upper serum layer; Step S23: Use a 1ml pipette to transfer the serum into a sterile EP tube and store at -80℃ for later use.

6. The method for detecting serum exosome proteins as described in claim 4, characterized in that, The steps for isolating serum exosomes from serum are as follows: Step S31: Bring the serum from -80℃ to room temperature, and use a pipette to draw 200 μl into a sterile EP tube, add 50 μl of the reagent for extracting serum exosomes, and gently pipette to mix. Step S32: Let stand overnight at 4°C, and centrifuge at 4°C and 1500g for 30 minutes the next day. Step S33: Discard the supernatant, add an appropriate amount of PBS, and centrifuge again at 4°C and 1500g for 30 min to wash the precipitate. Step S34: Discard the supernatant and add 50 to 100 μl of sterile 1xPBS to resuspend the exosome precipitate at the bottom of the tube; Step S35: Let stand at 4°C for several hours, then gently mix by blowing to obtain an exosome suspension.

7. The method for detecting serum exosome MZF1 protein as described in claim 4, characterized in that, The steps for detecting MZF1 protein in serum exosomes using ELISA are as follows: Step S41: Set up standard wells, sample wells, and blank wells on the microplate. Add 50 μL of standard and 50 μL of sample to the corresponding positions on the microplate, respectively. Do not add any sample to the blank wells. Gently shake the microplate. Step S42, incubation: After sealing the ELISA plate with sealing film, incubate at 37°C for 30 min; Step S43: Wash, remove the sealing film from the microplate, discard the liquid, shake dry, fill the blank wells, standard wells and sample wells with washing buffer, let stand for 30 seconds and then discard, repeat this process 5 times, and pat dry. Step S44: Add enzyme, add 50 μl of enzyme-labeled reagent to the standard well and the sample well to be tested respectively; Step S45, incubation: After sealing the ELISA plate with sealing film, incubate at 37°C for 30 min; Step S46: Wash, remove the sealing film, discard the liquid, spin dry, fill the blank well, standard well and sample well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry. Step S47: Add 50 μL of colorimetric reagent A to the blank well, standard well, and sample well, and then add 50 μL of colorimetric reagent B to each well. Gently shake to mix, and develop color at 37°C in the dark for 10 min. Step S48: Add 50 μL of stop solution to each blank well, standard well, and sample well to terminate the reaction; Step S49: Zero the aperture with a blank aperture and measure the absorbance of each aperture sequentially at a wavelength of 450nm.

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