Standardized quantitative fat cell lipolysis method

Abstract

The invention relates to the technical field of cell biology and metabolism research, in particular to a standardized quantitative fat cell lipolysis method, which comprises the following steps: separating and purifying rat epididymis primary fat cells, inoculating to a porous plate, treating by using a reagent, and quantifying fat cell lipolysis by using the specific gravity difference between the fat cells and an aqueous solution. The glycerin release analysis of the cell culture supernatant and the MTT cell activity analysis of the fat cells are sequentially completed in the same hole; and finally, calculating the ratio of the glycerin release light absorption value to the cell activity light absorption value to realize the standardized quantification of the lipolysis level. According to the method, cell activity is introduced as an internal reference, cytotoxicity and operation error interference are effectively eliminated, particularly, the consumption of cell samples is reduced through a detection mode completed in the same hole, limited primary adipocytes obtained from a single experimental animal can support more experimental groups or repetition times, and the detection accuracy is improved. The experimental efficiency and the data output are improved, and the method is suitable for high-throughput screening and evaluation of obesity and metabolic syndrome related drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell biology and metabolism research, and particularly relates to a method for standardizing and quantifying lipolysis of adipocytes. BACKGROUND

[0002] Lipolysis is a core physiological process of energy metabolism in adipocytes, which is characterized by the stepwise hydrolysis of intracellular stored triacylglycerol into free fatty acids and glycerol under the catalysis of lipase. The accurate quantification of this process is of great significance for revealing the regulation mechanism of fat metabolism and screening candidate drugs for obesity and metabolic syndrome, and has become an essential detection step in the development of related drugs.

[0003] At present, the "glycerol release amount" is generally used as a core indicator to evaluate the strength of lipolysis in the research field, and the lipolysis activity is indirectly reflected by measuring the glycerol concentration in the culture medium after cell culture. However, this traditional method has significant limitations in the application of rat epididymal primary adipocyte model:

[0004] Firstly, the interference of cell state is difficult to eliminate: primary adipocytes are abnormally sensitive to separation and digestion conditions and drug treatment, and the fluctuation of cell viability directly affects the reliability of the detection results. The traditional method only detects the total amount of glycerol, and cannot distinguish whether the source of glycerol is the normal lipolysis process or the leakage of intracellular contents caused by cell membrane damage, which is prone to false positive judgment;

[0005] Secondly, it is difficult to standardize the number of cells: due to the significant size heterogeneity (diameter range 20-200 μm) of primary adipocytes, it is difficult to accurately count using a conventional hemocytometer, resulting in a bias in the number of cells inoculated in different experimental groups, which directly affects the comparability and reproducibility of glycerol release amount;

[0006] Thirdly, the limitation of sample size restricts experimental design: the amount of epididymal adipose tissue and primary cells available from a single experimental animal is limited, and the traditional method requires glycerol detection and cell viability analysis in different wells, which consumes a large amount of samples, seriously limiting the number of experimental groups and the number of repetitions that can be set;

[0007] Fourthly, there is a lack of internal reference correction system: the existing method does not integrate cell metabolic activity indicators, which cannot correct the systematic bias introduced by compound toxicity, fluctuation of culture conditions or operation errors, resulting in insufficient specificity of the detection results, and making it difficult to accurately evaluate the specific regulation of drugs on the lipolysis pathway.

[0008] These defects seriously restrict the application value of the traditional glycerol release quantification method in precision medicine research. Therefore, there is an urgent need in the field to develop a detection method that can simultaneously correct the variation of cell viability and number, significantly improve the utilization rate of samples, and have the function of internal reference standardization, to provide reliable technical support for fat metabolism research. SUMMARY

[0009] The present application provides a method for standardizing quantification of adipocyte lipolysis to solve the technical problem of inaccurate quantification caused by cell viability fluctuations, operational errors, and the inability to distinguish between true lipolysis and cell membrane leakage in traditional methods.

[0010] The core of the application is to use the difference in specific gravity between adipocytes and aqueous medium to sequentially complete glycerol release detection and cell viability analysis in the same detection well, and use the ratio of the two as a standardization index, effectively correcting errors caused by cell toxicity, quantity differences, and non-specific leakage, significantly improving detection accuracy and sample utilization.

[0011] The technical solution of the present application includes the following steps:

[0012] Step 1: Isolation and purification of adipocytes:

[0013] Male Wistar rats aged 8 weeks or older and weighing ≥200g were taken after anesthesia to isolate the epididymal fat tissue. The tissue was washed with PBS, cut into small pieces, and digested with trypsin solution containing collagenase type II (10mg / 10mL) at 37°C for 2 hours. After digestion, the filtrate was filtered through a 70μm filter, centrifuged at 120×g for 3 minutes, and the upper layer of adipocytes was collected. After resuspension and washing with PBS, the cells were again centrifuged, and finally suspended in DMEM medium containing 10% FBS and 1% double antibody for ≤30 minutes of recovery culture.

[0014] Step 2: Cell treatment and drug incubation:

[0015] Purified adipocytes were seeded in 96-well plates (200μL / well) at a density of 10 4 -10 5 μL / well), and solvent control (0.05% DMSO) or test compound (e.g. 50μM statin or 0.05-5mM isoproterenol) was added, and incubated at 37°C with gentle shaking for 4 hours.

[0016] Step 3: Glycerol release detection:

[0017] After incubation, the lower layer of medium in each well was collected, and the glycerol detection kit (enzyme method) was used to determine the absorbance value at 570nm, which was recorded as the glycerol release absorbance value (A_glycerol).

[0018] Step 4: Cell viability detection:

[0019] MTT solution (final concentration 0.5 mg / mL) was added to the original hole, and after incubation at 37℃ for 2 hours, the upper adipose cells were collected, transferred to DMSO, vortexed to dissolve the formazan crystal, and the absorbance value at 570 nm was determined, which was recorded as the cell viability absorbance value (A_viability).

[0020] Step 5: Standardized calculation and statistical analysis:

[0021] The standardized glycerol release value was calculated as A_glycerol / A_viability. All experiments were independently repeated 3 times, with 4 replicate wells each time, and the data were expressed as mean ± standard deviation. t-test was performed using GraphPad Prism 6, and P<0.05 was considered as the threshold for significance.

[0022] The preferred embodiments of the present application further include any one or more of the following:

[0023] 1. Adaptive adjustment of digestion conditions:

[0024] For old rats or high-fat diet-induced obese model rats, the fat tissue mass and fibrosis degree are higher, and the concentration of type II collagenase needs to be increased in proportion to the actual isolated fat tissue mass (1.5-3g) to ensure the efficiency of tissue digestion and cell yield.

[0025] 2. Expansion and consistency control of experimental animal strains:

[0026] The Wistar rats can be replaced by Sprague-Dawley (SD) rats or Long-Evans rats and other standard strains, but a single strain must be strictly used in the same experimental batch to ensure consistent genetic background and avoid metabolic differences between strains interfering with experimental results.

[0027] 3. Optimization of cell seeding strategy:

[0028] The standard hemocytometer is used for density assisted adjustment during cell seeding, the core of which is to ensure uniform distribution of cell suspension between wells, rather than pursuing absolute accurate cell count, thereby effectively reducing the group error caused by uneven size of adipocytes.

[0029] 4. Alternative method for cell viability detection:

[0030] The MTT cell viability detection method can be replaced by XTT method, CCK-8 method or other similar cell viability detection methods based on mitochondrial dehydrogenase activity. When using different detection methods, the corresponding reagent concentration, incubation time and detection wavelength need to be adjusted according to the specific reagent instructions.

[0031] The present application successfully solves the three technical problems existing in the traditional method by integrating double-index detection and single-hole operation mode:

[0032] 1. unable to distinguish specific lipolysis from non-specific leakage;

[0033] 2. error introduced by cell number difference;

[0034] 3. single animal cell yield limits experimental design.

[0035] Especially suitable for high-throughput screening of drugs for obesity and related metabolic diseases.

[0036] Compared with the prior art, the present application has the following outstanding advantages:

[0037] 1. The detection accuracy and specificity are significantly improved: By introducing a cell viability index (such as the MTT method) as an internal reference, the ratio of glycerol release to cell viability is used to standardize the data, effectively correcting the systematic errors caused by cell viability fluctuations and cell number differences due to drug toxicity. This method can strictly distinguish between true lipolytic activity and non-specific glycerol leakage caused by cell membrane damage, fundamentally avoiding false positive / negative misjudgments. Experimental verification shows that compounds such as simvastatin do not show lipolytic activity in traditional methods, but after standardization by the present application, they show significant lipolysis-promoting effects (P < 0.05), demonstrating excellent discrimination ability.

[0038] 2. The sample utilization efficiency is significantly improved: Based on the innovative design of completing double-index detection in the same well, the present application greatly reduces the amount of cells required for single detection, making the number of experimental groups and the number of repetitions supported by primary adipocytes isolated from a single rat increase by more than 2 times. This feature greatly alleviates the limitations of the scarcity of primary cell samples, providing feasibility for multiple dose gradient screening, combination drug testing, and other research that requires a large number of parallel experiments.

[0039] 3. Simplify the experimental process and reduce operational errors: Take advantage of the difference in specific gravity between adipocytes and culture medium to complete glycerol release detection and cell viability analysis in the same well. This design not only reduces sample consumption by more than 50%, but also avoids operational errors and cell loss caused by repeated sample transfers, making it particularly suitable for high-precision analysis of primary scarce samples.

[0040] 4. Wide applicability of species and strains: The present application is not only suitable for different strains of rats such as Wistar, SD, and Long-Evans, but also can be extended to primary adipocytes from other species such as mice and humans for evaluation. Only the optimal cell seeding density and incubation conditions need to be calibrated in different species models to achieve standardized comparison of adipolysis activity across species, providing platform technology support for translational medicine research.

[0041] 5. High repeatability and standardization: The present application has systematically optimized and clearly defined the key experimental conditions (such as animal strain, collagenase dosage, centrifugation conditions, incubation time, etc.), and matched with standardized data processing procedures, which ensures that the method can obtain stable and repeatable results under different operators and laboratory conditions, meeting the strict requirements of preclinical drug research on the standardization and reproducibility of detection methods.

[0042] 6. High efficiency in supporting drug screening and mechanism research: The method can clearly distinguish the differential regulation of different structural compounds on lipolysis, providing a reliable and efficient in vitro evaluation tool for drug screening and mechanism analysis of obesity and related metabolic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 is the glycerol standard curve in the present application;

[0044] Figure 2 is the glycerol standard curve at 570 nm wavelength in the present application;

[0045] Figure 3 is the glycerol standard curve verification graph at 570 nm wavelength in the present application;

[0046] Figure 4 is the non-standardized glycerol release index column chart of different concentrations of isoproterenol treatment groups in the present application;

[0047] Figure 5 is the supplementary glycerol standard curve in the present application;

[0048] Figure 6 is the non-standardized glycerol release result column chart of statin treatment groups in the present application;

[0049] Figure 7 is the MTT cell viability detection result column chart of different treatment groups in the present application;

[0050] Figure 8 is the standardized glycerol release value column chart of different concentrations of isoproterenol treatment groups in the present application;

[0051] Figure 9 is the standardized glycerol release value column chart of statin treatment groups in the present application;

[0052] Figure 10 is the flowchart of the method in the present application. DETAILED DESCRIPTION

[0053] The embodiments of the present application will be further described in detail below with reference to the accompanying drawings and examples. The following examples are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0054] Please refer to Figures 1-10 The present application provides a standardized method for quantifying fat decomposition of fat cells, which aims to overcome the defects of traditional glycerol release quantification method that cannot exclude cell viability fluctuations and operation errors, and provides a standardized method based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability determination, which realizes precise quantification of fat decomposition of rat epididymal primary fat cells.

[0055] The core of the present application is to utilize the specific gravity difference between fat cells and aqueous solution, to sequentially complete glycerol release analysis and MTT cell viability analysis in the same well of the same 96-well plate, and to realize standardization through the ratio of glycerol release absorbance value / cell viability absorbance value, to exclude the interference of cell viability and quantity difference. The specific steps are as follows:

[0056] Experimental material preparation:

[0057] (1) Experimental animals:

[0058] Male Wistar rats aged more than 8 weeks and weighing more than 200 g are selected (if replaced by another strain, a single strain such as Sprague-Dawley rats or Long-Evans rats is required to ensure genetic consistency of fat tissue response);

[0059] The epididymal white adipose tissue mass of old rats (≥12 weeks old) or high-fat diet fed rats (fed with high-fat feed for more than 8 weeks) can reach 1.5-3 g, and the concentration of digestive enzymes needs to be adjusted according to the proportion of tissue mass.

[0060] (2) Reagents:

[0061] Digestive reagent: trypsin solution (0.25%, without EDTA), type II collagenase (powder, purity ≥95%);

[0062] Buffer and medium: phosphate buffer (PBS, pH 7.4, without calcium and magnesium ions), Dulbecco's modified Eagle's medium (DMEM, high-sugar type, containing 4.5 g / L glucose), fetal bovine serum (FBS, heat-inactivated, batch number needs to be consistent to reduce batch difference), penicillin-streptomycin solution (10000 U / mL penicillin + 10000 μg / mL streptomycin);

[0063] Detection reagents: MTT powder (purity≥98%), dimethyl sulfoxide (DMSO, cell culture grade), commercially available glycerol release detection kit (based on enzymatic reaction principle, specifically recognizes glycerol and converts it into 570nm detectable light signal), active reagents to be screened (such as 50μM statins: pravastatin, simvastatin, atorvastatin, lovastatin; or 0.05mM / 0.5mM / 5mM isoprenaline);

[0064] Control reagent: 0.05% DMSO (solvent control, consistent with the solvent concentration of the reagent to be screened).

[0065] (3) Instruments:

[0066] Animal surgical instruments: sterile surgical scissors, forceps, culture dishes (60mm);

[0067] Cell processing instruments: cell filter (70μm pore size, nylon membrane), 96-well cell culture plate (flat bottom, transparent), table centrifuge (supports 120×g speed, room temperature centrifugation), constant temperature shaking incubator (37℃, 5% CO2, shaking speed 100-150rpm);

[0068] Detection instruments: microplate reader (supports 570nm wavelength detection, detection range 0-4.0 OD value), hemocytometer (Neubauer counting chamber);

[0069] Data processing software: Microsoft Excel (for calculating mean and standard deviation), GraphPad Prism6 (for statistical analysis).

[0070] Isolation and purification of rat primary epididymal adipocytes:

[0071] (1) Animal anesthesia and tissue isolation:

[0072] According to the dose of 10mL / kg body weight, the male Wistar rats were injected intraperitoneally with 7% chloral hydrate solution (prepared in advance with distilled water, prepared and used immediately);

[0073] 10-15 minutes after injection, the complete anesthesia was confirmed by the disappearance of tail tip needle prick reflex; the rats were fixed supine, the abdomen was shaved and disinfected with 75% ethanol, a 1-2cm incision was made along the midline of the abdomen with sterile surgical scissors, and the epididymal fat pad in the abdominal cavity was exposed;

[0074] The visceral fat tissue on both sides of the epididymal fat pad was carefully separated with forceps (avoiding damage to blood vessels) and placed in a 60mm culture dish containing pre-cooled PBS, and immediately transferred to a clean bench.

[0075] (2) Tissue washing and digestion:

[0076] Rinse the adipose tissue with pre-cooled PBS twice, each time for 5 minutes, to remove surface-adherent blood vessels, blood clots, and other impurities; cut the cleaned tissue into small pieces (1-2 mm3) with sterile scissors (try to cut it into small pieces to ensure complete digestion);

[0077] Transfer the tissue pieces to a 50 mL centrifuge tube at a ratio of 10 mL of digestion solution per 0.6-0.8 g of adipose tissue, and add a trypsin solution containing 10 mg of collagenase type II (dissolve the collagenase type II powder in the trypsin solution in advance, and dissolve it in a 37°C water bath for 10 minutes to ensure that there is no precipitate);

[0078] Place the centrifuge tube in a constant-temperature shaking incubator and incubate it at 37°C with 120 rpm shaking for 2 hours (during the incubation process, gently invert the centrifuge tube every 30 minutes to promote uniform digestion).

[0079] (3) Cell filtration and purification:

[0080] After the incubation is complete, slowly add the digestion mixture to a 70 μm cell filter placed on a 50 mL centrifuge tube using a sterile pipette, gently press the tissue residue, and collect the filtrate (containing adipose cells and digestion solution);

[0081] Place the filtrate in a table-top centrifuge and centrifuge it at 120 x g for 3 minutes at room temperature; after centrifugation, the solution separates into three layers: the upper layer is white suspended adipose cells (low density, floating on the surface), the middle layer is transparent digestion solution, and the lower layer is cell debris and impurities;

[0082] Carefully aspirate the upper layer of white adipose cells with a pipette and transfer them to a new 50 mL centrifuge tube, add 10 mL of pre-cooled PBS, mix gently by blowing, and then centrifuge again at 120 x g for 3 minutes;

[0083] Discard the lower layer of PBS and resuspend the adipose cells in DMEM culture medium containing 10% FBS + 1% penicillin-streptomycin, and place it in a 37°C, 5% CO2 incubator for 20-30 minutes (allow the cells to recover, and the incubation time should not exceed 30 minutes to prevent cell adhesion).

[0084] Cell processing:

[0085] Count the purified adipose cells using a hemocytometer: take 10 μL of cell suspension and add it to a Neubauer counting chamber, and count the number of cells in the central large square (since the size of adipose cells varies greatly, there is no need for accurate counting, and the goal is to evenly distribute the cells in each well);

[0086] According to the ratio of 10 4 -10 5Cells were seeded in 96-well plates at a density of 5,000 cells / well, 200 μL / well, and the plates were gently shaken to ensure even distribution of the cells.

[0087] The treatment reagent was added to each well: 2 μL of 0.05% DMSO was added to the control group (to give a final concentration of 0.05%), and 2 μL of the active agent to be screened (e.g., 50 μM statin, which was diluted to the working concentration in advance using DMEM) was added to the drug group.

[0088] The 96-well plate was placed in a constant-temperature shaking incubator and incubated at 37°C, 5% CO2, 100 rpm, with light shaking for 4 hours (light shaking ensures that the reagent is in contact with the cells and prevents the cells from settling).

[0089] Glycerol release assay:

[0090] After the incubation, the 96-well plate was removed and allowed to stand at room temperature for 5 minutes (the difference in specific gravity allows the adipocytes to float on the surface, and the culture medium to sink to the bottom).

[0091] The lower red culture medium in each well was carefully pipetted using a 100 μL pipette (to avoid pipetting the upper adipocytes) and transferred to the corresponding well of a new 96-well plate.

[0092] The glycerol release assay kit was used according to the manufacturer's instructions: 100 μL of the reaction solution in the kit was added to each well, and the plate was incubated at 37°C for 30 minutes in the dark.

[0093] The absorbance value at 570 nm was measured using a microplate reader (using the blank culture medium as a blank control, and adjusting the zero before detection), and the glycerol release absorbance value in each well was recorded (denoted as A1).

[0094] MTT cell viability assay:

[0095] After the glycerol release assay, the remaining upper adipocytes in the original 96-well plate were retained, and 20 μL of MTT solution was added to each well (an MTT stock solution of 5 mg / mL was prepared in advance using distilled water, sterilized using a 0.22 μm filter, and stored at 4°C for no more than 1 week; the final concentration after addition was 0.5 mg / mL).

[0096] The 96-well plate was returned to the constant-temperature shaking incubator and incubated at 37°C, 5% CO2, for 2 hours (to allow the MTT to enter the adipocytes and be reduced to purple formazan crystals by mitochondrial dehydrogenase).

[0097] After the incubation, the upper purple adipocytes (containing formazan crystals) in each well were pipetted using a pipette and transferred to the corresponding well of a new 96-well plate containing 150 μL of DMSO.

[0098] Place the new 96-well plate on the vortex shaker, vortex at 1000 rpm for 10-30 seconds (until the purple aldehyde crystals are completely dissolved, no visible precipitate);

[0099] Determine the absorbance value at 570 nm with a microplate reader (blank control with blank DMSO, detection after zero adjustment), record the cell viability absorbance value of each well (recorded as A2).

[0100] Standardized calculation and statistical analysis:

[0101] (1) Standardized glycerol release value calculation:

[0102] For each sample in each treatment group, calculate the "standardized glycerol release value" according to the formula:

[0103] Standardized glycerol release value = glycerol release absorbance value (A1) / cell viability absorbance value (A2)

[0104] According to the design of three independent repeated experiments, each with 4 parallel samples (n=4), use Microsoft Excel to process the data:

[0105] Calculate the average value of each group: use the =AVERAGE(range) function to select the standardized values of all parallel samples in the same treatment group;

[0106] Calculate the standard deviation (SD) of each group: use the =STDEV.S(range) function to reflect the dispersion degree between samples.

[0107] (2) Statistical analysis of differences between groups:

[0108] Use GraphPad Prism 6 software for unpaired two-tailed Student's t-test: compare the standardized glycerol release values of each drug group with the control group (0.05% DMSO) pairwise;

[0109] P<0.05 as the statistical significance criterion: P<0.05 indicates that the difference is statistically significant, P<0.01 indicates that the difference is extremely significant, and P>0.05 indicates that the difference is not statistically significant.

[0110] Key considerations:

[0111] Collagenase concentration adjustment: when the adipose tissue mass of old rats or high-fat diet rats is 1.5-3 g, adjust the concentration of the digestive solution according to the proportion of 1 mg type II collagenase per 0.1 g tissue (such as 1.5 g tissue corresponding to 15 mg collagenase, 3 g tissue corresponding to 30 mg collagenase), to avoid under-digestion or over-digestion;

[0112] MTT solution preparation: MTT powder needs to be completely dissolved, avoid repeated freeze-thaw after sterilization; if the solution appears blue precipitate, need to reconfigure;

[0113] Cell seeding uniformity: If large cells (diameter > 50 μm) are encountered during blood cell counting, they can be ignored, and the focus is on ensuring uniform distribution by ensuring that the volume of cell suspension per well is consistent and the culture plate is gently shaken after seeding;

[0114] Morpholine crystal dissolution: vortexing time needs to be controlled within 10-30 seconds, if there is still precipitate, put it in a 37℃ incubator for 5 minutes and then vortex again to ensure the accuracy of the absorbance detection.

[0115] By introducing cell metabolic activity internal reference through MTT method, effectively exclude cell viability fluctuations (such as drug toxicity caused by decreased viability), cell number interference, distinguish "real lipolysis activation" and "non-specific membrane leakage", for example, simvastatin has no significant effect on original glycerol release data, but after standardization, it shows significant lipolysis activity (P<0.05);

[0116] Using the difference in specific gravity between adipocytes and aqueous solution, the glycerol release and MTT detection can be completed in the same well of the same 96-well plate, without transferring cells or changing culture plates, reducing the operation steps (traditional method needs to detect two batches of wells), reducing sample loss and operation error;

[0117] It can accurately evaluate the effect of different compounds on fat decomposition, such as distinguishing the difference between statins (pravastatin and simvastatin significantly promote lipolysis, while atorvastatin and lovastatin have no effect), providing an efficient screening tool for obesity and metabolic syndrome drug research and development;

[0118] Specifically stipulate animal strain, reagent specification, operation parameters (such as centrifugal speed, incubation time) and data processing method, ensure that different laboratories can repeat the experimental results, meet the standardization requirements of drug research and development.

[0119] The key control points of operation need to be quantified, the adipose tissue needs to be light yellow without vascular silk, and the cutting needs to be less than 1 mm³; the centrifuge needs to be calibrated according to the actual parameters of the centrifuge radius, to ensure that the upper cell layer is clear and the middle layer is transparent; the shaking amplitude is 2 mm at 37℃ for 4 h, and MTT incubation appears obvious purple particles in the cells.

[0120] In terms of interference factors, glycerol detection needs to set up a blank group without cells, and the actual A_g needs to be corrected by A_g detection-A blank.

[0121] If the drug to be screened has absorption at 570 nm, pre-experiment of drug-MTT reaction needs to be done, and if necessary, adjust the detection wavelength or use CCK-8 method;

[0122] The cell suspension was blown 10 times before cell seeding to ensure uniform cell number by volume normalization. The method validation should meet the requirements of batch CV <10%, batch-to-batch CV <15%, glycerol recovery rate 85%-115%, and 0.1% Triton X-100 as a membrane leakage positive control to distinguish between true lipolysis and leakage.

[0123] When the application was extended to mouse epididymal primary adipocytes, 6-8-week-old male C57BL / 6 mice were selected, the collagenase concentration was 15 mg / 10 mL, the seeding density was 5×10 4 3.5 h;

[0124] When the application was extended to human subcutaneous primary adipocytes, it needed to be approved by ethics, the digestion time was 2.5-3 h, the culture medium was added with 10 μg / mL insulin and 5 μg / mL transferrin, and the detection wavelength was 560 nm.

[0125] According to the safety regulations, the animals were anesthetized by slow injection of 7% chloral hydrate and prepared with naloxone awakening agent, and the corpses were autoclaved and professionally treated;

[0126] The operation of DMSO was carried out in a fume hood and nitrile gloves were worn, and MTT waste was collected as hazardous chemical waste.

[0127] If the cell yield after digestion is low in the experiment, the collagenase can be replaced, the tissue can be cut, or the digestion time can be extended;

[0128] If the MTT absorbance is low, the separation step can be optimized, the MTT concentration can be increased, or the incubation time can be extended;

[0129] If the CV of the standardized glycerol release value is high, the cell seeding uniformity needs to be ensured, the centrifugal force needs to be calibrated, or a multichannel pipettor needs to be used for sample addition.

[0130] Example 1: Determination of the effect of statins on the lipolysis of rat epididymal primary adipocytes

[0131] 1. Experimental materials:

[0132] Animals: 8-week-old male Wistar rats, weighing 220-250 g, a total of 12 (divided into 3 batches, 4 in each batch, corresponding to 3 independent repeated experiments);

[0133] Drugs: pravastatin, simvastatin, atorvastatin, and lovastatin (all diluted to 50 μM with DMEM, and sterilized with a 0.22 μm filter membrane);

[0134] Control: 0.05% DMSO (prepared with DMEM).

[0135] 2. Experimental steps:

[0136] According to the isolation and purification method of rat primary epididymal adipocytes, about 2.4-3.2 g of adipose tissue was isolated from 4 rats in each batch, and purified adipocytes were obtained after digestion, and inoculated into 96-well plates (10 cells per well, 200 μL / well); 4

[0137] Divided into 5 groups, 8 parallel samples in each group (n=8, including 4 test holes + 4 standby holes): control group (0.05% DMSO), pravastatin group (50 μM), simvastatin group (50 μM), atorvastatin group (50 μM), lovastatin group (50 μM);

[0138] After incubation at 37℃ for 4 hours, the absorbance value was detected by glycerol release analysis and MTT cell viability analysis, and the standardized glycerol release value was calculated and statistically analyzed.

[0139] 3. Experimental results:

[0140]

[0141] 4. Results analysis:

[0142] The original glycerol release data show that only the pravastatin group is significantly higher than the control group (P<0.05), and the simvastatin group has no significant difference;

[0143] The standardized data show that the pravastatin group (P<0.01) and the simvastatin group (P<0.05) are significantly higher than the control group, and the lipolytic activity of pravastatin is stronger; the atorvastatin and lovastatin groups have no difference with the control group;

[0144] The MTT results show that the cell viability of the pravastatin and simvastatin groups is significantly lower than that of the control group (about 40% reduction), indicating that the lipolytic activity of simvastatin in the original data is masked by the decrease in cell viability, and the standardized data can accurately reflect the true lipolytic effect.

[0145] The core design of MTT method for standardizing glycerol release solves the accuracy defect of the traditional fat decomposition quantitative method, and provides a set of simple and reliable detection scheme. The method can not only be used for the exploration of fat metabolism mechanism in basic research, but also can be efficiently applied to the screening and evaluation of obesity and metabolic syndrome related drugs, and has important scientific research and application value.

[0146] ​The embodiments of the present application are presented by way of example and description, and are not intended to be exhaustive or to limit the application to the form disclosed. Many modifications and variations will be apparent to those skilled in the art. Embodiments are chosen and described in order to best explain the principles of the application and its practical application, and to thereby enable others skilled in the art to best utilize the application in various embodiments and with various modifications as are suited to the particular use contemplated.

Claims

1. A standardized and quantitative method for adipocyte fat decomposition, characterized in that, Includes the following steps: (a) Provide isolated and purified adipocytes; (b) Adipocytes were seeded in multi-well plates and incubated with the treatment reagent; (c) After incubation, collect the cell culture medium from the lower layer of the multi-well plate; (d) Detect the amount of glycerol released in the culture medium collected in step (c) to obtain a glycerol release detection value; (e) Add cell viability assay reagent to the same well of the multi-well plate after collecting the culture medium in step (c), incubate, and collect the upper layer of suspended fat cells; (f) Detect the cell viability of the adipocytes collected in step (e) to obtain a cell viability test value; (g) Divide the glycerol release detection value by the corresponding cell viability detection value to calculate a standardized glycerol release value; The study utilized the difference in specific gravity between adipocytes and aqueous culture medium to sequentially detect glycerol release and cell viability in the same well of a multi-well plate, and used standardized glycerol release values ​​to quantify the true level of lipolysis in adipocytes.

2. The method according to claim 1, characterized in that, The adipocytes mentioned in step (a) are primary adipocytes of the rat epididymis.

3. The method according to claim 2, characterized in that, Step (a) includes: taking epididymal adipose tissue from male rats, digesting it with collagenase, filtering it, and purifying it by centrifugation to obtain the adipocytes.

4. The method according to claim 1, characterized in that, The processing reagents described in step (b) include candidate drug compounds to be screened.

5. The method according to claim 1, characterized in that, The glycerol release detection value mentioned in step (d) is the absorbance value measured by an enzyme-linked immunosorbent assay (ELISA) reader.

6. The method according to claim 1, characterized in that, The cell viability test value mentioned in step (f) is the absorbance value obtained by the MTT method, XTT method, CCK-8 method or other similar cell viability test methods based on mitochondrial dehydrogenase activity.

7. The method according to claim 1, characterized in that, The standardized glycerol release value described in step (g) is used to correct for the effect of cell number differences caused by cytotoxicity or operational errors on glycerol release, thereby distinguishing between true lipolysis activity and nonspecific cell membrane leakage.

8. A detection system for quantitative fat decomposition, characterized in that, The system is configured to perform the method described in any one of claims 1 to 7.

9. The use of the method according to any one of claims 1 to 7 in screening drugs that regulate lipid metabolism.

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