Application method of miR-30e-5p level of adipocyte in atherosclerosis disease

Abstract

The invention relates to the technical field of biomedicine, and provides an application method of miR-30e-5p level of fat cells in atherosclerosis, which comprises an auxiliary diagnosis method and specifically comprises the following steps: step 1, collecting a blood sample of a to-be-detected individual; 3, detecting the expression level of miR-30e-5p in the serum by adopting a real-time quantitative PCR (Polymerase Chain Reaction) technology, and carrying out standardization treatment by using Cel-miR-39 selected from nematodes as a reference gene; step 4, comparing the miR-30e-5p standardized expression level of the individual to be detected with a preset miR-30e-5p expression level threshold value of a normal control group; and if the expression level of the individual to be detected is higher than the threshold value, performing auxiliary diagnosis on the risk of atherosclerosis of the individual. The atherosclerosis risk is diagnosed and prognosis is evaluated in an auxiliary manner by detecting the miR-30e-5p level in serum, and all systems form a closed loop, so that the whole process optimization from target screening to decision support is realized, and a personalized treatment scheme can be provided for a patient.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases. Background Technology

[0002] Atherosclerosis, a cardiovascular disease that seriously threatens human health, has a complex pathogenesis and insidious disease progression, often leading to fatal complications such as myocardial infarction and stroke. With the increasing aging of the global population and changes in lifestyle, the incidence of atherosclerosis is showing an upward trend year by year. Therefore, achieving early and accurate diagnosis, prognostic assessment, and personalized treatment of this disease is of vital clinical significance for reducing the incidence of cardiovascular events and improving patients' quality of life.

[0003] Currently, the diagnosis and treatment of atherosclerosis still face numerous technical bottlenecks. At the diagnostic level, traditional serological markers (such as blood lipids and C-reactive protein) lack tissue specificity, making it difficult to accurately reflect early pathological changes in vascular endothelial damage and plaque formation. This leads to missed or misdiagnosed cases, delaying treatment for some patients. In terms of prognostic assessment, existing methods are mostly based on clinical symptoms and imaging examinations, lacking molecular-level quantitative indicators and failing to dynamically monitor disease progression and treatment response, thus limiting the accuracy of prognostic judgments. Furthermore, current treatment systems lack efficient data interaction and integration mechanisms in areas such as target screening, treatment plan evaluation, efficacy monitoring, and personalized decision-making. This makes it difficult to form a closed-loop precision treatment system, resulting in treatment plan optimization lagging behind disease progression and failing to meet the needs of personalized medicine. Fundamentally, current technologies have not fully explored the regulatory role of adipocyte-derived microRNAs (such as miR-30e-5p) in the development of atherosclerosis, lacking diagnostic markers and therapeutic targets centered on this molecule, thus hindering the breakthrough of the limitations of traditional diagnostic and treatment models. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases, solving the problem that traditional diagnosis and treatment lacks molecular indicators and closed-loop systems, making it difficult to achieve precise diagnosis and treatment of atherosclerosis.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases, comprising:

[0006] The auxiliary diagnostic methods specifically include the following steps:

[0007] Step 1: Collect blood samples from the individual to be tested.

[0008] Step 2: Separate serum from the blood sample;

[0009] Step 3: The expression level of miR-30e-5p in the serum was detected by real-time quantitative PCR, and the expression level was standardized using a gene selected from nematode Cel-miR-39 as an internal reference.

[0010] Step 4: Compare the standardized expression level of miR-30e-5p of the individual to be tested with the preset miR-30e-5p expression level threshold of the normal control group; if the expression level of the individual to be tested is higher than the threshold, it can be used to help diagnose the risk of atherosclerosis in the individual.

[0011] The prognostic assessment method specifically includes the following steps:

[0012] S1. Obtain blood samples from patients with atherosclerosis;

[0013] S2. Separate serum and detect the standardized expression level of miR-30e-5p in the patient's serum according to steps two and three;

[0014] S3. Calculate the patient's relative miR-30e-5p risk index according to the formula: R = [(EN) / N] × 100%;

[0015] S4. The patient's prognosis is graded and assessed based on the calculated relative miR-30e-5p risk index, where a higher risk index indicates a worse prognosis. The grades include mild, moderate, and severe adverse outcomes.

[0016] Preferably, the miR-30e-5p expression level threshold in the normal control group is determined through the following steps:

[0017] T1. Collect blood samples from no fewer than 30 healthy individuals clinically diagnosed without atherosclerosis;

[0018] T2. Perform steps two and three of claim 1 on the blood samples of the healthy individuals to obtain the normalized expression level of miR-30e-5p for each healthy individual;

[0019] T3. Calculate the mean and standard deviation of the standardized expression level of miR-30e-5p in the healthy individuals, and set the threshold to M+3SD.

[0020] Preferably, N in S3 is the average expression level of miR-30e-5p in the normal control group.

[0021] Preferably, the system includes an atherosclerosis treatment target screening system, an atherosclerosis treatment plan evaluation system based on miR-30e-5p antagonism, a system for monitoring the atherosclerosis treatment progress using adipocyte miR-30e-5p levels, and an atherosclerosis personalized treatment decision support system based on adipocyte miR-30e-5p. These systems are interconnected through data exchange to form a closed-loop connection, as detailed below:

[0022] The system for screening treatment targets for atherosclerosis is connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism. The system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is also connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism. Furthermore, the system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is connected to the system for personalized atherosclerosis treatment decision support based on adipocyte miR-30e-5p. Finally, the system for personalized atherosclerosis treatment decision support based on adipocyte miR-30e-5p is also connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism.

[0023] Preferably, the atherosclerosis treatment target screening system includes:

[0024] Adipocyte simulation unit: used to construct a cell model simulating adipocytes in an obese state, capable of regulating intracellular lipid metabolism parameters, including fatty acid synthase activity and lipolysis rate;

[0025] miR-30e-5p regulatory unit: connected to the adipocyte mimic unit, used to precisely regulate the expression level of miR-30e-5p in the adipocyte mimic unit through transfection technology, with the regulation range being ±100% overexpression or inhibition of expression.

[0026] Endothelial cell effect monitoring unit: connected to the adipocyte simulation unit, used to receive and apply exosomes containing miR-30e-5p released by the adipocytes to the endothelial cells, and to monitor the physiological function indicators of the endothelial cells in real time, including cell proliferation rate, migration ability, oxidative stress level and permeability, with a detection accuracy of not less than 95% for each indicator;

[0027] Data analysis and processing unit: connected to the endothelial cell effect monitoring unit, used to process the acquired endothelial cell function index data, establish a quantitative relationship model between miR-30e-5p level and endothelial cell function index through multiple linear regression analysis, and predict the degree of endothelial cell damage and the probability of atherosclerosis under different miR-30e-5p levels based on the model.

[0028] Preferably, the endothelial cell effect monitoring unit includes:

[0029] Cell proliferation detection module: Using CCK-8 or EdU staining, endothelial cell proliferation is detected every 24 hours for 72 consecutive hours to obtain cell proliferation curves and proliferation rate data. The proliferation rate calculation error is less than 5%.

[0030] Cell migration detection module: Using scratch assay combined with fluorescent labeling technology, cell migration images are captured at 0, 6, 12 and 24 hours after scratching. Cell migration distance and migration speed are calculated by image analysis software. The measurement error of migration speed does not exceed 10% μm / h.

[0031] Oxidative stress detection module: Quantitatively detects intracellular reactive oxygen species (ROS) levels using the DCFH-DA probe combined with flow cytometry, with a detection sensitivity of 10 nM;

[0032] Cell permeability detection module: Based on the permeability of fluorescently labeled FITC-dextran through an endothelial cell monolayer, cell permeability is calculated by measuring fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a microplate reader. The detection limit is 10 pg / cm³. 2 ·s.

[0033] Preferably, the miR-30e-5p antagonist-based atherosclerosis treatment regimen evaluation system includes:

[0034] Animal model construction module: used to cultivate animal models of atherosclerosis, including Apoe- / - mice, ob / ob mice, and C57BL / 6 mice induced by AAV8-PCSK9D377Y and fed a high-fat diet; this module can precisely control the dietary composition of the animal models (the fat content of the high-fat diet can be adjusted within the range of 21%-60% w / w) and the temperature and humidity of the rearing environment;

[0035] Antagonist delivery module: used to administer miR-30e-5p antagonist to the animal model via tail vein injection, intraperitoneal injection or local administration, with the dosage accurate to ±0.1 mg / kg body weight, and the administration frequency can be set to once daily, once every other day or three times a week;

[0036] Treatment efficacy monitoring module: used to evaluate treatment effectiveness, including:

[0037] The area of ​​atherosclerotic plaques in the aorta was determined using Oil Red O staining.

[0038] Serum glutamate and cystine levels and their ratio were detected using enzyme-linked immunosorbent assay (ELISA).

[0039] Perform histopathological examination to assess morphological changes in vascular endothelial cells;

[0040] The coefficient of variation for repeatability of each indicator is less than 15%;

[0041] Data evaluation and feedback module: Based on the data obtained by the efficacy monitoring module, the multi-level fuzzy comprehensive evaluation method is used to quantitatively evaluate the effect of the treatment plan. The evaluation indicators include plaque area reduction rate, degree of improvement of metabolite level, and endothelial cell repair index. The final output is a treatment plan effectiveness score ranging from 0 to 100 points, with a scoring error of no more than 10 points.

[0042] The efficacy monitoring module also includes:

[0043] Vascular function testing submodule: used to evaluate vascular endothelial function in animal models, including using a vascular tonometer to detect the percentage of vasodilation induced by acetylcholine in vascular rings (representing vasodilation function, with a detection error of less than 8%), and using the Griess reagent colorimetric method to determine the level of nitric oxide (NO) release (detection limit of 1 μM).

[0044] The inflammatory factor detection submodule is used to quantitatively detect inflammation-related cytokines (such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) in serum and vascular tissue. It uses an ELISA kit, with a detection range of 10 pg / ml to 10 ng / ml and a detection accuracy of over 90%.

[0045] Preferably, the monitoring of atherosclerosis treatment progress using adipocyte miR-30e-5p levels includes:

[0046] Sample collection unit: Equipped with a multi-functional blood collection needle and disposable blood collection tubes, suitable for collecting arterial blood, venous blood and microvascular blood samples. The blood collection volume is adjustable from 0.1ml to 10ml. The blood collection process meets aseptic operation standards and the blood collection success rate is higher than 98%.

[0047] On-site testing unit: Integrates a portable nucleic acid extractor and a real-time fluorescence quantitative PCR instrument, which can complete the entire process of detecting miR-30e-5p levels in serum from blood samples within 60 minutes after blood collection. The detection limit is as low as 10 copies / ml serum, and the relative standard deviation of the test results is less than 12%.

[0048] Data transmission unit: The miR-30e-5p horizontal data acquired by the field detection unit is encrypted in real time (AES-256-bit) and transmitted to the remote medical terminal via Bluetooth 5.0 or Wi-Fi technology, with a transmission success rate of over 99%.

[0049] Remote analysis unit: Deployed on a cloud server, it uses machine learning algorithms to comprehensively analyze the transmitted miR-30e-5p level data, combined with the patient's pre-treatment baseline data and medication records during treatment, to generate and update a dynamic evaluation report of treatment effect every 72 hours.

[0050] Preferably, the on-site detection unit includes:

[0051] Quality control module: Built-in positive and negative controls and internal reference gene standards, automatically performing quality control tests during each miR-30e-5p test; if the positive and negative control results deviate from the preset standard range or the internal reference gene Ct value fluctuates by more than ±2, the test is deemed invalid; the quality control coverage reaches 100% of the test batches;

[0052] Environmental adaptability module: It has the functions of sensing and compensating for ambient temperature (5℃-40℃) and humidity (10%-90% relative humidity), and the deviation of environmental factors on the test results is controlled within ±8%.

[0053] Preferably, the personalized treatment decision support system for atherosclerosis based on adipocyte miR-30e-5p includes:

[0054] Patient information integration module: used to collect and integrate basic patient information, disease-related information and treatment history information, with a data integrity verification accuracy rate of over 95%;

[0055] The miR-30e-5p multidimensional analysis module is used to jointly analyze the miR-30e-5p level in peripheral blood, the miR-30e-5p expression profile in adipose tissue, and the miR-30e-5p load in serum exosomes. It also combines bioinformatics analysis to predict the targeting strength of miR-30e-5p to the SLC7A11 gene in patients. The confidence interval of the analysis results is 90%-95%.

[0056] Treatment plan recommendation module: Based on the output results of the patient information integration module and the miR-30e-5p multi-dimensional analysis module, it uses deep learning algorithms to select personalized treatment plans from the treatment plan database; the recommended plans cover drug therapy, lifestyle intervention, and surgical treatment options, and quantitatively evaluate the expected efficacy, risk probability, and treatment cost of each plan; the clinical validation accuracy of the recommended plans is over 80%;

[0057] The dynamic treatment plan adjustment module is used to adjust the treatment plan in real time during the treatment process based on the changes in miR-30e-5p levels and the improvement of clinical indicators as regularly monitored by the patient. The adjustment strategies include increasing or decreasing drug dosage, changing combination drug regimens, and strengthening intervention measures. The effective rate of the adjusted plan is increased by no less than 20% compared with the initial plan.

[0058] This invention provides a method for applying the miR-30e-5p level in adipocytes to atherosclerotic diseases. It has the following beneficial effects:

[0059] 1. This invention uses real-time quantitative PCR technology to detect the expression level of miR-30e-5p in serum. Comparison with a preset threshold can aid in the diagnosis of whether an individual has a risk of atherosclerosis. This method has a standardized operating procedure; from blood sample collection to result generation, each step adheres to strict standards and quality control, ensuring diagnostic reliability. Simultaneously, by calculating the patient's relative miR-30e-5p risk index using a formula, the patient's prognosis can be graded, providing important reference for doctors to assess disease progression and formulate treatment plans.

[0060] 2. This invention comprises an arteriosclerosis treatment target screening system, a treatment plan evaluation system, a treatment progress monitoring system, and a personalized treatment decision support system, with each system forming a closed loop through data interaction. For example, the treatment target screening system constructs a model of adipocytes simulating an obese state, regulates miR-30e-5p expression, and monitors endothelial cell function, providing potential target data for the treatment plan evaluation system; the treatment progress monitoring system detects miR-30e-5p levels in real time and transmits the data, providing a basis for the decision support system to optimize treatment plans, thus achieving full-process optimization from target screening, plan evaluation, progress monitoring to decision support.

[0061] 3. This invention integrates multi-dimensional patient information through a personalized treatment decision support system, including basic information, disease-related information, treatment history, and miR-30e-5p levels in peripheral blood, adipose tissue, and serum exosomes. It uses deep learning algorithms to select personalized treatment plans from a treatment plan database, covering options such as medication, lifestyle interventions, and surgery, and quantitatively assesses efficacy, risks, and costs. Simultaneously, the plan is adjusted in real-time based on changes in miR-30e-5p levels and clinical indicators during the patient's treatment process, improving treatment effectiveness and providing patients with more precise and personalized treatment plans. Attached Figure Description

[0062] Figure 1 This is a flowchart of the auxiliary diagnostic method of the present invention;

[0063] Figure 2 This is a flowchart of the prognostic assessment method of the present invention. Detailed Implementation

[0064] The technical solutions of the embodiments of the present invention 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.

[0065] Example:

[0066] Please see the appendix Figure 1 and attached Figure 2 This invention provides a method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases, comprising:

[0067] The auxiliary diagnostic methods specifically include the following steps:

[0068] Step 1: Collect blood samples from the individuals to be tested. In the morning, on an empty stomach, use a multi-functional blood collection device to collect 5ml of blood from both the test group and the normal control group. Strict aseptic technique must be followed during the blood collection process to ensure a success rate of over 98%.

[0069] Step 2: Separate serum from the blood sample. Centrifuge the collected blood sample at 3000 rpm for 10 minutes at room temperature to separate the serum. Immediately store the separated serum sample in a -80℃ freezer for subsequent testing.

[0070] Step 3: The expression level of miR-30e-5p in the serum was detected using real-time quantitative PCR. A gene selected from nematode Cel-miR-39 was used as an internal reference for standardization. Total RNA was extracted from the serum using Trizol reagent, following the reagent instructions. The concentration and purity of the extracted RNA sample were determined using a Nanodrop spectrophotometer, ensuring the A260 / A280 ratio was between 1.8 and 2.2. The extracted RNA was reverse transcribed into cDNA using miR-30e-5p-specific reverse transcription primers. The reaction system was 20 μl, including 5 μl RNA sample, 10 μl reverse transcription buffer, 2 μl reverse transcriptase, and 3 μl miR-30e-5p reverse transcription primers. The reaction conditions were: 16℃ for 30 minutes, 42℃ for 60 minutes, and 85℃ for 5 minutes. The sample was then stored at 4℃. The cDNA obtained from the reverse transcription was used as a template for real-time quantitative PCR amplification of miR-30e-5p. The reaction volume was 20 μl, containing 10 μl SYBR Green PCR Master Mix, 2 μl cDNA template, 0.8 μl upstream primer, 0.8 μl downstream primer, and 6.4 μl lddH2O. Nematode Cel-miR-39 was used as an internal control gene for standardization, with the same reaction volume and primers as miR-30e-5p. PCR conditions were: 95℃ pre-denaturation for 5 minutes, 40 cycles (95℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds), followed by melting curve analysis to ensure the specificity of the amplified products.

[0071] Step 4: Compare the standardized miR-30e-5p expression level of the individual to be tested with a preset miR-30e-5p expression level threshold for the normal control group. If the expression level of the individual to be tested is higher than the threshold, it is used to assist in the diagnosis that the individual has a risk of atherosclerosis. The miR-30e-5p expression level threshold for the normal control group is determined through the following steps: blood samples are collected from 30 healthy individuals in the normal control group; serum is separated and miR-30e-5p expression levels are detected according to the above steps; the mean (M) and standard deviation (SD) of the standardized miR-30e-5p expression level in the normal control group are calculated; and the threshold is set to M+3SD. If the standardized miR-30e-5p expression level of the individual to be tested is higher than this threshold, it is used to assist in the diagnosis that the individual has a risk of atherosclerosis.

[0072] T1. Collect blood samples from no fewer than 30 healthy individuals clinically diagnosed without atherosclerosis;

[0073] T2. Perform steps two and three of claim 1 on the blood samples of the healthy individuals to obtain the normalized expression level of miR-30e-5p for each healthy individual;

[0074] T3. Calculate the mean and standard deviation of the standardized expression level of miR-30e-5p in the healthy individuals, and set the threshold to M+3SD;

[0075] The prognostic assessment method specifically includes the following steps:

[0076] S1. Obtain blood samples from patients with atherosclerosis. Separate serum and detect the standardized expression level of miR-30e-5p. The procedure is the same as the steps above.

[0077] S2. Following steps two and three, separate serum and detect the standardized expression level of miR-30e-5p in the patient's serum. Calculate the relative miR-30e-5p risk index of the patient using the formula. Where N is the average miR-30e-5p expression level in the normal control group.

[0078] S3. Calculate the relative miR-30e-5p risk index of the patient according to the formula: R=[(EN) / N]×100%, where N in S3 is the average miR-30e-5p expression level of the normal control group;

[0079] S4. Patient prognosis is graded and assessed based on the calculated relative miR-30e-5p risk index, with a higher risk index indicating a worse prognosis. The grading includes mild, moderate, and severe adverse outcomes. The grading criteria are: mild adverse (risk index < 2), moderate adverse (2 ≤ risk index < 3), and severe adverse (risk index ≥ 3). Statistical analysis is performed on the auxiliary diagnostic results to calculate the sensitivity, specificity, and accuracy of the method to evaluate its diagnostic performance. Simultaneously, patients are followed up for one year to record clinical outcomes (such as the occurrence of cardiovascular events like myocardial infarction and stroke) to verify the accuracy and reliability of the prognostic grading. The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the invention in any way. Those skilled in the art can make appropriate modifications and variations to the above embodiments without departing from the principles of the invention to adapt to different needs and application scenarios.

[0080] The system for assessing adipocyte miR-30e-5p levels in atherosclerotic diseases includes an atherosclerosis treatment target screening system, an atherosclerosis treatment regimen evaluation system based on miR-30e-5p antagonism, a system for monitoring atherosclerosis treatment progress using adipocyte miR-30e-5p levels, and a personalized atherosclerosis treatment decision support system based on adipocyte miR-30e-5p. These systems are interconnected through data exchange, forming a closed-loop connection, as detailed below:

[0081] The system for screening treatment targets for atherosclerosis is connected to the system for evaluating treatment plans for atherosclerosis based on miR-30e-5p antagonism. The system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is connected to the system for evaluating treatment plans for atherosclerosis based on miR-30e-5p antagonism. The system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is connected to the system for personalized treatment decision support for atherosclerosis based on adipocyte miR-30e-5p. The system for personalized treatment decision support for atherosclerosis based on adipocyte miR-30e-5p is connected to the system for evaluating treatment plans for atherosclerosis based on miR-30e-5p antagonism.

[0082] In the data exchange process between systems, a unified data interface standard should be established. A standardized data format based on HL7 FHIR should be developed to ensure accurate transmission and parsing of data on adipocyte miR-30e-5p levels, treatment target screening results, treatment plan evaluation data, and personalized treatment decision information across different systems. This will eliminate interaction barriers caused by inconsistent data formats, improve data exchange efficiency and accuracy, establish a secure data sharing platform, and clearly define the permissions and scope of data sharing between systems. For example, the atherosclerosis treatment target screening system can share the data of potential targets with the miR-30e-5p antagonism-based atherosclerosis treatment plan evaluation system for use in designing new treatment plans. Simultaneously, real-time data collected by the adipocyte miR-30e-5p level monitoring system for atherosclerosis treatment progress can be fed back to the personalized treatment decision support system, providing a basis for dynamically adjusting treatment plans. During the data sharing process, blockchain technology should be used to ensure data immutability and traceability, guaranteeing data security and reliability.

[0083] The atherosclerosis treatment target screening system includes: an adipocyte simulation unit: used to construct a cell model simulating adipocytes in an obese state, capable of regulating intracellular lipid metabolism parameters, including fatty acid synthase activity and lipolysis rate; a miR-30e-5p regulation unit: connected to the adipocyte simulation unit, used to precisely regulate the expression level of miR-30e-5p in the adipocyte simulation unit through transfection technology, with a regulation range of ±100% overexpression or inhibition; and an endothelial cell effect monitoring unit: connected to the adipocyte simulation unit, used to receive and apply exosomes containing miR-30e-5p released by the adipocytes to endothelial cells, and to monitor the physiological function indicators of the endothelial cells in real time, including cell proliferation rate, migration ability, oxidative stress level, and permeability, with a detection accuracy of not less than 95% for each indicator. The endothelial cell effect monitoring unit includes: cells The cell proliferation detection module uses CCK-8 assay or EdU staining to detect endothelial cell proliferation every 24 hours for 72 consecutive hours, obtaining cell proliferation curves and proliferation rate data with a calculation error of less than 5%. The cell migration detection module uses a scratch assay combined with fluorescent labeling technology to capture cell migration images at 0, 6, 12, and 24 hours after scratching. Image analysis software is used to calculate cell migration distance and speed, with a migration speed measurement error of no more than 10% μm / h. The oxidative stress detection module uses the DCFH-DA probe combined with flow cytometry to quantitatively detect intracellular reactive oxygen species (ROS) levels, with a detection sensitivity of 10 nM. The cell permeability detection module uses the permeability of fluorescently labeled FITC-dextran through an endothelial cell monolayer. A microplate reader is used to measure fluorescence intensity at 485 nm excitation wavelength and 520 nm emission wavelength to calculate cell permeability, with a detection limit of 10 pg / cm³. 2 •s; Data analysis and processing unit: connected to the endothelial cell effect monitoring unit, used to process the acquired endothelial cell function index data, establish a quantitative relationship model between miR-30e-5p level and endothelial cell function index through multiple linear regression analysis, and predict the degree of endothelial cell damage and the probability of atherosclerosis under different miR-30e-5p levels based on the model.

[0084] In the data analysis and processing unit, in addition to multiple linear regression analysis, machine learning algorithms such as random forests and support vector machines are introduced to more accurately model and analyze the complex relationship between miR-30e-5p levels and endothelial cell functional indicators. Simultaneously, by combining proteomics and metabolomics data, the potential signaling pathways regulated by miR-30e-5p are explored in depth, providing a more comprehensive basis for therapeutic target screening.

[0085] In addition, a target validation module is added to validate potential therapeutic targets in vitro and in vivo. In vitro, gene editing technologies (such as CRISPR-Cas9) are used to knock down or overexpress target genes to observe their effects on endothelial cell function and miR-30e-5p regulation. In vivo, gene knockout or transgenic animal models are constructed to assess the role of targets in the development and progression of atherosclerosis, thereby improving the reliability of therapeutic targets.

[0086] The miR-30e-5p antagonist-based atherosclerosis treatment evaluation system includes: an animal model construction module for cultivating atherosclerosis animal models, including Apoe- / - mice, ob / ob mice, and C57BL / 6 mice induced by AAV8-PCSK9D377Y and a high-fat diet; this module can precisely control the dietary composition of the animal models (the fat content of the high-fat diet is adjustable from 21% to 60% w / w) and the temperature and humidity of the rearing environment; and an antagonist delivery module for administering miR... The -30e-5p antagonist was administered to the animal models via tail vein injection, intraperitoneal injection, or local administration, with a dosage accurate to ±0.1 mg / kg body weight. The dosing frequency could be set to once daily, every other day, or three times weekly. The efficacy monitoring module was used to assess treatment effectiveness, including: detecting aortic atherosclerotic plaque area using Oil Red O staining; detecting serum glutamate and cysteine ​​levels and their ratio using enzyme-linked immunosorbent assay (ELISA); performing histopathological examination to assess vascular endothelial cell morphological changes; and detecting various indicators. The repeatability coefficient of variation is less than 15%; the data evaluation and feedback module is used to quantitatively evaluate the treatment effect of the treatment plan based on the data obtained by the efficacy monitoring module using a multi-level fuzzy comprehensive evaluation method. The evaluation indicators include plaque area reduction rate, degree of improvement in metabolite level, and endothelial cell repair index. The final output is a treatment plan effectiveness score ranging from 0 to 100 points, with a scoring error of no more than 10 points; the efficacy monitoring module also includes: a vascular function detection submodule: used to evaluate the vascular endothelial function of animal models, including using a vascular tension meter to detect the percentage of vasodilation induced by acetylcholine in vascular rings (representing vasodilation function, with a detection error of less than 8%), and using the Griess reagent colorimetric method to determine the nitric oxide (NO) release level (detection limit is 1 μM); an inflammatory factor detection submodule: used to quantitatively detect inflammatory-related cytokines (such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) in serum and vascular tissue using an ELISA kit, with a detection range covering 10 pg / ml-10 ng / ml and a detection accuracy of more than 90%;

[0087] In the data evaluation and feedback module, patient-reported outcomes (PROs), such as symptom improvement and quality of life scores, are incorporated into the treatment efficacy evaluation system. A mixed-effects model is used to comprehensively analyze clinical test data and PROs data to more fully assess the effectiveness of the treatment plan and patient satisfaction.

[0088] Simultaneously, a drug safety assessment submodule was added to monitor adverse reactions of miR-30e-5p antagonists in animal models and subsequent clinical trials, including changes in liver and kidney function indicators and hematological parameters. An adverse reaction prediction model was used to provide early warnings of potential serious adverse reactions, offering a reference for optimizing treatment plans.

[0089] The method for monitoring the progress of atherosclerosis treatment using adipocyte miR-30e-5p levels includes: a sample collection unit equipped with a multifunctional blood collection needle and disposable blood collection tubes, suitable for collecting arterial blood, venous blood, and microvascular blood samples, with an adjustable blood volume range of 0.1ml-10ml. The blood collection process conforms to aseptic operation standards, with a success rate of over 98%. An on-site testing unit integrating a portable nucleic acid extractor and a real-time quantitative PCR instrument is used to complete the entire process of detecting miR-30e-5p levels in serum from blood samples within 60 minutes after blood collection. The detection limit is as low as 10 copies / ml serum, and the relative standard deviation of the test results is less than 12%. The on-site testing unit includes: a quality control module with built-in positive and negative controls and internal reference gene standards, automatically performing quality control tests during each miR-30e-5p detection process; if the positive and negative control results... If the deviation from the preset standard range or the fluctuation of the internal reference gene Ct value exceeds ±2, the test is deemed invalid; the quality control coverage reaches 100% of the test batches; the environmental adaptability module has the functions of sensing and compensating for ambient temperature (5℃-40℃) and humidity (10%-90% relative humidity), and the deviation of environmental factors on the test results is controlled within ±8%; the data transmission unit transmits the miR-30e-5p level data acquired by the on-site testing unit in real time with encryption (AES-256 bits) to the remote medical terminal via Bluetooth 5.0 or Wi-Fi technology, with a transmission success rate of over 99%; the remote analysis unit is deployed on a cloud server and is used to use machine learning algorithms to comprehensively analyze the transmitted miR-30e-5p level data, combined with the patient's pre-treatment baseline data and medication records during the treatment process, to generate and update a dynamic evaluation report of treatment effect every 72 hours;

[0090] In the remote analysis unit, in addition to analyzing patients' pre-treatment baseline data and medication records, the system integrates patients' genetic polymorphism data and comorbidity information to construct a personalized treatment outcome prediction model. Artificial intelligence algorithms are used to perform stratified predictions of patients' treatment responses, providing clinicians with more targeted treatment recommendations.

[0091] In addition, a data visualization module has been added to display the trend of miR-30e-5p level changes and the dynamic evaluation results of treatment effects in an intuitive chart format, making it easier for doctors and patients to quickly understand the treatment process and effects.

[0092] The personalized treatment decision support system for atherosclerosis based on adipocyte miR-30e-5p includes: a patient information integration module, used to collect and integrate basic patient information, disease-related information, and treatment history information, with a data integrity verification accuracy rate of over 95%; and a miR-30e-5p multidimensional analysis module, used to jointly analyze the patient's peripheral blood miR-30e-5p level, adipose tissue miR-30e-5p expression profile, and serum exosome miR-30e-5p load, and combined with bioinformatics analysis to predict the targeting strength of miR-30e-5p to the SLC7A11 gene in the patient, with a confidence interval of 90%-95% for the analysis results.

[0093] Treatment plan recommendation module: Based on the outputs of the patient information integration module and the miR-30e-5p multi-dimensional analysis module, it uses deep learning algorithms to select personalized treatment plans from the treatment plan database. The recommended plans cover drug therapy, lifestyle interventions, and surgical treatment options, and the expected efficacy, risk probability, and treatment cost of each plan are quantitatively assessed. The clinical validation accuracy of the recommended plans exceeds 80%. Dynamic treatment plan adjustment module: During treatment, it adjusts the treatment plan in real time based on changes in the patient's miR-30e-5p levels and improvements in clinical indicators from regular follow-up examinations. Adjustment strategies include increasing or decreasing drug dosage, changing combination therapy regimens, and strengthening intervention measures. The effectiveness of the adjusted plan compared to the initial plan is improved by at least 20%.

[0094] In the treatment plan recommendation module, a dynamic linkage mechanism is established with clinical guidelines and expert consensus. The treatment plan database is updated regularly to ensure that recommended personalized treatment plans conform to the latest clinical standards and research progress. Simultaneously, a treatment plan comparison analysis function has been added, providing doctors and patients with detailed comparative information on different treatment plans to help them make more rational treatment decisions. In the dynamic treatment plan adjustment module, reinforcement learning algorithms are introduced to automatically optimize treatment plan adjustment strategies based on patient feedback during treatment. Through continuous learning and accumulation of clinical experience, the accuracy and effectiveness of treatment plan adjustments are improved.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases, characterized in that... include: The auxiliary diagnostic methods specifically include the following steps: Step 1: Collect blood samples from the individual to be tested. Step 2: Separate serum from the blood sample; Step 3: The expression level of miR-30e-5p in the serum was detected by real-time quantitative PCR, and the expression level was standardized using a gene selected from nematode Cel-miR-39 as an internal reference. Step 4: Compare the standardized expression level of miR-30e-5p of the individual to be tested with the preset miR-30e-5p expression level threshold of the normal control group; if the expression level of the individual to be tested is higher than the threshold, it can be used to help diagnose the risk of atherosclerosis in the individual. The prognostic assessment method specifically includes the following steps: S1. Obtain blood samples from patients with atherosclerosis; S2. Separate serum and detect the standardized expression level of miR-30e-5p in the patient's serum according to steps two and three; S3. Calculate the patient's relative miR-30e-5p risk index according to the formula: R = [(EN) / N] × 100%; S4. The patient's prognosis is graded and assessed based on the calculated relative miR-30e-5p risk index, where a higher risk index indicates a worse prognosis. The grades include mild, moderate, and severe adverse outcomes.

2. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 1, characterized in that, The miR-30e-5p expression level threshold in the normal control group was determined through the following steps: T1. Collect blood samples from no fewer than 30 healthy individuals clinically diagnosed without atherosclerosis; T2. Perform steps two and three of claim 1 on the blood samples of the healthy individuals to obtain the normalized expression level of miR-30e-5p for each healthy individual; T3. Calculate the mean and standard deviation of the standardized expression level of miR-30e-5p in the healthy individuals, and set the threshold to M+3SD.

3. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 2, characterized in that, In S3, N represents the average expression level of miR-30e-5p in the normal control group.

4. A system for analyzing adipocyte miR-30e-5p levels in atherosclerotic diseases, using the method of applying the adipocyte miR-30e-5p levels in atherosclerotic diseases as described in any one of claims 1-3, characterized in that... The system includes a target screening system for atherosclerosis treatment, a treatment plan evaluation system for atherosclerosis based on miR-30e-5p antagonism, a system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels, and a personalized treatment decision support system for atherosclerosis based on adipocyte miR-30e-5p. These systems are interconnected through data exchange, forming a closed-loop connection, as detailed below: The system for screening treatment targets for atherosclerosis is connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism. The system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is also connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism. Furthermore, the system for monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels is connected to the system for personalized atherosclerosis treatment decision support based on adipocyte miR-30e-5p. Finally, the system for personalized atherosclerosis treatment decision support based on adipocyte miR-30e-5p is also connected to the system for evaluating atherosclerosis treatment plans based on miR-30e-5p antagonism.

5. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 4, characterized in that, The atherosclerosis treatment target screening system includes: Adipocyte simulation unit: used to construct a cell model simulating adipocytes in an obese state, capable of regulating intracellular lipid metabolism parameters, including fatty acid synthase activity and lipolysis rate; miR-30e-5p regulatory unit: connected to the adipocyte mimic unit, used to precisely regulate the expression level of miR-30e-5p in the adipocyte mimic unit through transfection technology, with the regulation range being ±100% overexpression or inhibition of expression. Endothelial cell effect monitoring unit: connected to the adipocyte simulation unit, used to receive and apply exosomes containing miR-30e-5p released by the adipocytes to the endothelial cells, and to monitor the physiological function indicators of the endothelial cells in real time, including cell proliferation rate, migration ability, oxidative stress level and permeability, with a detection accuracy of not less than 95% for each indicator; Data analysis and processing unit: connected to the endothelial cell effect monitoring unit, used to process the acquired endothelial cell function index data, establish a quantitative relationship model between miR-30e-5p level and endothelial cell function index through multiple linear regression analysis, and predict the degree of endothelial cell damage and the probability of atherosclerosis under different miR-30e-5p levels based on the model.

6. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 5, characterized in that, The endothelial cell effect monitoring unit includes: Cell proliferation detection module: Using CCK-8 or EdU staining, endothelial cell proliferation is detected every 24 hours for 72 consecutive hours to obtain cell proliferation curves and proliferation rate data. The proliferation rate calculation error is less than 5%. Cell migration detection module: Using scratch assay combined with fluorescent labeling technology, cell migration images are captured at 0, 6, 12 and 24 hours after scratching. Cell migration distance and migration speed are calculated by image analysis software. The measurement error of migration speed does not exceed 10% μm / h. Oxidative stress detection module: Quantitatively detects intracellular reactive oxygen species (ROS) levels using the DCFH-DA probe combined with flow cytometry, with a detection sensitivity of 10 nM; Cell permeability detection module: Based on the permeability of fluorescently labeled FITC-dextran through an endothelial cell monolayer, cell permeability is calculated by measuring fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a microplate reader. The detection limit is 10 pg / cm³. 2 ·s.

7. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 4, characterized in that, The miR-30e-5p antagonist-based atherosclerosis treatment regimen evaluation system includes: Animal model construction module: used to cultivate animal models of atherosclerosis, including Apoe- / - mice, ob / ob mice, and C57BL / 6 mice induced by AAV8-PCSK9D377Y and fed a high-fat diet; this module can precisely control the dietary composition of the animal models (the fat content of the high-fat diet can be adjusted within the range of 21%-60% w / w) and the temperature and humidity of the rearing environment; Antagonist delivery module: used to administer miR-30e-5p antagonist to the animal model via tail vein injection, intraperitoneal injection or local administration, with the dosage accurate to ±0.1 mg / kg body weight, and the administration frequency can be set to once daily, once every other day or three times a week; Treatment efficacy monitoring module: used to evaluate treatment effectiveness, including: The area of ​​atherosclerotic plaques in the aorta was determined using Oil Red O staining. Serum glutamate and cystine levels and their ratio were detected using enzyme-linked immunosorbent assay (ELISA). Perform histopathological examination to assess morphological changes in vascular endothelial cells; The coefficient of variation for repeatability of each indicator is less than 15%; Data evaluation and feedback module: Based on the data obtained by the efficacy monitoring module, the multi-level fuzzy comprehensive evaluation method is used to quantitatively evaluate the effect of the treatment plan. The evaluation indicators include plaque area reduction rate, degree of improvement of metabolite level, and endothelial cell repair index. The final output is a treatment plan effectiveness score ranging from 0 to 100 points, with a scoring error of no more than 10 points. The efficacy monitoring module also includes: Vascular function testing submodule: used to evaluate vascular endothelial function in animal models, including using a vascular tonometer to detect the percentage of vasodilation induced by acetylcholine in vascular rings (representing vasodilation function, with a detection error of less than 8%), and using the Griess reagent colorimetric method to determine the level of nitric oxide (NO) release (detection limit of 1 μM). The inflammatory factor detection submodule is used to quantitatively detect inflammation-related cytokines (such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) in serum and vascular tissue. It uses an ELISA kit, with a detection range of 10 pg / ml to 10 ng / ml and a detection accuracy of over 90%.

8. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 4, characterized in that, The method of monitoring the treatment progress of atherosclerosis using adipocyte miR-30e-5p levels includes: Sample collection unit: Equipped with a multi-functional blood collection needle and disposable blood collection tubes, suitable for collecting arterial blood, venous blood and microvascular blood samples. The blood collection volume is adjustable from 0.1ml to 10ml. The blood collection process meets aseptic operation standards and the blood collection success rate is higher than 98%. On-site testing unit: Integrates a portable nucleic acid extractor and a real-time fluorescence quantitative PCR instrument, which can complete the entire process of detecting miR-30e-5p levels in serum from blood samples within 60 minutes after blood collection. The detection limit is as low as 10 copies / ml serum, and the relative standard deviation of the test results is less than 12%. Data transmission unit: The miR-30e-5p horizontal data acquired by the field detection unit is encrypted in real time (AES-256-bit) and transmitted to the remote medical terminal via Bluetooth 5.0 or Wi-Fi technology, with a transmission success rate of over 99%. Remote analysis unit: Deployed on a cloud server, it uses machine learning algorithms to comprehensively analyze the transmitted miR-30e-5p level data, combined with the patient's pre-treatment baseline data and medication records during treatment, to generate and update a dynamic evaluation report of treatment effect every 72 hours.

9. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 8, characterized in that, The on-site detection unit includes: Quality control module: Built-in positive and negative controls and internal reference gene standards, automatically performing quality control tests during each miR-30e-5p test; if the positive and negative control results deviate from the preset standard range or the internal reference gene Ct value fluctuates by more than ±2, the test is deemed invalid; the quality control coverage reaches 100% of the test batches; Environmental adaptability module: It has the functions of sensing and compensating for ambient temperature (5℃-40℃) and humidity (10%-90% relative humidity), and the deviation of environmental factors on the test results is controlled within ±8%.

10. The method for applying the miR-30e-5p level of adipocytes in atherosclerotic diseases according to claim 4, characterized in that, The personalized treatment decision support system for atherosclerosis based on adipocyte miR-30e-5p includes: Patient information integration module: used to collect and integrate basic patient information, disease-related information and treatment history information, with a data integrity verification accuracy rate of over 95%; The miR-30e-5p multidimensional analysis module is used to jointly analyze the miR-30e-5p level in peripheral blood, the miR-30e-5p expression profile in adipose tissue, and the miR-30e-5p load in serum exosomes. It also combines bioinformatics analysis to predict the targeting strength of miR-30e-5p to the SLC7A11 gene in patients. The confidence interval of the analysis results is 90%-95%. Treatment plan recommendation module: Based on the output results of the patient information integration module and the miR-30e-5p multi-dimensional analysis module, it uses deep learning algorithms to select personalized treatment plans from the treatment plan database; the recommended plans cover drug therapy, lifestyle intervention, and surgical treatment options, and quantitatively evaluate the expected efficacy, risk probability, and treatment cost of each plan; the clinical validation accuracy of the recommended plans is over 80%; The dynamic treatment plan adjustment module is used to adjust the treatment plan in real time during the treatment process based on the changes in miR-30e-5p levels and the improvement of clinical indicators as regularly monitored by the patient. The adjustment strategies include increasing or decreasing drug dosage, changing combination drug regimens, and strengthening intervention measures. The effective rate of the adjusted plan is increased by no less than 20% compared with the initial plan.

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