CN-121975902-A - Method for detecting metabolism function of peripheral blood mononuclear cells by SeaHorse bioenergy analysis technology and application

Abstract

The application discloses a method for detecting the metabolic function of Peripheral Blood Mononuclear Cells (PBMC) by using a SeaHorse bioenergy analysis technology and application thereof. The application firstly determines the linear interval for detecting each index of the metabolism function of the peripheral blood mononuclear cells by the SeaHorse bioenergy analyzer, provides a basis for establishing a standardized detection flow, is favorable for the comparison analysis among different samples and the unification of cross-center data, lays a solid technical foundation for the diagnosis, the curative effect evaluation and the prognosis prediction of metabolic diseases, and further establishes the corresponding relation between the bioenergy parameters of the peripheral blood mononuclear cells and the clinical state by selecting different disease types for analysis, thereby remarkably improving the accuracy of the diagnosis, the curative effect monitoring and the prognosis prediction of related diseases.

Inventors

• HUANG GUORUI
• NING GUANG

Assignees

• 上海交通大学医学院附属瑞金医院

Dates

Publication Date

20260505

Application Date

20260126

Claims (6)

1. 1. A method for detecting the metabolism function of Peripheral Blood Mononuclear Cells (PBMC) of a sample by using a Seahorse bioenergy analysis method is characterized by comprising the steps of inoculating PBMC on a detection plate, setting the inoculation density range to be 8×10 4 -50×10 4 cells/hole, sequentially adding oligomycin, carbonyl cyanide-4-trifluoromethoxybenzohydrazone and a rotenone/antimycin A mixed inhibitor, measuring the mitochondrial oxygen consumption rate by using a Seahorse bioenergy analyzer and calculating various respiration indexes including basic respiration, ATP generation, maximum respiration and reserve respiration, and measuring the glycolysis function, wherein the PBMC is inoculated on the detection plate, the inoculation density range is set to be 8×10 4 -50×10 4 cells/hole, the oligomycin, the 2-deoxy-D-glucose and a detection buffer solution are sequentially added, the proton exorate is measured by using the Seahorse bioenergy analyzer and calculated, and the various glucose indexes including basic glycolysis, basic exogenesis and basic glycolysis and reserve glycolysis are supplemented with 2, 374, and the basic glycolysis buffer is supplemented with 2, and mM.
2. 2. Use of a detection reagent for the preparation of a product for early screening of a disease, evaluation and dynamic monitoring of a disease-related metabolic state, disease typing and risk stratification, efficacy evaluation and monitoring, disease severity or disease activity evaluation and prognosis prediction, characterized in that the detection reagent is used for detecting the metabolic function of peripheral blood mononuclear cells of a sample according to the method of claim 1.
3. 3. The use according to claim 2, wherein the detection reagent is a reagent used in the method of claim 1.
4. 4. The use according to claim 2, wherein the diseases include neoplasms, endocrinopathy, metabolic diseases, inflammation and cardiovascular diseases.
5. 5. The use according to any one of claims 2 to 4, wherein the product comprises a kit, system or device.
6. 6. A disease evaluation system is characterized by comprising a detection module and an evaluation module, wherein the detection module is used for detecting metabolic function indexes of peripheral blood mononuclear cells of a sample, the evaluation module comprises a memory, a processor and a computer program which is stored in the memory and can run on the processor, and the processor realizes the following steps when executing the program, 1) acquiring all metabolic function indexes detected by the detection module, and 2) judging the disease state of the sample according to the metabolic function indexes of 1).

Description

Method for detecting metabolism function of peripheral blood mononuclear cells by SeaHorse bioenergy analysis technology and application Technical Field The application relates to a method for detecting the metabolic function of Peripheral Blood Mononuclear Cells (PBMC) by using a SeaHorse bioenergy analysis technology and application thereof, belonging to the technical fields of in-vitro diagnosis and metabonomics detection. Background Mitochondria are the core sites of cellular energy metabolism, and their functional status plays a key role in the occurrence, progression and prognosis of a variety of diseases. In recent years, a great deal of basic research has demonstrated that mitochondrial dysfunction is not only closely related to tumors, metabolic diseases (such as diabetes, obesity, thyroid diseases) and nervous system diseases, but also has important connection with pathological processes such as immune dysfunction, abnormal inflammatory response and the like. Therefore, how to accurately evaluate the mitochondrial function and the overall metabolic state of a patient becomes an urgent need in clinical research and disease diagnosis. SeaHorse bioenergy analysis technology is an internationally accepted real-time cell metabolism detection platform, and can comprehensively reflect mitochondrial functions and glycolysis levels of cells by measuring indexes such as Oxygen Consumption Rate (OCR), proton Excretion Rate (PER) and the like. The technology is widely applied in the basic research field, and is used for analyzing dynamic changes of cell energy metabolism, exploring the action mechanism of medicines and finding new molecular targets of diseases. However, the technology is currently limited mainly to laboratory studies and has not been truly transformed into clinical detection means. In clinical practice, disease diagnosis and efficacy assessment typically rely on biochemical indicators, hormone level detection, imaging examinations, and molecular genetic analysis. Although valuable, these methods often suffer from the disadvantages of (1) inability to directly reflect the metabolic status of the patient's cells, (2) invasive or reliance on tissue samples, inconvenience for repeated detection, and (3) inadequate predictive sensitivity to early changes or recurrence of the disease. Therefore, a simple, sensitive and repeatable detection method capable of dynamically monitoring the energy metabolism state of a patient is not yet available clinically. Peripheral Blood Mononuclear Cells (PBMCs) are a widely available and convenient clinical routine sample that represents an important component of the immune system of the body. Numerous studies have demonstrated that the metabolic profile of PBMCs changes in a variety of diseases, e.g., PBMCs in tumor patients often manifest as mitochondrial dysfunction, metabolic disease patients have reprogramming of glycolysis and oxidative phosphorylation, and specific disorders of PBMC metabolism in neurological and immune diseases. This provides a solid basis for using PBMCs as windows for metabolic function detection. Therefore, if the SeaHorse bioenergy analysis technology can be applied to the detection of PBMC of patients, a set of standardized and generalized clinical detection method is established, so that the defect of the existing detection means can be overcome, and the transformation from basic research results to clinical practice can be realized. By detecting the metabolic function of PBMC, the method is expected to be used for early diagnosis, typing, curative effect evaluation and prognosis prediction of diseases, and further provides a new basis for accurate medical treatment and personalized treatment. Based on the method, the invention provides a PBMC metabolic function detection method based on SeaHorse bioenergy analysis technology, realizes the transformation application of the technology in the clinical level for the first time, and provides a new thought and tool for diagnosis and prognosis management of various diseases. Disclosure of Invention The application aims at solving the problems that the existing clinical detection means can not directly, dynamically and noninvasively reflect the energy metabolism function of patient cells, and especially lacks indexes with sensitivity to early diagnosis, curative effect monitoring and prognosis prediction of diseases. The application aims to provide a Peripheral Blood Mononuclear Cell (PBMC) metabolism detection method based on SeaHorse bioenergy analysis technology, which establishes a standardized metabolism index system and a risk scoring model to realize conversion from a scientific research detection platform to a clinical diagnosis tool. In order to achieve the above purpose, the present application adopts the following technical scheme: In a first aspect, the present application provides a method for detecting a metabolic function of peripheral blood mononuclear cells of a sample by a