Adipose tissue metabolism health related gene for evaluating fat health state and detection kit and application thereof

Abstract

The invention relates to adipose tissue metabolism health related genes for evaluating the health state of fat and a detection kit and application of the adipose tissue metabolism health related genes. Specifically, PLA2G5, GPR120 and downstream signal channel genes thereof can be used as markers for evaluating the health state of adipose tissue metabolism; the kit realizes direct and rapid evaluation of adipose tissues by detecting expression of genes related to metabolism health of the adipose tissues; according to the detection kit, the technical requirements on operators are reduced, the operation steps are simplified, and the detection kit can be applied on a larger scale, so that the early detection and management treatment effects of obesity-related diseases are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection and its related clinical medical application, and in particular to adipose tissue metabolic health-related genes and detection kits and applications thereof for evaluating the health status of fat. Background Art

[0002] Recently, the number of obese people has increased significantly worldwide, and (obesity) has become a worldwide epidemic disease, causing huge social and economic burdens. The diagnosis and prevention of obesity require the detection of multiple biomarkers to comprehensively evaluate the individual's metabolic status and inflammation level. In blood tests, fasting blood glucose, insulin levels, and lipid profiles (including cholesterol and triglycerides) are usually measured by biochemical analyzers. In addition, leptin and adiponectin levels are evaluated by enzyme-linked immunosorbent assay (ELISA) to assess body fat regulation, while high-sensitivity C-reactive protein (CRP) testing is used to assess the degree of inflammation. In urine testing, creatinine and microalbumin levels can be used to assess renal function through biochemical analysis. For tissue testing, gene expression analysis of adipose tissue is performed by real-time fluorescence quantitative PCR and RNA sequencing to help understand the mechanisms of lipid metabolism and inflammation. Imaging methods such as dual-energy X-ray absorptiometry (DEXA) and magnetic resonance imaging (MRI) are used to accurately measure body fat distribution. Emerging technologies such as metabolomics and microbiome analysis have further expanded our understanding of the mechanisms of obesity; metabolomics studies metabolite changes through nuclear magnetic resonance and mass spectrometry, while microbiome analysis studies the relationship between intestinal flora and obesity. Together, these detection technologies provide a scientific basis for personalized obesity intervention and help develop effective prevention and treatment strategies.

[0003] In addition to the above indirect indicators, there are also studies that directly detect the health status of adipose tissue. Currently, the metabolic markers for monitoring the health status of adipose tissue mainly include free fatty acids, leptin, adiponectin, adenosine, and some inflammatory factors such as C-reactive protein (CRP) and tumor necrosis factor α (TNF-α). Some studies have extracted RNA from adipose tissue and used real-time quantitative PCR (qPCR) or RNA sequencing technology (RNA-seq) to analyze the expression levels of specific genes. Commonly concerned genes include genes related to metabolism, inflammation, and adipocyte differentiation, such as PPARγ, FABP4, TNF-α, etc. By comparing the gene expression data of different groups (such as the healthy group and the obesity-related metabolic syndrome group), the metabolic health status of adipose tissue is evaluated, and statistical methods are used to determine the significance of differences in gene expression.

[0004] However, the current detection methods have the following shortcomings: (1) Traditional obesity-related prevention and treatment marker detection, such as blood drawing, gene screening, fecal microbial flora analysis, etc., are complicated to operate, and these methods are mainly based on indirect markers or metabolic risk assessment, lacking direct assessment and detection of adipose tissue to reflect the patient's obesity-related pathological state indicators. (2) Although there are also studies on direct detection of genes related to fat metabolism health in adipose tissue, the detection indicators in these studies are relatively conventional and lack innovation; moreover, the corresponding studies are mostly used in the field of scientific research, and there is no complete set of kits to detect the expression of these related genes in adipose tissue; furthermore, the current methods for sampling and analyzing adipose tissue are usually complicated to operate, requiring high professional skills of operators, and are difficult to use on a large scale in clinical work, limiting their practicality and popularity.

[0005] Adipose tissue, as a core energy storage organ, is directly and closely related to the occurrence and development of obesity. Experimental studies have shown that the collection of abdominal wall adipose tissue samples can provide valuable data to study adipocyte function, changes in inflammatory markers, and gene expression. Different from traditional indirect detection of blood indicators, direct and rapid detection of adipose tissue can not only provide more intuitive information on metabolic health status, but also has important significance for understanding the mechanism of obesity.

[0006] Although the existing technology has obtained adipose tissue samples from the subcutaneous abdomen, such as detecting the levels of HMGA2 and PPAR-γ genes, predicting and / or diagnosing diseases such as obesity, diabetes, cardiovascular disease and metabolic syndrome, and detecting the expression of FABP4 gene in adipose tissue of cynomolgus monkeys based on real-time fluorescence quantitative method, there is still a lack of complete gene detection kits related to fat metabolism health in adipose tissue.

[0007] G protein-coupled receptor 120 (GPR120) belongs to the medium-chain fatty acid receptor family. It is involved in regulating a series of metabolic processes in the body, such as hormone secretion, cell proliferation and lipid production. Studies have found that GPR120 is involved in sugar and lipid metabolism, improving insulin resistance and inhibiting inflammatory response. GPR120 plays a certain role in the treatment of osteoporosis, bone metabolism homeostasis and anti-tumor. GPR120 can be used to predict the aggressiveness and poor prognosis of ovarian cancer. PLA2G5 is a member of the phospholipase A2 family. It is expressed in both immune cells and non-immune cells and participates in the occurrence and development of various biological events. Studies have found that secreted PLA2G5 plays an important role in inflammatory response, atherosclerosis and tumorigenesis. Some literature has studied new genes related to adipose tissue health (Genome-wide discovery of genetic loci that uncouple excess adiposity from its comorbidities), but it does not involve genes related to PLA2G5, GPR120 and their downstream signaling pathways. Therefore, there are currently no studies reporting the relationship between PLA2G5, GPR120 and their downstream signaling pathway-related genes and the metabolic health status of adipose tissue. Summary of the invention

[0008] In order to overcome at least one problem existing in the prior art, the present invention conducts in-depth research on genes related to healthy metabolism of adipose tissue, and finds that PLA2G5, GPR120 and its downstream signaling pathway-related genes, leptin and adiponectin can be used as markers of healthy metabolic adipose tissue; the present invention also provides a rapid detection kit for the expression of genes related to healthy metabolism of adipose tissue, which directly uses adipose tissue as a sample, thereby realizing a direct and rapid evaluation of adipose tissue. The above research and constructed detection kit effectively overcome the following technical problems: (1) Traditional obesity-related prevention and treatment marker detection is mainly based on indirect markers or metabolic risk assessment, lacking the evaluation of adipose tissue directly related to obesity; (2) Traditional adipose tissue metabolic health research methods are relatively complex to operate and analyze, with high costs, requiring high professional level of operators and analysts, and are limited to laboratory research and difficult to promote clinically; (3) Direct detection of genes related to healthy metabolism of adipose tissue is currently limited to scientific research, lacks a complete set of kits, and is difficult to use on a large scale in clinical work; (4) Existing research indicators for direct detection of genes related to healthy metabolism of adipose tissue are relatively conventional.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention is to provide the application of adipose tissue metabolic health-related genes as markers for assessing fat health status, wherein the adipose tissue metabolic health-related genes include at least one of PLA2G5 (group V phospholipase A2), GPR120 (G protein coupled receptor 120) and their downstream signal pathway genes.

[0011] Furthermore, the downstream signaling pathway genes of GPR120 include GLUT4 (glucose transporter 4), INSR (insulin receptor, wherein the insulin receptor substrate IRS is the effector protein of INSR), PI3K (phosphatidylinositol 3-kinase), AKT (serine / threonine protein kinase B), and ERK1 / 2 (extracellular regulated protein kinase 1 / 2).

[0012] Furthermore, the adipose tissue metabolic health-related genes also include adipose factor genes.

[0013] Furthermore, the adipokine genes include leptin and adiponectin.

[0014] Furthermore, the adipose tissue metabolic health-related genes also include genes related to lipid metabolism and fat decomposition.

[0015] Furthermore, the lipolysis-related genes include FASN (fatty acid synthase), SREBF1 (sterol regulatory element binding transcription factor 1), PPARγ (peroxisome proliferator-activated receptor γ), LPL (lipoprotein lipase), HSL (hormone-sensitive lipase), ATGL (attenuated triglyceride lipase), and FABP4 (fatty acid binding protein 4).

[0016] Further, the adipose tissue is selected from orbital fat, abdominal subcutaneous fat, and adipose tissue in serum. Preferably, the adipose tissue is adipose tissue in abdominal subcutaneous fat.

[0017] The second aspect of the present invention is to provide a kit for detecting the expression of adipose tissue metabolic health-related genes, wherein the adipose tissue metabolic health-related genes are any adipose tissue metabolic health-related genes described in the first aspect.

[0018] Furthermore, the kit includes adipose tissue lysate, qPCR premix, primer preformed plate, and plate sealing film. The kit may also include instructions for explaining the components of each reagent and the operating steps of the kit.

[0019] Furthermore, the kit detects the expression of genes related to adipose tissue metabolic health by polymerase chain reaction.

[0020] Furthermore, the kit also includes standards and positive and negative controls to ensure the reliability of the test results.

[0021] Furthermore, the kit also includes a fat tissue collection device, which can be a biopsy needle, anesthesia biopsy needle, etc. conventionally used in the art. The fat tissue used can be directly used for the detection of fat tissue lipid metabolites after sample pretreatment.

[0022] Specifically, the detection method of the above kit is: through rapid RNA purification, impurities are removed, and then the RNA is reverse transcribed into cDNA using a high-efficiency reverse transcriptase and specifically designed primers; next, PCR amplification is performed on a fluorescent quantitative PCR instrument, and Taq polymerase and specific primer pairs are responsible for amplifying lipid metabolism-related genes, and PCR products are detected in real time using fluorescent dyes or probes, and the gene amplification process is monitored by fluorescent signals.

[0023] The third aspect of the present invention is to provide an application of any kit described in the second aspect, which is selected from at least one of the following applications: application in evaluating the expression level of genes related to obesity and metabolic syndrome; application in evaluating the metabolic health status of adipose tissue of a subject; application in screening drugs for preventing and treating diseases related to obesity and metabolic syndrome.

[0024] Specifically, the application areas of the above-mentioned kit include: research on adipose tissue metabolism in the scientific research field, rapid assessment of gene expression levels related to obesity and metabolic syndrome in clinical screening, and assessment of the effects of therapeutic interventions on gene expression in drug development.

[0025] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0026] (1) The present invention innovatively discovered that PLA2G5, GPR120 and their downstream signaling pathways can be used as marker genes for assessing the health status of fat and achieve rapid detection thereof;

[0027] (2) In view of the complexity of the detection of obesity-related markers in the prior art and the lack of assessment of adipose tissue directly related to obesity, the present invention introduces a rapid kit for direct assessment of adipose tissue. By directly detecting the expression of metabolism-related genes in adipose tissue, a direct and rapid assessment of adipose tissue is provided, generating more accurate individual pathological status data, which is more clinically practical and has more diagnostic value than indirect indicators.

[0028] (3) In view of the operational complexity and high cost of the direct detection method of adipose tissue in the prior art, the kit constructed by the present invention reduces the technical requirements for operators and simplifies the steps so that it can be applied on a larger scale, thereby improving the early detection and management treatment of obesity-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0030] Figure 1 Schematic diagram of immunohistochemical staining of CD68, a marker of adipose tissue macrophages, in orbital fat (OF) and abdominal subcutaneous fat (SF) of obese people in one embodiment of the present invention;

[0031] Figure 2 Schematic diagram of the comparison of the expression of GPR120 protein and downstream signaling pathway proteins in the adipose tissue of the orbital fat (OF) and the abdominal subcutaneous fat (SF) of obese people in one embodiment of the present invention;

[0032] Figure 3 This is a schematic diagram of the gene expression of GPR120 and its downstream signaling pathway in adipose tissue of obese mice induced by a high-fat diet in one embodiment of the present invention;

[0033] Figure 4 This is a schematic diagram of protein expression of GPR120 and its downstream signaling pathway in adipose tissue of obese mice induced by a high-fat diet in one embodiment of the present invention;

[0034] Figure 5 Schematic diagram of PLA2G5 gene mRNA expression in orbital fat (OF) and abdominal subcutaneous fat (SF) of obese people in one embodiment of the present invention;

[0035] Figure 6 Schematic diagram of PLA2G5 mRNA expression in adipose tissue of obese mice induced by a high-fat diet in one embodiment of the present invention;

[0036] Figure 7 Schematic diagram of the mRNA expression of adipokine genes leptin and adiponectin in adipose tissue of obese mice induced by a high-fat diet in one embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0038] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0039] Example 1 - Screening of genes related to adipose tissue metabolic health for use as markers for assessing adipose health status

[0040] This embodiment analyzes the expression of genes related to adipose tissue metabolic health to determine markers for assessing the health status of adipose tissue, specifically including the following steps:

[0041] (A) GPR120 (G protein-coupled receptor 120) and downstream signaling pathway genes;

[0042] (1) In adipose tissue with healthy metabolism, GPR120 protein and downstream signaling pathways are active;

[0043] Collection and post-processing steps of orbital fat (OF) and abdominal subcutaneous fat (SF) of obese patients: In clinical practice, patients' excess orbital fat tissue is mainly collected during blepharoplasty, and patients' subcutaneous fat tissue is collected during abdominal wall plastic surgery and liposuction for subsequent experiments. The collection process follows a strict and sterile operating procedure. First, the patient's informed consent and the approval of the ethics committee were obtained. Then, an appropriate amount of adipose tissue was taken out under a sterile environment, and the sample was gently washed with saline under sterile conditions to remove blood and impurities, and then placed in a sterile clean EP tube. The fat samples used for subsequent immunohistochemical observation were fixed in formalin and stored at room temperature to maintain the cell structure. The samples used for the subsequent mRNA expression and protein expression determination steps were kept cold by dry ice, quickly transferred to the laboratory, and kept on ice during transportation. Precautions include maintaining sterile operation, shortening the collection and processing time as much as possible to reduce the risk of sample degradation, and recording each step and any abnormal phenomena observed in detail.

[0044] 1) Immunohistochemical staining of CD68 was used to detect macrophages in adipose tissue and compare the inflammatory conditions of periorbital fat (OF) and abdominal fat (SF) to establish a research model for metabolic health of periorbital adipose tissue. Antigen retrieval was performed at high temperature using sodium citrate buffer to expose antigen epitopes. Then, the sections were treated with hydrogen peroxide to block endogenous peroxidase activity. Next, normal goat serum was added to the sections to block nonspecific binding sites. Subsequently, the primary antibody for CD68 was applied and incubated under appropriate conditions. After washing, the corresponding biotinylated secondary antibody was added and incubated again. After that, horseradish peroxidase-labeled streptavidin and DAB were used for color development to show brown positive signals. Finally, hematoxylin was used for counterstaining to facilitate microscopic observation of cell nuclei, and the sections were dehydrated with gradient alcohol and sealed with neutral gum. In this way, the distribution and density of macrophages can be observed and analyzed under a microscope. The above test results are shown in Figure 1 . .

[0045] like Figure 1 As shown, compared with abdominal subcutaneous fat (SF), orbital fat (OF) of obese people has smaller adipocytes and mild adipose inflammation, which is a metabolically healthy adipose tissue; while SF has larger adipocytes, significant CD68-positive macrophage infiltration (red arrows), and obvious macrophage crown structures (red triangles), indicating chronic inflammation of adipose tissue. The results show that orbital adipose tissue is a good model for studying healthy fat metabolism.

[0046] 2) The expression of target proteins in adipose tissue is mainly determined by Western Blot experiments. First, total protein is extracted from adipose tissue. The homogenized adipose tissue is placed in a lysis buffer containing protease and phosphatase inhibitors. After incubation on ice and sufficient lysis of the tissue, the supernatant is separated as a protein sample by high-speed centrifugation. Subsequently, protein quantification is performed using the BCA method. During the electrophoresis stage, an appropriate amount of protein sample is mixed with a sample buffer containing a reducing agent, boiled and denatured, and then loaded into an SDS-PAGE gel for electrophoretic separation. The separated proteins are transferred to a PVDF membrane by wet transfer or semi-dry transfer. After transfer, nonspecific binding sites on the membrane are blocked using 5% non-fat milk powder in TBST buffer. After blocking, the membrane is incubated in a specific primary antibody solution (anti-GPR120, GLUT4, NF-κb, (p)ERK1 / 2, (p)Akt) at an appropriate dilution overnight at 4°C. After incubation, the membrane is washed with TBST buffer to remove unbound primary antibodies. Next, the membrane was incubated in HRP-labeled secondary antibody solution at room temperature for 1 hour, and then washed again with TBST. Finally, the membrane was immersed in enhanced chemiluminescence (ECL) color development solution, and the protein signal was detected and recorded using a chemiluminescence imaging system. The band intensity of the target protein was quantitatively analyzed by standardization with the internal reference protein GAPDH to compare the differences in protein expression levels and phosphorylation levels in OF and SF samples. The above test results are shown in Figure 2 .

[0047] like Figure 2 As shown, GPR120 and its downstream signaling pathways GLUT4, (p)ERK1 / 2, and (p)Akt were significantly activated in OF.

[0048] (2) further validate GPR120 and downstream signaling pathway genes in high-fat diet-induced obese mice;

[0049] Construction of a high-fat diet-induced obese mouse model. C57BL / 6 mice were used and fed with a high-fat diet for 8 weeks. First, healthy 4-week-old C57BL / 6 mice were selected and fed under normal diet conditions for 1 week to adapt them to the laboratory environment. Then the mice were randomly divided into a normal diet group (control group) and a high-fat diet group (experimental group). The experimental group began to feed a high-fat diet containing 45%-60% fat, while the control group continued to feed a normal low-fat diet. The feed was sourced from Jiangsu Collaborative Biological Co., Ltd. During this period, the mice were supplied with sufficient fresh feed and water every day, and maintained a 12-hour light / dark cycle, a temperature of 22±2°C, and a humidity of 40%-60%. At the same time, the mice were weighed weekly and weight changes were recorded, and the food intake and daily behavioral health status were observed. After 8 weeks of high-fat diet feeding, the weight and length of the mice were measured, the mice were anesthetized, and adipose tissue sampling and blood index determination were performed to evaluate the obesity effect and metabolic status, thereby successfully constructing a high-fat diet-induced obese mouse model.

[0050] After 8 weeks, the mice were anesthetized and the epididymal adipose tissue was collected. The eyeballs were removed using sterile forceps, and the blood that flowed out was quickly and carefully collected into a clean EP tube for subsequent separation of serum. Next, the mice were killed by cervical dissection and dissected to expose the abdominal cavity. The target epididymal adipose tissue was carefully separated using sterile scissors and forceps and quickly placed in a pre-cooled sterile centrifuge tube. The adipose tissue samples were further analyzed or stored at -80°C to maintain their biological activity and integrity for subsequent molecular biology experiments. The above operations must be performed under the premise of ensuring animal welfare and experimental guidelines.

[0051] 1) The qRT-PCR method was used to determine the mRNA expression levels of GPR120 and its downstream GLUT4, IRS1, PI3K, ERK1, ERK2, and Akt in mouse epididymal adipose tissue. The method is briefly described as follows: First, total RNA was extracted from adipose tissue. Commonly used methods include TRIzol reagent or other RNA extraction reagents. After extraction, the concentration and purity of RNA were determined to verify the quality of the extracted RNA. Next, the extracted RNA was reverse transcribed into cDNA using a reverse transcription reagent, and the reaction was carried out under conditions containing reverse transcriptase and oligo dT primers. After synthesizing cDNA, a qPCR reaction system was prepared, which included a cDNA template, specifically designed primers, and SYBR Green fluorescent dye. The reaction system was added to a qPCR plate and amplified on a real-time fluorescence quantitative PCR instrument. During the amplification process, the cycle threshold (Ct value) was determined by detecting changes in the fluorescence signal for quantitative analysis. Normalization was performed using an internal reference gene. Finally, the 2^-△△Ct method was used to calculate the relative expression of the target gene. The above test results are shown in Figure 3 .

[0052] like Figure 3 As shown, compared with mice fed a normal diet, the mRNA expression of GPR120 and its downstream signaling pathway genes in the epididymal adipose tissue of mice fed a high-fat diet-induced obesity was decreased.

[0053] 2) Similarly, the Western Blot method was used to determine the expression of GPR120, GLUT4, and (p)ERK1 / 2 proteins in mouse epididymal adipose tissue. The specific method is as described above. The above test results are shown in Figure 4 .

[0054] like Figure 4 As shown in the figure, compared with mice fed a normal diet, the expression of GPR120 and its downstream signaling pathway proteins in epididymal adipose tissue of high-fat diet-induced obese mice decreased. The above results also prove that GPR120 and downstream signaling pathway genes can be used as markers of healthy metabolic adipose tissue.

[0055] (ii) PLA2G5 (group V phospholipase A2);

[0056] (1) The PLA2G5 mRNA expression level in adipose tissue was determined by qRT-PCR. The specific method was as described above. Figure 5 .

[0057] like Figure 5 As shown, the expression level of PLA2G5 gene in orbital fat (OF) was significantly higher than that in abdominal subcutaneous fat (SF).

[0058] (2) The expression of PLA2G5 mRNA in epididymal adipose tissue of obese and normal diet mice was determined by qRT-PCR method. The specific method is as described above. Figure 6 .

[0059] like Figure 6 As shown, compared with mice fed a normal diet, the mRNA expression of PLA2G5 in the epididymal adipose tissue of mice fed a high-fat diet was decreased, indicating that PLA2G5 can be used as a marker to assess the health status of fat.

[0060] (iii) adipokine genes - leptin and adiponectin;

[0061] The mRNA expression of adiponectin and leptin in epididymal adipose tissue of obese and normal diet mice was determined by qRT-PCR method, and the specific method was as described above. Figure 7 .

[0062] like Figure 7As shown in the figure, compared with mice on a normal diet, the mRNA expression of the adipokine genes leptin and adiponectin in the epididymal adipose tissue of mice with high-fat diet-induced obesity changed significantly. Under obese conditions, adiponectin mRNA expression decreased significantly, while leptin expression increased significantly. This suggests that leptin and adiponectin can be used as markers to assess the health of fat.

[0063] Example 2 - Rapid Detection Kit for Adipose Tissue Metabolic Health-Related Gene Expression

[0064] Based on the research of Example 1, this example constructs a rapid detection kit for the expression of genes related to adipose tissue metabolism health, which can directly and rapidly detect the expression of genes related to adipose tissue metabolism, wherein the gene detection combination includes: 1) group V phospholipase A2 (PLA2G5); 2) G protein-coupled receptor 120 (GPR120) and its downstream signaling pathway genes: glucose transporter 4 (GLUT4), insulin receptor (INSR) and its effector protein insulin receptor substrate (IRS), phosphatidylinositol 3-kinase (PI3K), serine / threonine protein kinase B (AKT), extracellular regulated protein kinase 1 / 2 (ERK1 / 2); 3) adipokine genes: leptin and adiponectin; 4) lipid metabolism and lipolysis related genes: fatty acid synthase (FASN), sterol regulatory element binding transcription factor 1 (SREBF1), peroxisome proliferator-activated receptor γ (PPARγ), lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), fatty acid binding protein 4 (FABP4). This kit mainly detects the expression of the above genes through the polymerase chain reaction (PCR) method, and the results will be used to evaluate the metabolic health status of the subjects' adipose tissue.

[0065] The above-mentioned gene expression rapid detection kit mainly uses the PCR Array method, also known as the PCR array. It fixes the target gene primers in the wells and can quantitatively and qualitatively detect multiple genes at the same time, with high sensitivity and high specificity. Detection principle: Real-time quantitative fluorescence PCR is to achieve quantitative and qualitative analysis of the starting template by real-time detection of the fluorescent signal corresponding to each cycle amplification product of PCR. It adopts the green fluorescent dye method. In the PCR reaction system, green fluorescent dye is added. After the green fluorescent dye is specifically incorporated into the DNA double strand, it emits a fluorescent signal, and the dye molecules that are not incorporated into the chain will not emit any fluorescent signals, thereby ensuring that the increase in the fluorescent signal is completely synchronized with the increase in the PCR product.

[0066] Specifically, the above kit mainly includes: 1) 48 / 96-well primer preformed plate and sealing film; 2) qPCR premix; 3) RNase-free ddH2O; 4) ROX Reference Dye; 5) instruction manual; 6) adipose tissue collection device (the adipose tissue obtained through it can be directly used to detect adipose tissue lipid metabolites after sample pretreatment); 7) standard control cDNA library.

[0067] The operating steps of the above kit are briefly described as follows:

[0068] (1) Obtain adipose tissue using the adipose tissue collection device provided with the kit;

[0069] (2) Using a conventional RNA extraction kit (self-prepared), RNA was extracted from the obtained adipose tissue;

[0070] (3) Use a conventional reverse transcription kit (self-prepared) to reverse transcribe the extracted RNA into cDNA;

[0071] (4) Mix the cDNA obtained in the previous step with the qPCR premix, enzyme-free ddH2O water, and ROX solution provided in this kit to form a qPCR reaction system. Use the same method to mix the standard control cDNA library in the kit with the qPCR premix, enzyme-free ddH2O water, and ROX solution. Then, transfer the solution to the PCR 48 / 96-well primer preformed plate in this kit.

[0072] (3) Use the new sealing film in the product packaging to cover the qPCR reaction plate, making sure that the sealing film is adhered flat and tightly to prevent light refraction and evaporation loss, and centrifuge briefly to remove bubbles.

[0073] (4) Set up the qPCR reaction program and perform the PCR reaction.

[0074] (5) Use the software provided with the PCR instrument to calculate the cycle threshold (Ct) of each gene in the PCR chip.

[0075] (6) Using 2^ -△△Ct The method calculates the expression changes of specific genes.

[0076] (7) Using the gene expression levels of the control samples provided by the kit as a reference for comparison (the specific range can be determined by further in-depth research), the relative expression levels of the genes related to the health of the patient's adipose tissue are evaluated.

[0077] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. The application of genes related to adipose tissue metabolism health as markers for assessing fat health status, characterized in that: The adipose tissue metabolic health-related genes include at least one of PLA2G5, GPR120 and downstream signaling pathway genes thereof.

2. The use according to claim 1, characterized in that: The downstream signaling pathway genes of GPR120 include GLUT4, INSR, PI3K, AKT, and ERK1 / 2.

3. The use according to claim 1, characterized in that: The adipose tissue metabolic health-related genes also include adipokine genes, lipid metabolism and fat decomposition-related genes; wherein the adipokine genes include leptin and adiponectin; the lipid metabolism and fat decomposition-related genes include FASN, SREBF1, PPARγ, LPL, HSL, ATGL, and FABP4.

4. A kit for detecting the expression of genes related to adipose tissue metabolic health, characterized in that: The adipose tissue metabolic health-related genes include at least one of PLA2G5, GPR120 and downstream signaling pathway genes thereof.

5. The kit according to claim 4, characterized in that The downstream signaling pathway genes of GPR120 include GLUT4, INSR, PI3K, AKT, and ERK1 / 2.

6. The kit according to claim 4, characterized in that The adipose tissue metabolic health-related genes also include adipokine genes, lipid metabolism and fat decomposition-related genes; wherein the adipokine genes include leptin and adiponectin; the lipid metabolism and fat decomposition-related genes include FASN, SREBF1, PPARγ, LPL, HSL, ATGL, and FABP4.

7. The kit according to claim 4, characterized in that The kit comprises adipose tissue lysate, qPCR premix, primer preformed plate and plate sealing film.

8. The kit according to claim 4, characterized in that The kit detects the expression of genes related to adipose tissue metabolism health by polymerase chain reaction method.

9. A use of a kit as claimed in any one of claims 4 to 8, characterized in that: The application is selected from at least one of the following applications: application in evaluating the expression level of genes related to obesity and metabolic syndrome; application in evaluating the metabolic health status of adipose tissue of a subject; application in screening drugs for preventing and treating obesity and metabolic syndrome related diseases.

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