Use method and application of mitochondrial membrane potential probe

Abstract

The invention relates to a use method and application of a mitochondrial membrane potential probe. The use method comprises the following steps: (a) separating human peripheral blood mononuclear cells by adopting a Ficoll method; (b) carrying out flow antibody staining; (c) centrifugally collecting the cells, cleaning with a PBS buffer solution, and adding a cell culture medium to resuspend the cells; (d) adding a fluorescent probe JC-1 into the culture medium, incubating in a 5% CO2 cell incubator at 37 DEG C for 15-30 minutes, centrifuging, precipitating cells, and discarding the supernatant; adding resuspended cells, centrifuging, precipitating the cells, and discarding the supernatant; and (e) resuspending the cells in a precooled PBS buffer solution, and measuring and analyzing the membrane potential in a flow cytometer. The progress of the liver disease can be analyzed according to the significant difference, the accuracy and specificity of diagnosis are significantly improved, and a reliable basis is provided for clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical diagnostic technology, specifically relating to the method and application of a mitochondrial membrane potential probe. Background Technology

[0002] Acute-on-chronic liver failure (ACLF) is a syndrome characterized by rapid deterioration of liver function triggered by acute injury factors on the basis of chronic liver disease. Clinical manifestations include deepening jaundice, coagulation dysfunction, and multiple organ failure. The course of ACLF is rapid, but effective early diagnostic tools and monitoring methods are currently lacking. Clinical manifestations of ACLF often only appear when liver damage is already severe, thus hindering effective early diagnosis. In the early stages, the disease may not be fully apparent, often leading to delayed clinical identification and intervention. While clinical methods and techniques for diagnosing ACLF offer some assistance, they still have limitations and shortcomings. Currently used serum biomarkers have some diagnostic value for ACLF, but their sensitivity and specificity are insufficient and may be influenced by various factors. Existing indicators (such as ALT, AST, and total bilirubin) primarily reflect hepatocellular necrosis and loss of function, while the core driving factors of ACLF are systemic inflammation and immune metabolic disorders. Current serum biomarkers cannot accurately quantify the intensity and nature of this inflammatory response, nor can they effectively assess hepatocellular regeneration, a crucial prognostic process. Therefore, it is necessary to find a specific biomarker that can identify the disease as early as possible before clinical symptoms become apparent, providing an opportunity for early intervention and thus improving patient prognosis. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a method for using a mitochondrial membrane potential probe.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: a method for using a mitochondrial membrane potential probe, comprising the following steps: (a) Human peripheral blood mononuclear cells were isolated using the Ficoll method; (b) Perform flow cytometry antibody staining; (c) Collect cells by centrifugation, wash with PBS buffer, and resuspend cells in cell culture medium; (d) Add fluorescent probe JC-1 to the culture medium, incubate at 37℃ and 5% CO2 for 15-30 min, centrifuge, precipitate the cells, and discard the supernatant; add resuspended cells, centrifuge, precipitate the cells, and discard the supernatant; (e) The cells were resuspended in pre-cooled PBS buffer and the membrane potential was measured and analyzed by flow cytometer.

[0005] Optimally, in step (c), CCCP is added to the culture medium of the positive control wells and incubated in a 37°C, 5% CO2 cell culture incubator for 15-30 min as a positive control group.

[0006] Optimally, step (a) includes: (a1) Dilute the blood sample in a centrifuge tube using PBS buffer at a ratio of at least 1:1; (a2) Prepare Ficoll of the same volume as the whole blood sample before dilution and add it to the bottom of the centrifuge tube; (a3) Centrifuge at 400g for 20 minutes at room temperature; (a4) Collect human peripheral blood mononuclear cells at the junction of PBS buffer and Ficoll layer into a new test tube; (a5) Add an appropriate amount of PBS buffer to the centrifuge tube and wash the cells; (a6) Centrifuge the cells at 300-400g for 4-5 minutes at 2-8℃ and discard the supernatant.

[0007] Further, step (b) specifically involves: adding 100 μl of PBS buffer containing 2 μl of antibody to the EP tube for resuspending, incubating at room temperature for 15-20 minutes, protecting from light, centrifuging the sample at 3000 rpm for 5 minutes at room temperature, and discarding the supernatant.

[0008] Further, step (c) specifically involves: centrifuging the product from step (b) (after discarding the supernatant) at 300g for 5 min to collect the cells, washing twice with 1×PBS buffer; discarding the 1×PBS buffer, resuspending the cells in 1mL of cell culture medium at a cell density of 1×10⁻⁶ cells / mL. 6 pcs / mL

[0009] Optimized, step (d) is as follows: Add 10 μL of fluorescent probe JC-1 to the culture medium, mix thoroughly, and incubate at 37°C and 5% CO2 in a cell culture incubator for 15-30 min; after incubation at 37°C, centrifuge at 400g and 4°C for 3-4 min to precipitate the cells; discard the supernatant, taking care not to aspirate the cells; add 1 mL of pre-cooled 1×PBS to resuspend the cells, centrifuge at 400g and 4°C for 3-4 min to precipitate the cells, discard the supernatant, and repeat the washing once.

[0010] Optimally, step (e) specifically involves resuspending the cells in 500 μL of pre-cooled 1×PBS and measuring and analyzing the membrane potential using a flow cytometer.

[0011] Another object of the present invention is to provide an application of a mitochondrial membrane potential probe in the preparation of a tool to differentiate between cirrhosis and acute-on-chronic liver failure, wherein the mitochondrial membrane potential probe shows significant differences in the mitochondrial membrane potential of peripheral blood lymphocyte subsets in cirrhosis and acute-on-chronic liver failure, the peripheral blood lymphocyte subsets including CD8 + T, CD4 + T, CD19 + B and CD56 + NK.

[0012] Optimally, it includes the following steps: The mitochondrial membrane potential of peripheral blood lymphocyte subsets in the sample was detected to determine and predict whether the liver was in the stage of cirrhosis or acute-on-chronic liver failure. The proportion of JC-1 probes in CD56+ NK cells showed good discriminatory power between patients with liver fibrosis and healthy controls, with a discriminatory AUC of 0.8060; the proportion of JC-1 probes in CD8+ T cells showed good discriminatory power between patients with acute-on-chronic liver failure and patients with liver fibrosis, with a discriminatory AUC of 0.8425.

[0013] Ideally, the tool includes detection reagents, detection kits, and detection chips.

[0014] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art: The method and application of the mitochondrial membrane potential probe of the present invention, by testing the mitochondrial membrane potential of peripheral blood lymphocyte subsets, can analyze the progression of liver disease based on its significant differences, which significantly improves the accuracy and specificity of diagnosis and provides a reliable basis for clinical diagnosis. Attached Figure Description

[0015] Figure 1 The diagram shows the membrane potential changes in the healthy control group (HC), the cirrhosis group (LC), and the acute-on-chronic liver failure group (ACLF) as measured by the mitochondrial membrane potential probe of this invention: CD8 + T(A), CD4 + T(B), CD19 + B(C), CD56 + NK(D); Figure 2 Diagnostic graphs of mitochondrial membrane potentials in healthy control group (HC) and cirrhosis group (LC) measured by the mitochondrial membrane potential probe of this invention: CD8 + T(A), CD4 + T(B), CD19 + B(C), CD56 + NK(D); Figure 3Diagnostic graphs of mitochondrial membrane potentials measured by the mitochondrial membrane potential probe of this invention in the cirrhosis group (LC) and the acute-on-chronic liver failure group (ACLF): CD8 + T(A), CD4 + T(B), CD19 + B(C), CD56 + NK(D). Detailed Implementation

[0016] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0017] The method of using the mitochondrial membrane potential probe of the present invention includes the following steps: (a) Human peripheral blood mononuclear cells were isolated using the Ficoll method; Step (a) includes: (a1) Dilute the blood sample in a centrifuge tube using PBS buffer at a ratio of at least 1:1; (a2) Prepare Ficoll of the same volume as the whole blood sample before dilution and add it to the bottom of the centrifuge tube; (a3) Centrifuge at 400g for 20 minutes at room temperature; (a4) Collect human peripheral blood mononuclear cells at the junction of PBS buffer and Ficoll layer into a new test tube; (a5) Add an appropriate amount of PBS buffer to the centrifuge tube and wash the cells; (a6) Centrifuge the cells at 300-400g for 4-5 minutes at 2-8℃ and discard the supernatant.

[0018] (b) Perform flow cytometry antibody staining; Step (b) is as follows: Add 100 μl of PBS buffer containing 2 μl of antibody to the EP tube for resuspending, incubate at room temperature for 15-20 minutes, protect from light, centrifuge the sample at 3000 rpm for 5 minutes at room temperature, and discard the supernatant.

[0019] (c) Collect cells by centrifugation, wash with PBS buffer, and resuspend cells in cell culture medium; Step (c) specifically involves: centrifuging the product from step (b) (after discarding the supernatant) at 300g for 5 min to collect cells, washing twice with 1×PBS buffer; discarding the 1×PBS buffer, adding 1 mL of cell culture medium to resuspend cells at a cell density of 1×10⁻⁶ cells / mL. 6 per mL.

[0020] CCCP was also added to the culture medium in the positive control wells, and the cells were incubated at 37°C in a 5% CO2 cell culture incubator for 15–30 min to serve as the positive control group. (d) Add fluorescent probe JC-1 to the culture medium, incubate at 37℃ and 5% CO2 for 15-30 min, centrifuge, precipitate cells, and discard the supernatant; add resuspended cells, centrifuge, precipitate cells, and discard the supernatant; Step (d) is as follows: Add 10 μL of fluorescent probe JC-1 to the culture medium, mix thoroughly, and incubate at 37℃ and 5% CO2 for 15-30 min; after incubation at 37℃, centrifuge at 400g and 4℃ for 3-4 min to precipitate cells; discard the supernatant, and be careful not to aspirate cells; add 1 mL of pre-cooled 1×PBS to resuspend cells, centrifuge at 400g and 4℃ for 3-4 min to precipitate cells, discard the supernatant, and repeat the washing once.

[0021] (e) Resuspend the cells in pre-chilled PBS buffer and measure and analyze the membrane potential using a flow cytometer; step (e) specifically involves: resuspending the cells in 500 μL of pre-chilled 1×PBS and measuring and analyzing the membrane potential using a flow cytometer, such as... Figure 1 As shown.

[0022] The aforementioned mitochondrial membrane potential probes have been used in the preparation of tools (including detection reagents, detection kits, and detection chips) to differentiate between cirrhosis and acute-on-chronic liver failure. The probes revealed significant differences in mitochondrial membrane potential among peripheral blood lymphocyte subsets in cirrhosis and acute-on-chronic liver failure, including CD8+. + T, CD4 + T, CD19 + B and CD56 + NK.

[0023] The above application includes the following steps: Detecting the mitochondrial membrane potential of peripheral blood lymphocyte subsets in the test sample, such as... Figure 2 and Figure 3 To determine and predict whether a patient is in the stage of cirrhosis or acute-on-chronic liver failure, it was found that the proportion of JC-1 probes on CD56+NK cells had a good distinguishing effect between patients with liver fibrosis and healthy controls (AUC=0.8060); the proportion of JC-1 probes on CD8+T cells had a good distinguishing effect between patients with acute-on-chronic liver failure and patients with liver fibrosis (AUC=0.8425).

[0024] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method of using a mitochondrial membrane potential probe, comprising: Comprising the following steps: (a) Isolate human peripheral blood mononuclear cells by Ficoll method; (b) Perform flow antibody staining; (c) Centrifuge to collect cells, wash with PBS buffer, and resuspend cells in cell culture medium; (d) Add fluorescent probe JC-1 to the culture medium, incubate at 37°C in a 5% CO2 cell incubator for 15-30 min, centrifuge and precipitate the cells, discard the supernatant; resuspend the cells, centrifuge and precipitate the cells, discard the supernatant; (e) Resuspend the cells in pre-cooled PBS buffer and measure and analyze the membrane potential on a flow cytometer.

2. The method of using a mitochondrial membrane potential probe according to claim 1, wherein: In step (c), add CCCP to the culture medium in the positive control well, incubate at 37°C in a 5% CO2 cell incubator for 15-30 min, as a positive control group.

3. The method of claim 1, wherein the mitochondrial membrane potential probe is JC-1. Step (a) includes: (a1) Dilute the blood sample with PBS buffer at a ratio of at least 1:1 in a centrifuge tube; (a2) Prepare the same volume of Ficoll as the whole blood sample before dilution and add it to the bottom of the centrifuge tube; (a3) Centrifuge at 400g for 20 minutes at room temperature; (a4) Collect human peripheral blood mononuclear cells at the interface between the PBS buffer and the Ficoll layer into a new test tube; (a5) Add an appropriate amount of PBS buffer to the centrifuge tube to wash the cells; (a6) Centrifuge the cells at 300-400g for 4-5 minutes at 2-8°C, and discard the supernatant.

4. The method of claim 3, wherein the mitochondrial membrane potential probe is JC-1. Step (b) specifically: resuspend 100 μl of PBS buffer containing 2 μl of antibody in an EP tube, incubate at room temperature for 15-20 minutes, avoid light, centrifuge the sample at 3000 rpm at room temperature for 5 minutes, and discard the supernatant. ​ 5. The method of claim 4, wherein the mitochondrial membrane potential probe is JC-1. Step (c) is specifically: collect the cells by centrifuging the product in step (b) at 300g for 5 min, wash the cells twice with 1x PBS buffer; discard the 1x PBS buffer, resuspend the cells with 1 mL of cell culture medium, and the cell density is 1x10 6 per mL. ​ 6. The method of claim 1, wherein the mitochondrial membrane potential probe is JC-1. Step (d) specifically: add 10 μL of fluorescent probe JC-1 to the culture medium, mix well, and incubate at 37°C in a 5% CO2 cell incubator for 15-30 min; after incubation at 37°C, centrifuge at 400g at 4°C for 3-4 min to precipitate the cells; discard the supernatant, taking care not to aspirate the cells; resuspend the cells with 1 mL of pre-cooled 1x PBS, centrifuge at 400g at 4°C for 3-4 min to precipitate the cells; discard the supernatant and repeat the washing once. ​ 7. The method of claim 1, wherein the mitochondrial membrane potential probe is JC-1. Step (e) specifically: resuspend the cells in 500 μL of pre-cooled 1x PBS, and measure and analyze the membrane potential on a flow cytometer.

8. Use of a mitochondrial membrane potential probe in the manufacture of a tool for differentiating between cirrhosis and acute-on-chronic liver failure, characterized in that: The mitochondrial membrane potential probe has significant differences in mitochondrial membrane potential in peripheral blood lymphocyte subsets of liver cirrhosis and acute-on-chronic liver failure, including CD8 + T, CD4 + T, CD19 + B and CD56 + NK.

9. Use according to claim 8, characterized in that, Comprising the following steps: Detecting the mitochondrial membrane potential of peripheral blood lymphocyte subsets in the sample to be tested to determine and predict whether the patient is in the liver cirrhosis or acute-on-chronic liver failure stage; The ratio of JC-1 probe of CD56+ NK cells has good discrimination between liver fibrosis patients and healthy controls, with a discrimination AUC of 0.8060; the ratio of JC-1 probe of CD8+ T cells has good discrimination between acute-on-chronic liver failure patients and liver fibrosis patients, with a discrimination AUC of 0.8425.

10. Use according to claim 8, characterized in that: The tools include detection reagents, detection kits, and detection chips.

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