Detection method for stimulating cell fat synthesis by insulin analogue and product and application thereof

Abstract

The invention belongs to the field of biological activity detection, and particularly relates to a detection method for stimulating cell fat synthesis by an insulin analogue and a product and application thereof. The detection method provided by the invention comprises the step of applying cultured 3T3-L1 precursor fat cells to insulin analogue synthesis fat so as to evaluate the biological activity of the insulin analogue. The insulin analogue is diluted by using a diluent; the diluent contains dexamethasone and rosiglitazone, and the diluent contains dexamethasone and rosiglitazone; the diluent also contains a GM culture medium. The diluent provided by the invention can well cooperate with stimulation of gradient series insulin analogues, so that the detection method can be quantified. The detection method provided by the invention also has the advantages of good stability and reproducibility, high sensitivity, simple operation, no toxicity and no harm.

Description

Technical Field

[0001] The invention belongs to the field of biological activity detection, and specifically relates to a detection method for insulin analogs to stimulate cell fat synthesis, and a product and application thereof. Background Art

[0002] Biological activity is an important indicator of product quality and clinical effectiveness. During the research and development stage of new drugs, it is necessary to establish biological activity analysis methods that are the same or related to the product's in vivo mechanism of action for the functional activity research of the product, which has a large number of applications throughout the product life cycle. The methods used for biological activity determination mainly include animal-based biological activity detection methods and cell culture-based biological activity detection methods. Cell biological activity determination uses the reactivity of cells cultured in vitro to samples to evaluate the biological activity of the product.

[0003] With the establishment of high-throughput drug screening platforms and the expansion of production scale, as well as the deepening understanding of the 3R (Reduction, Refinement, Replacement) principle, people are increasingly seeking alternatives to animal experiments. Cell-based in vitro bioactivity analysis methods have the advantages of high throughput, high efficiency, and high precision.

[0004] In general, biological activity is expressed in potency, which is the unit of activity obtained by comparison with a reference substance. Quantitative biological activity assays should select reaction systems with low background and high signal-to-noise ratio to obtain better reproducible dose-response curves.

[0005] Insulin can increase the activity of lipoprotein lipase, increase the uptake of glucose, fatty acids and triglycerides by the liver and adipose tissue, increase fat synthesis and store it in fat cells. Therefore, the biological activity of insulin can be reflected by detecting the content of fat (triglycerides).

[0006] Fully differentiated 3T3-L1 adipocytes are currently considered to have good stability and high sensitivity to insulin, and are suitable as a cell model for insulin-stimulated cellular lipid synthesis. However, the inventors found in previous studies that no matter how the adipocyte differentiation induction method and sample detection process were adjusted, the final result was that the adipocytes had too high adipose content (i.e., high background), resulting in unclear or unstable insulin stimulation results, and thus unable to effectively accept insulin stimulation to produce fat.

[0007] In addition, there are currently no reports on the effects of insulin stimulation on fat synthesis under different insulin concentration gradients, that is, the detection index cannot be quantified. Moreover, the two common technologies for detecting fat, Oil Red O staining and radioactive element method, both have great limitations. Oil Red O staining can only be used as a qualitative test. The radioactive element method is inconvenient to implement due to the incorporation of radioactive elements, and has high requirements for the experimental environment and experimental personnel, and cannot meet the needs of general companies for biological activity detection.

[0008] Therefore, there is an urgent need to develop a detection method that can stably quantify insulin-stimulated fat synthesis. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a method for detecting the biological activity of insulin analogs. The method uses precursor adipocytes as a model and quantifies insulin analogs by detecting the content of fat (triglycerides).

[0010] In one aspect, the present invention provides a method for detecting insulin analogs stimulating cellular fat synthesis, the method comprising the following steps:

[0011] S1, culturing preadipocytes to obtain cell A;

[0012] S2. Add insulin analogs to cell A and continue culturing to obtain cell B

[0013] S3, lyse cell B to obtain lysate;

[0014] S5, detecting the fat content in the lysate;

[0015] Step S2: the insulin analog is diluted with a diluent; the diluent contains dexamethasone and rosiglitazone.

[0016] Specifically, the final concentration of dexamethasone in step S2 is 0.5-2 μM; the final concentration of rosiglitazone is 2.2-3.4 mM.

[0017] More specifically, the final concentration of dexamethasone in step S2 is 1 μM; the final concentration of rosiglitazone is 2.8 mM.

[0018] Specifically, the diluent in step S2 also includes growth medium (GM medium).

[0019] More specifically, the GM culture medium includes DMEM, fetal bovine serum and penicillin-streptomycin.

[0020] Preferably, the GM culture medium comprises, by volume, 83.5-91.5% DMEM, 8-15% fetal bovine serum and 0.5-1.5% penicillin-streptomycin.

[0021] Further preferably, the GM culture medium comprises, by volume, 89% DMEM, 10% fetal bovine serum and 1% penicillin-streptomycin.

[0022] Specifically, the insulin analogs described in step S2 include, but are not limited to: insulin glargine, insulin degludec, insulin lispro, insulin degludec, insulin aspart or insulin detemir.

[0023] Preferably, the insulin analog in step S2 may be insulin glargine or insulin degludec.

[0024] Specifically, the precursor fat cells in step S1 can be derived from embryonic development, adipose tissue, stem cell differentiation, cell lines, and in vitro culture or induced pluripotent stem cells.

[0025] Preferably, the preadipocytes in step S1 can be derived from cell lines and cultured in vitro.

[0026] More preferably, the preadipocytes are 3T3-L1 preadipocytes.

[0027] Specifically, the culture medium used in the culture in step S1 is a cell culture medium (CM culture medium).

[0028] More specifically, the CM culture medium includes DMEM, calf serum and penicillin-streptomycin.

[0029] Preferably, the CM culture medium comprises, by volume, 83.5-91.5% DMEM, 8-15% calf serum and 0.5-1.5% penicillin-streptomycin.

[0030] More preferably, the CM culture medium comprises, by volume, 89% DMEM, 10% calf serum and 1% penicillin-streptomycin.

[0031] Specifically, the method for detecting the fat content in step S5 includes, but is not limited to: spectroscopy, chromatography, mass spectrometry or lipid oxidation detection.

[0032] More specifically, the method for detecting fat content comprises the following steps:

[0033] (1) Lysing cells to obtain lysate;

[0034] (2) taking the lysate, adding lipoprotein lipase solution and incubating;

[0035] (3) adding triglyceride detection reagent and incubating;

[0036] (4) measuring the luminescent signal on an ELISA reader;

[0037] (5) Analyze the luminescence signal, use the four-parameter regression of the constraint model to fit the standard curve and the sample curve, and calculate the relative potency of the sample.

[0038] More specifically, in step (1), the lysate is produced by cell lysis stimulated by different concentrations of insulin analog standards and / or test samples.

[0039] More specifically, analyzing the luminescent signal in (5) of the detection method comprises the following steps:

[0040] 1) Use the four-parameter regression of the unconstrained model to fit each curve and evaluate the system suitability:

[0041] Analysis window = D / A

[0042] Maximum signal ratio = A TS / A RS

[0043] Dynamic Range Ratio = (DA) TS / (DA) RS

[0044] Effective slope ratio = [B × (DA) TS ] / [B×(DA) RS ]

[0045] In the formula, A represents the lower limit of absorbance, which represents the lower asymptote of the S-shaped curve; D represents the upper limit of absorbance, which represents the upper asymptote of the S-shaped curve; B is the absorbance increase rate parameter, which is equivalent to the slope of the curve; subscript TS represents the sample; subscript RS represents the standard.

[0046] 2) The four-parameter regression of the constraint model was used to fit the standard curve and the sample, and the relative potency of the sample was calculated as follows:

[0047] Relative potency = C RS / C TS

[0048] In the formula, C is the concentration at which the absorbance growth rate begins to change, that is, the half-effective reaction concentration (EC 50 );Subscript TS stands for sample;Subscript RS stands for standard。

[0049] In yet another aspect, the present invention provides a kit, comprising the reagents or culture medium in the aforementioned detection method.

[0050] In another aspect, the present invention provides the use of the aforementioned detection method or kit in detecting insulin analogs stimulating fat synthesis.

[0051] The technical effects achieved by the present invention are:

[0052] (1) The use of 3T3L1 precursor cells avoids the process of adipocyte differentiation induction, reduces the introduction of variables, and greatly shortens the duration of the entire detection process.

[0053] (2) The detection method of the present invention can be quantified and compared.

[0054] (3) The detection method of the present invention is stable and has good reproducibility.

[0055] (4) The detection method of the present invention is easy to operate and will not cause fat (triglyceride) loss to affect the results.

[0056] (5) The method provided by the present invention has good sensitivity and specificity and has few restrictions on samples, that is, it can detect low-concentration samples and is not affected by other components in the sample.

[0057] (6) The detection method of the present invention avoids the use of radioactive elements, and the entire experiment is non-toxic and harmless and will not cause harm to the body of the experimenter. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of fat detection principle in a basic embodiment.

[0059] Figure 2 It is a curve chart of the test results of Example 1, wherein the hollow circles represent the standard, the hollow squares represent the samples with a theoretical potency of 1, the triangles represent the samples with a theoretical potency of 0.5, and the diamonds represent the samples with a theoretical potency of 0.75; the solid circles represent the samples with a theoretical potency of 1.25, and the solid squares represent the samples with a theoretical potency of 1.5.

[0060] Figure 3 This is the linear graph of Example 4.

[0061] Figure 4 It is a curve chart of the test results of Example 5, where the circle represents the standard, the square represents the sample with a theoretical potency of 1, the triangle represents the sample with batch number F20240303, and the diamond represents the sample with batch number DBJL054B.

[0062] Figure 5 It is a curve chart of the test results of Example 8, wherein the hollow circles represent the standard, the hollow squares represent the samples with a theoretical potency of 1, the triangles represent the samples with a theoretical potency of 0.5, and the diamonds represent the samples with a theoretical potency of 0.75; the solid circles represent the samples with a theoretical potency of 1.25, and the solid squares represent the samples with a theoretical potency of 1.5.

[0063] Figure 6It is a curve chart of the test results of Comparative Example 1, where the circle represents the standard, the square represents the sample with a theoretical potency of 1, the triangle represents the sample of the excipient for glargine insulin injection (F20240302); and the diamond represents the BSA sample. DETAILED DESCRIPTION

[0064] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0065] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary explanation of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0066] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0067] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Products from different manufacturers have no significant effect on the effects.

[0068] The term "theoretical potency level (relative potency theoretical value)" refers to the potency value obtained under specific conditions based on theoretical calculations or standard regulations, which is used to compare with the actual measured potency value to evaluate the accuracy and reliability of the measurement.

[0069] The term "relative potency measurement value" refers to the comparison of the biological response of the test article with the response produced by a known standard in the biological activity / potency assay of biological products, thereby quantitatively determining the potency of the test article relative to the standard. The relative potency theoretical value is dimensionless.

[0070] Basic Example

[0071] 1. Fat detection process

[0072] 3T3 L1 precursor cells were inoculated into 96-well plates and cultured in cell culture medium (CM) for 3 days. A certain concentration of synergistic factors (final concentration of 1 μM dexamethasone and 2.8 mM rosiglitazone) was added to growth medium (GM) to dilute the insulin analog injection sample (taking glargine insulin injection as an example, each milliliter of glargine insulin injection contains 3.64 mg of glargine insulin main component, 30 μg zinc ion, 2.7 mg of m-cresol and 17 mg of glycerol) into 8 concentration gradients, with specific concentrations of 6000 nM, 2000 nM, 666.67 nM, 222.22 nM, 74.074 nM, 14.8 nM, 2.96 nM, and 0.593 nM, respectively. Then, the samples were added into 96-well plates to stimulate cells to synthesize fat. After 4 days of stimulation, the cells were lysed, and the intracellular fat content was detected using a fat detection kit (manufacturer: Biyuntian, catalog number: S0219S). The principle of fat detection is that triglycerides are hydrolyzed into glycerol and fatty acids by lipoprotein lipase, and the generated glycerol is phosphorylated into glycerol-3-phosphate and ADP under the action of ATP and glycerol kinase. The generated glycerol-3-phosphate undergoes oxidation reaction with oxygen under the action of glycerol phosphate oxidase to produce dihydroacetone phosphate (DHAP) and H2O2. The light signal intensity of resorufin, the reaction product of H2O2 and Amplex Red (fluorescent red dye), is then detected to finally detect the triglyceride content. The light signal intensity of resorufin is proportional to the triglyceride content in the sample, see Figure 1 .

[0073] 2. Preparation of main solution

[0074] (1) 0.012 M HCl solution: Add 10 μL of 12 M HCl to 10 mL of water and mix thoroughly to obtain 0.012 M HCl.

[0075] (2) 2.5 mM dexamethasone solution: Weigh 10 mg of dexamethasone, add anhydrous ethanol filtered through a 0.22 μm syringe filter to dissolve, and dilute to 10 mL. Store at 2-8°C, with a shelf life of one month.

[0076] (3) 250 mM IBMX solution: Weigh 555 mg of 3-isobutyl-1-methylxanthine, add dimethyl sulfoxide to dissolve, dilute to 10 mL, dispense 0.15 mL per tube, store at -20°C, valid for one year.

[0077] (4) 10 mg / mL rosiglitazone solution: Add 1 mL of dimethyl sulfoxide to 10 mg / bottle of rosiglitazone to dissolve. Dispense into 10 μL vials and store at -20°C. The shelf life is one year.

[0078] (5) Lipoprotein lipase solution: Take 6400 μL of triglyceride detection buffer, add 320 μL of lipoprotein lipase, and mix well. Or prepare according to the proportion. Prepare and use immediately.

[0079] (6) Triglyceride detection working solution: Take 7040 μL triglyceride detection buffer, 320 μL Amplex red, 320 μL enzyme mixture, and 320 μL auxiliary factor, and mix well. Or prepare according to the proportion. Prepare and use immediately.

[0080] 3. Culture medium composition (percentages are by volume)

[0081] (1) Cell culture medium (CM): 89% DMEM (ATCC), 10% calf serum and 1% penicillin-streptomycin.

[0082] (2) Growth medium (GM): 89% DMEM (Gibco), 10% fetal bovine serum and 1% penicillin-streptomycin.

[0083] (3) Sample dilution medium (SDM): 50 mL GM, 20 μL 2.5 mM dexamethasone solution, 5 μL 10 mg / mL rosiglitazone solution, or prepared in proportion.

[0084] 4. 3T3-L1 precursor cell culture

[0085] (1) Count the cells and add cell culture medium (CM) to adjust the cell density. 4 The cells were seeded at a density of 100 μL / well in a 96-well culture plate and placed in a humidified box and cultured in a 37°C, 5% CO2 incubator for 3 days.

[0086] (2) Sample dilution: The standard (RS) and test sample (TS) were diluted from the stock solution to 6000 nM. The samples were diluted with sample dilution medium (SDM) to make a total of 8 gradient dilution concentration points (6000 nM, 2000 nM, 666.67 nM, 222.22 nM, 74.074 nM, 14.8 nM, 2.96 nM, 0.593 nM).

[0087] Note: In general, biological activity is expressed in potency, which is an activity unit obtained by comparison with a reference substance. Therefore, in order to obtain a quantifiable potency value, both the standard and the sample need to be prepared simultaneously in each experiment.

[0088] 5. Fat (triglyceride) determination

[0089] (1) Aspirate the cell supernatant, add 200 μL PBS to each well for washing, then add 50 μL isopropanol and lyse for 10 minutes.

[0090] (2) Add 42 μL of lipoprotein lipase solution to the test plate, and take 8 μL of the sample prepared with isopropanol into the corresponding position of the test plate according to the plate layout. Incubate for 20 minutes.

[0091] (3) Add 50 μL of triglyceride detection working solution to each well and incubate for 60 minutes.

[0092] (4) Measure the luminescent signal on an ELISA reader.

[0093] 6. Analysis

[0094] (1) The four-parameter regression of the unconstrained model was used to fit each curve and the applicability of the system was evaluated as follows:

[0095] Analysis window = D / A

[0096] Maximum signal ratio = A TS / A RS

[0097] Dynamic Range Ratio = (DA) TS / (DA) RS

[0098] Effective slope ratio = [B × (DA) TS ] / [B×(DA) RS ]

[0099] In the formula, A represents the lower limit of absorbance, which represents the lower asymptote of the S-shaped curve; D represents the upper limit of absorbance, which represents the upper asymptote of the S-shaped curve; B is the absorbance increase rate parameter, which is equivalent to the slope of the curve; subscript TS represents the sample; subscript RS represents the standard.

[0100] (2) Applicability of curve system:

[0101] R of standards and samples 2 Not less than 0.95;

[0102] The analysis window is not less than 2;

[0103] The maximum signal ratio is between 0.6 and 1.4;

[0104] The dynamic range ratio is between 0.6-1.4;

[0105] The effective slope ratio is between 0.6 and 1.4.

[0106] (3) The four-parameter regression of the constraint model was used to fit the standard curve and the sample, and the relative potency and potency recovery rate of the sample were calculated as follows:

[0107] Relative potency = C RS / C TS

[0108] Potency recovery rate = (relative potency measured value / relative potency theoretical value) × 100%

[0109] In the formula, C is the concentration value at which the absorbance growth rate begins to change, that is, the half-effective reaction concentration (EC50); the subscript TS represents the sample; and the subscript RS represents the standard.

[0110] Example 1 Establishment of a method for detecting lipogenesis in 3T3L1 precursor cells stimulated by glargine insulin

[0111] According to the record in the validation of the ICHQ2 (R2) analytical method, five relative potency levels (0.5, 0.75, 1, 1.25, 1.5; that is, the theoretical potency is 0.5 times, 0.75 times, 1 times, 1.25 times and 1.5 times of RS, respectively, and the specific operation is to dilute the standard to 3000nM (0.5), 4500nM (0.75), 6000nM (1), 7500nM (1.25), 9000nM (1.5) as the starting concentration, and then perform gradient dilution simultaneously, the same below) are determined by the method of the basic embodiment. The test result curve is shown in FIG. Figure 2 The curve system suitability results are shown in Table 1, and the relative potency determination values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 2.

[0112] Table 1 Curve system applicability results

[0113]

[0114] Table 2 Relative potency determination values ​​and potency recovery results

[0115] Relative potency theoretical value Relative potency determination value Titer recovery rate RS N / A N / A 0.5 0.377 75.4％ 0.75 0.788 105.1％ 1 1.101 110.1％ 1.25 1.237 99.0％ 1.5 1.184 78.9％

[0116] Result analysis:

[0117] The results show that R 2 All of them can reach above 0.99 and close to 1, and the concentration points of the upper platform, lower platform and intermediate transition period of the curve are evenly distributed, indicating that the four-parameter curve fit is excellent. The analysis window, maximum signal ratio, dynamic range ratio, effective slope ratio and relative activity all meet the system adaptability requirements, indicating that the method can be used for comparative research and quantitative indicators.

[0118] Example 2 Confirmation of the accuracy of the detection method for glargine insulin stimulating 3T3L1 precursor cell fat synthesis

[0119] In order to further confirm the accuracy of the fat synthesis detection method, the accuracy of the fat detection method was verified according to the accuracy requirements in the verification of the ICHQ2 (R2) analytical method. Two experimenters performed relative potency determinations on five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) according to the method of the basic embodiment at different time points.

[0120] Relative bias (RB), geometric mean (GM) of relative activity for each titer level and across all titer levels were calculated.

[0121] Relative bias calculation formula:

[0122] Relative bias (RB) = [(relative potency measured value / relative potency theoretical value) - 1] × 100%

[0123] The accuracy results are shown in Table 3:

[0124] Table 3 Accuracy results analysis

[0125]

[0126]

[0127] Result analysis:

[0128] The results showed that the relative bias (RB) of the titer measurement value at each titer level was between -40% and 40%; the geometric mean (GM) of the relative activity at each titer level and the overall level was between 0.60 and 1.40, indicating that the detection method had good accuracy.

[0129] Example 3 Confirmation of the precision of the detection method for glargine insulin stimulating lipogenesis in 3T3L1 precursor cells

[0130] In order to further confirm the fat synthesis detection method, based on the experiment of Example 2, according to the precision requirements in the verification of the ICHQ2 (R2) analytical method, the precision of the fat detection method was analyzed, mainly based on the relative activity to calculate the geometric standard deviation (GSD) and geometric coefficient of variation (GCV). The calculation method is as follows:

[0131] GSD=antilog(SD)

[0132] GCV=(GSD-1)×100%

[0133] Where: SD is the logarithmic standard deviation of the relative activity at each titer level and the overall level.

[0134] The precision results are shown in Table 4:

[0135] Table 4 Precision results analysis

[0136]

[0137]

[0138] Result analysis:

[0139] The results showed that the geometric standard deviation (GSD) of the relative activity of each titer level and the overall level was between 0.6 and 1.4; the geometric coefficient of variation (GCV) of the relative activity of each titer level and the overall level was no more than 30%. This indicated that the detection method had good precision.

[0140] Example 4 Confirmation of linearity and range of the detection method for glargine insulin-stimulated 3T3L1 precursor cell lipogenesis

[0141] In order to further confirm the fat synthesis detection method, based on the experiment of Example 2, according to the requirements for linearity and range in the validation of the ICHQ2 (R2) analytical method, the linearity and range of the fat synthesis detection method were evaluated using the data of the five potency levels of all test samples. The correlation coefficient R was obtained by linear fitting with the theoretical potency value as the X-axis and the measured potency value as the Y-axis. 2 , and report the equation and graph at the same time. Linearity and range results are shown in Tables 5 and Figure 3 .

[0142] Table 5 Linearity and range results analysis

[0143]

[0144] Result analysis:

[0145] The results show that the linear regression equation correlation coefficient R 2 ≥0.95. This indicates that the linearity of the fat synthesis detection method is good, and the detection range is between 50% and 150%.

[0146] Example 5 Confirmation of the detection method of glargine insulin injection from different sources and batches to stimulate 3T3L1 precursor cell fat synthesis

[0147] To further confirm the applicability of the method, different batches of glargine insulin from different sources were used to confirm the lipogenesis method, and the operation method was the same as in Example 1. The test result curve is shown in FIG. Figure 4 The curve system suitability results are shown in Table 6, and the relative potency determination values ​​and recovery results are shown in Table 7.

[0148] Table 6 Curve system applicability results

[0149]

[0150] Table 7 Relative potency determination values ​​and potency recovery results

[0151]

[0152] Note: The manufacturer of insulin glargine injection with batch number F20240303 is Yifan Pharmaceutical; the manufacturer of insulin glargine injection with batch number DBJL054B is Sanofi of France.

[0153] Result analysis:

[0154] The test results of glargine insulin fat synthesis from different batches from different sources showed good curves, and the results were consistent with the system suitability, indicating that the fat synthesis detection method is suitable for glargine insulin, and the method is stable and reproducible.

[0155] Example 6

[0156] The difference between Example 6 and Example 1 is that the final concentration of dexamethasone in GM is 0.5 μM, the final concentration of rosiglitazone is 2.2 mM, and the rest is the same as Example 1. The curve system applicability results are shown in Table 8, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 9.

[0157] Table 8 Curve system applicability results

[0158]

[0159] Table 9 Relative potency determination values ​​and potency recovery results

[0160] Relative potency theoretical value Relative potency determination value Titer recovery rate RS N / A N / A 0.5 0.452 90.4％ 0.75 0.570 76.0％ 1 0.716 71.6％ 1.25 0.944 75.5％ 1.5 1.014 67.6％

[0161] The results showed that when the final concentration of dexamethasone in GM was adjusted to 0.5μM and the final concentration of rosiglitazone was 2.2mM, the glargine insulin fat synthesis detection result curve was still good, and the results met the system suitability requirements, indicating that the fat synthesis detection method has a certain durability, and the fluctuation of the final concentration of dexamethasone in the range of 0.5-1μM and the final concentration of rosiglitazone in the range of 2.2-2.8mM will not affect the detection results.

[0162] Example 7

[0163] The difference between Example 7 and Example 1 is that the final concentration of dexamethasone in GM is 2.0 μM, the final concentration of rosiglitazone is 3.4 mM, and the rest is the same as Example 1. The curve system applicability results are shown in Table 10, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 11.

[0164] Table 10 Curve system applicability results

[0165]

[0166] Table 11 Relative potency determination values ​​and potency recovery results

[0167] Relative potency theoretical value Relative potency determination value Titer recovery rate RS N / A N / A 0.5 0.519 103.8％ 0.75 0.776 103.5％ 1 0.884 88.4％ 1.25 1.298 103.8％ 1.5 1.380 92.0％

[0168] The results showed that when the final concentration of dexamethasone in GM was adjusted to 2.0μM and the final concentration of rosiglitazone was 3.4mM, the glargine insulin fat synthesis detection result curve was still good, and the results met the system suitability requirements, indicating that the fat synthesis detection method has a certain durability, and the fluctuation of the final concentration of dexamethasone in the range of 1-2μM and the final concentration of rosiglitazone in the range of 2.8-3.4mM will not affect the detection results.

[0169] Example 8 Establishment of a method for detecting the lipogenesis of T3L1 precursor cells stimulated by degludec and insulin aspart injection

[0170] In order to further determine the applicable scope of the fat synthesis method, the detection method of Example 1 was used to detect the insulin degludec aspart injection. The detection result curve is as follows: Figure 5 As shown, the curve system suitability results are shown in Table 12, and the relative potency measurement values ​​and recovery results of five relative potency levels (0.5, 0.75, 1, 1.25, 1.5) are shown in Table 13.

[0171] Table 12 Curve system applicability results

[0172]

[0173] Table 13 Relative potency determination values ​​and potency recovery results

[0174] Relative potency theoretical value Relative potency determination value Titer recovery rate RS N / A N / A 0.5 0.365 73.0％ 0.75 0.531 70.8％ 1 0.722 72.2％ 1.25 0.889 71.1％ 1.5 1.332 88.8％

[0175] Result analysis:

[0176] The results show that R 2 All of them can reach above 0.99 and close to 1, and the concentration points of the upper platform, lower platform and intermediate transition period of the curve are evenly distributed, indicating that the four-parameter curve fitting is excellent. The analysis window, maximum signal ratio, dynamic range ratio, effective slope ratio and relative activity all meet the system adaptability requirements, indicating that the method can also be applied to degludec and insulin aspart injection.

[0177] Comparative Example 1

[0178] A comparative example is set with reference to Example 1. The difference between the comparative example and Example 1 is shown in Table 14, and the test results are shown in Tables 15 and Figure 6 .

[0179] Table 14 Experimental conditions of comparative example 1

[0180]

[0181] Table 15 Comparative Example 1 Curve System Applicability Results

[0182]

[0183] Result analysis:

[0184] The results of different concentrations of BSA were comparable to the lowest concentration of RS (0.593 nM), indicating that the non-related protein BSA had no dose response. The results of different dilution multiples of the excipients of insulin glargine injection were comparable to the lowest concentration of RS (0.593 nM), indicating that it also had no dose response. Overall, the method has good specificity and is specifically suitable for the detection of insulin glargine.

[0185] Comparative Example 2

[0186] A comparative example was set with reference to Example 1. The difference between the comparative example and Example 1 and the test results are shown in Table 16:

[0187] Table 16 Experimental conditions and experimental results of comparative example 2

[0188]

[0189] Comparative Example 2 Preparation of main reagents and culture medium:

[0190] 10 mg / mL insulin (human) solution: Add 1 mL of 0.012 M HCl solution to 10 mg / vial of insulin (human) to dissolve it.

[0191] Cell differentiation medium (DM): 89% DMEM (Gibco), 10% fetal bovine serum, 1% penicillin-streptomycin, 1 μM dexamethasone, 0.5 mM IBMX and 1 μg / mL human insulin.

[0192] Adipocyte maintenance medium (IM): 89% DMEM (Gibco), 10% fetal bovine serum, 1% penicillin-streptomycin and 1 μg / mL human insulin.

[0193] Result analysis:

[0194] The fully induced adipocytes can respond to insulin stimulation of fat synthesis to a certain extent, but because they are adipocytes with a high fat content, and the whole cell is treated for too long (17 days), the cells are easily disturbed, resulting in unstable results. However, the semi-induced cells are not yet fully differentiated into adipocytes, and in the state of precursor cells, the stimulation of dexamethasone, IBMX and insulin will induce a certain amount of fat to be generated in the cells, and the cells in this state will be insensitive to insulin stimulation. Only by using dexamethasone and rosiglitazone to stimulate 3T3L1 precursor cells in coordination with a series of gradient insulin can the curve of cell fat synthesis in response to insulin stimulation be obtained.

[0195] Comparative Example 3

[0196] A comparative example was set with reference to Example 1. The difference between the comparative example and Example 1 and the test results are shown in Table 17:

[0197] Table 17 Experimental conditions and experimental results of comparative example 3

[0198]

[0199]

[0200] Results and Discussion:

[0201] Compared with Example 1, which uses GM, dexamethasone and rosiglitazone as sample diluents to coordinate the stimulation of the gradient series of insulin, this comparative example shows that without adding factors, single factor stimulation and superposition of other factors cannot well reflect the stimulation of insulin, indicating that only the specific use of GM, dexamethasone and rosiglitazone sample diluents can effectively coordinate the stimulation of insulin and then carry out fat synthesis detection.

Claims

1. A method for detecting insulin analogs stimulating cellular fat synthesis, characterized in that: The detection method comprises the following steps: S1, culturing preadipocytes to obtain cell A; S2, adding insulin analogs to cell A and continuing culturing to obtain cell B; S3, lyse cell B to obtain lysate; S5, detecting the fat content in the lysate; Step S2: the insulin analog is diluted with a diluent; the diluent contains dexamethasone and rosiglitazone.

2. The detection method according to claim 1, characterized in that: The final concentration of dexamethasone in step S2 is 0.5-2 μM; the final concentration of rosiglitazone is 2.2-3.4 mM.

3. The detection method according to claim 1, characterized in that: The diluent in step S2 also includes a growth medium.

4. The detection method according to claim 3, characterized in that: The growth medium includes, by volume, 83.5-91.5% DMEM, 8-15% fetal bovine serum and 0.5-1.5% penicillin-streptomycin.

5. The detection method according to claim 1, characterized in that: The insulin analogue in step S2 is at least one of insulin glargine, insulin degludec, insulin degludec, insulin lispro, insulin aspart or insulin detemir.

6. The detection method according to claim 1, characterized in that: The precursor adipocytes described in step S1 are derived from embryonic development, adipose tissue, stem cell differentiation, cell lines, and in vitro culture or induced pluripotent stem cells.

7. The detection method according to claim 6, characterized in that: The preadipocytes are 3T3-L1 preadipocytes.

8. The detection method according to claim 1, characterized in that: The culture medium used in the culture in step S1 includes 83.5-91.5% DMEM, 8-15% calf serum and 0.5-1.5% penicillin-streptomycin by volume.

9. The detection method according to claim 1, characterized in that: The fat content detection method in step S5 is spectroscopy, chromatography, mass spectrometry or lipid oxidation detection.

10. The detection method according to claim 9, characterized in that: The fat content detection method in step S5 comprises the following steps: (1) Lysing cells to obtain lysate; (2) taking the lysate, adding lipoprotein lipase solution and incubating; (3) adding triglyceride detection reagent and incubating; (4) measuring the luminescent signal; (5) Analyze the luminescence signal, use the four-parameter regression of the constraint model to fit the standard curve and the sample curve, and calculate the relative potency of the sample.

11. A kit, characterized in that: The kit comprises the reagent or culture medium in the detection method according to any one of claims 1 to 10.

12. Use of the detection method according to any one of claims 1 to 10 or the kit according to claim 11 in detecting the stimulation of fat synthesis by insulin analogs.

Images