Method for determining in vitro activity of human insulin and analogues based on mouse myoblasts C2C12

Abstract

The invention relates to the technical field of insulin biological detection, and discloses an in vitro activity determination method for human insulin and analogues based on mouse myoblasts C2C12, which comprises the following steps: adding human insulin DMEM-H to domesticate C2C12 cells, inducing and differentiating the C2C12 cells into myotubes, and detecting the activity of the myotubes. Then adding human insulin reference products and human insulin and analogue test products with different concentration gradients to incubate myotubes, and calculating the in-vitro biological activity of the human insulin and analogue test products by comparing glucose consumption and utilization rates and carrying out parameter fitting after incubation is finished. Compared with an existing insulin activity detection method which is long in period, high in difficulty and poor in repeatability, the method in the scheme has the advantages that the sensitivity of cells to insulin is effectively improved through domestication, and the detection period is greatly shortened; and compared with a primary adipose cell method, the operation difficulty is reduced, the repeatability is better, and the method is conveniently popularized and applied to detection of the biological activity of various insulin and analogues.

Description

Technical Field

[0001] The present invention relates to the technical field of insulin bioassay, and in particular to a method for determining the in vitro activity of human insulin and the like based on mouse myoblast C2C12. Background Art

[0002] Insulin is a protein hormone secreted by pancreatic β cells in response to endogenous or exogenous substances. It is the only hormone in the body that lowers blood sugar. With the rapid changes in the pace of modern life, and due to the improvement of living standards, irregular diet, and reduced exercise, patients with hyperglycemia are becoming younger and more common. In the process of using insulin to control patients' blood sugar, insulin mainly promotes the uptake and utilization of glucose by liver cells, muscle cells, and fat cells, and promotes the cell membrane carriers of target cells such as muscle and adipose tissue to transport glucose in the blood into cells and convert it into fatty acids for storage as glycogen. At the same time, it accelerates the oxidation of glucose into high-energy phosphate compounds as a source of energy, and promotes the synthesis of glycogen, fat, and protein. This lowers blood sugar levels.

[0003] At present, insulin preparations on the market are mainly divided into three categories according to the speed of insulin action and the duration of insulin action: (1) rapid-acting human insulin analogs: including insulin aspart, insulin lispro and insulin glulisine; (2) intermediate-acting insulin: including regular insulin and neutral insulin; (3) long-acting human insulin: including insulin glargine, insulin detemir and insulin degludec. In order to meet the treatment needs of patients with diabetes / hyperglycemia of different constitutions and different degrees of illness, short-, medium- and long-acting insulin preparations can be used alone, or auxiliary treatment preparations (protamine, zinc ions) can be added, or different types of insulin can be premixed in proportion to form insulin combination preparations to improve the targeted treatment and treatment effect. Therefore, rapid determination of the biological activity of insulin preparations or insulin combination preparations before treatment can not only achieve targeted administration of drugs to different patients, but also effectively improve the treatment effect.

[0004] However, the current international method for determining the bioavailability of human insulin and its analogs or conjugates is an in vivo method. For example, CN105092490A discloses an in vitro method for determining the biological activity of human insulin and its analogs or conjugates. After rat preadipocytes are induced into adipocytes in vitro, the glucose oxidase method is used to determine the consumption of glucose in the culture supernatant under the action of insulin to determine the biological activity of insulin. It still has the following problems: (1) The rat adipocyte primary cell culture used in the prior art is difficult to operate, has a long cycle, poor repeatability, and reduces the detection efficiency and accuracy; (2) Other existing detection technologies (such as in-cell Western, time-resolved fluorescence immunoassay, etc.) require methods for determining biological activity based on the phosphorylation level of the human insulin receptor, which have the problems of high cost and cumbersome operation.

[0005] In summary, the development of an in vitro activity determination method for human insulin and analogs based on mouse myoblasts C2C12 with high detection efficiency and accuracy can not only effectively make up for the shortcomings of the existing technology, but also quickly determine the in vitro activity of insulin and analogs, and realize targeted drug delivery for different patients, which is of great significance. Summary of the invention

[0006] The present invention aims to provide an in vitro activity assay method for human insulin and the like based on mouse myoblasts C2C12, so as to solve the technical problems that the rat adipocyte primary cell culture used in the prior art is difficult to operate, has a long cycle, and has poor repeatability, resulting in low detection efficiency and accuracy.

[0007] To achieve the above-mentioned object, the present invention adopts the following technical scheme: an in vitro activity determination method of human insulin and analogs based on mouse myoblasts C2C12, firstly acclimating C2C12 cells with a high-glucose DMEM medium supplemented with human insulin, then inducing differentiation of C2C12 cells into myotubes, plating the myotube solution, adding human insulin reference substances and human insulin and analogs test substances of different concentration gradients to incubate the myotubes, detecting the glucose consumption utilization rate after the incubation, performing parameter fitting according to the dose-effect relationship between the human insulin and analogs test substances and the human insulin reference substance and the dilution concentration, and calculating the in vitro biological activity of the human insulin and analogs test substances.

[0008] Preferably, as an improvement, the human insulin and analogs test samples include human insulin, aspart insulin, degludec insulin, and elabortin insulin.

[0009] Beneficial effects:

[0010] 1. Compared with the existing insulin activity detection methods with long cycles, high difficulty and poor repeatability, this scheme provides a new in vitro biological activity determination method for human insulin and analogs. Through domestication, it effectively improves the sensitivity of cells to insulin and greatly shortens the detection cycle. Compared with the primary adipocyte method, it reduces the difficulty of operation and has better repeatability, which is convenient for promotion and application to the detection of various types of insulin and analogs biological activity.

[0011] 2. Compared with the narrow scope of application of existing insulin, the in vitro detection method of this scheme is suitable for the in vitro biological activity determination of human insulin, aspart insulin, degludec insulin, and eleucidin insulin, and has a wider scope of application.

[0012] 3. Compared with the existing technology that uses in-cell Western, time-resolved fluorescence immunoassay, etc., which are methods for determining biological activity based on the phosphorylation level of human insulin receptor, the detection method used in the present invention can quickly calculate the biological activity of insulin and analogs by measuring the residual glucose content in the culture medium. The operation is simpler and more economical. In actual detection, the same 96-well plate can detect glucose concentrations at multiple time points, and the cost is also lower.

[0013] Preferably, as an improvement, the method comprises the following steps:

[0014] Step 1, cell acclimation and plating stage: acclimating cells with high-glucose DMEM medium containing human insulin and 10% fetal bovine serum FBS, centrifuging cells in logarithmic growth phase, resuspending cells with DMEM complete medium to obtain cell fluid, adjusting the cell density in the cell fluid, inoculating the cell fluid into a cell culture plate, and culturing for later use;

[0015] Step 2, induction stage: replace the culture medium in the cell culture plate with the induction medium, and keep it for later use after induction culture;

[0016] Step 3, drug action and detection stage: replace the culture medium in the cell culture plate with a drug-added culture medium, then add human insulin reference products and human insulin and analog test products of different dilution concentrations into the cell culture plate induced as described above, and during the cell incubation process, aspirate the supernatant in different wells at different times to detect the glucose concentration value;

[0017] Step 4, result analysis stage: according to the glucose concentration values ​​of human insulin and analogs test samples and human insulin reference samples obtained in the above tests and the dose-effect relationship between the dilution concentration, four-parameter fitting is performed to calculate the in vitro biological activity of the test samples.

[0018] Beneficial effects: This scheme adopts the above scheme to effectively and quickly detect the in vitro biological activity of insulin and analogs, shorten the activity detection cycle of insulin and analogs, and the test results have good repeatability, which can effectively improve the accuracy of the test results.

[0019] Preferably, as an improvement, in step 1, the concentration of human insulin in the high-glucose DMEM medium is 1 μg / mL when acclimating the cells.

[0020] Beneficial effects: This scheme adopts the above settings, which effectively accelerates the cell's response to insulin, shortens the detection cycle, and has good repeatability. The inventors found through long-term experiments that when the untamed group and the tamed group were compared, under the same drug-added culture medium, the time required for the untamed group to reach the same glucose consumption was significantly longer than that required for the tamed group. Specifically, when the same "2% HS+DMEM-M+0.5% FBS" drug-added culture medium and the same drug concentration of 5.8ug / mL were used, the time required for the unacclimated group to consume only 6.4% of glucose was 195.5h, while the time required for the acclimated group to consume only 5.8% of glucose was 49h. In comparison, after C2C12 cells were acclimated and cultured in high-glucose DMEM culture medium supplemented with insulin, C2C12 cells were more sensitive to insulin, and the myotubes induced by the cells were used to detect insulin activity, which had a faster reaction speed, not only effectively shortening the detection cycle, but also with good repeatability. In addition, the present scheme only requires advance acclimation to shorten the detection cycle, which reduces the difficulty of operation and is convenient for widespread promotion in the rapid detection of in vitro activity of various insulins and their analogs.

[0021] Preferably, as an improvement, in step 1, the cell density in the cell solution is adjusted to 7000 to 9000 cells / mL -1 .

[0022] Beneficial effect: This solution adopts the above settings to effectively increase the glucose consumption in the same time, thereby improving the accuracy of the test results. The inventors found through long-term experiments that if the number of cells is too small, the glucose consumption will not be obvious, which will reduce the accuracy of the test results; and if the cell concentration is too high, there will be too many cells and they will fall off, resulting in unclear glucose consumption, which will reduce the accuracy of the test results.

[0023] Preferably, as an improvement, in step 2, the induction medium is a high-glucose DMEM medium containing 2-4% horse serum.

[0024] Preferably, as an improvement, in step 2, the induction time is 2 to 3 days.

[0025] Beneficial effect: This scheme adopts the above-mentioned setting, which is convenient for increasing the number of C2C12 cells transformed into myotubes, effectively increasing the glucose consumption, and thus improving the accuracy of the test results. The inventors found through long-term experiments that when the HS in the induction medium is the same as 4%, the dosing medium remains unchanged, and the induction time is too long (such as 5 days of induction), the glucose consumption rate is slower, which is not conducive to rapid detection; the inventors analyzed that the reason is that the excessively high HS concentration in the induction medium leads to too few myotubes obtained by induced cell differentiation, which in turn leads to a decrease in glucose consumption and reduces the accuracy and efficiency of the test results.

[0026] Preferably, as an improvement, in step three, the drug-added culture medium is a medium-sugar DMEM culture medium supplemented with 2-4% horse serum.

[0027] Beneficial effect: This scheme adopts the above-mentioned setting, which is convenient for improving the consumption rate of glucose in the culture medium by myotubes and accelerating the detection efficiency of glucose. The inventors found through long-term experiments that when using high-sugar DMEM culture medium (i.e. DMEM-H), medium-sugar DMEM culture medium (i.e. DMEM-M) or low-sugar DMEM culture medium (i.e. DMEM-L) to detect the biological activity of insulin, the change pattern of glucose content in some high-sugar or medium-sugar culture media is more suitable for detecting insulin activity, and a more accurate insulin activity detection effect is obtained. If the sugar content in the drug-added culture medium is too low (such as medium-sugar DMEM culture medium with 2% HS added or low-sugar DMEM culture medium with 2% HS added), the glucose content decreases too fast after the same concentration of insulin acts, and the insulin activity cannot be accurately detected; considering the cost, the medium-sugar DMEM culture medium with 2-4% horse serum is used to detect insulin activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the fitting curve of ordinary human insulin in the embodiment of the present invention.

[0029] Figure 2 It is the fitting curve of insulin aspart in the embodiment of the present invention.

[0030] Figure 3 It is the fitting curve of degludec insulin in the embodiment of the present invention.

[0031] Figure 4 It is the fitting curve of insulin echo in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0033] Example

[0034] This protocol provides a method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12, and determines the in vitro biological activity of recombinant human insulin using C2C12 cells. First, C2C12 cells are acclimated with a high-glucose DMEM medium supplemented with human insulin, and then C2C12 cells are induced to differentiate into myotubes. After the myotube solution is plated, human insulin reference substances and human insulin and its analogs test substances of different concentration gradients are added to incubate the myotubes. After the incubation, the glucose consumption utilization rate is detected, and parameter fitting is performed based on the dose-effect relationship between the human insulin and its analogs test substances and the human insulin reference substances and the dilution concentration, and the in vitro biological activity of the human insulin and its analogs test substances is calculated.

[0035] Among them, human insulin and analogs test products include human insulin, insulin aspart, insulin degludec, insulin lispro, insulin glulisine, and insulin modified with fatty acids. As a reference, the national standard of recombinant human insulin (derived from the China Food and Drug Inspection Institute, 25 mg / vial), ordinary human insulin (Novolin, purchased from Novo Nordisk, specification is 400 IU / 10 ml / vial), insulin aspart purchased from Novo Nordisk (NovoRapid, specification is 300 IU / 3 ml / vial), insulin degludec purchased from Novo Nordisk (Novoda, specification is 300 IU / 3 ml / vial), and insulin lispro was developed by our company.

[0036] The specific steps include:

[0037] Step 1: Cell acclimation and plating stage: Use high-glucose DMEM medium containing 1 μg / mL human insulin and 10% fetal bovine serum (FBS) to acclimate the cells. Centrifuge the cells in the logarithmic growth phase at 1000 rpm·min-1 for 10 minutes, resuspend the cells in DMEM complete medium to obtain cell fluid, and adjust the cell density in the cell fluid to 7000-9000 cells·mL -1 , and then inoculate the cell solution into a 96-well cell culture plate and culture it for later use; as a reference, inoculate 100 μL per well of a 96-well cell culture plate and place it at 37°C, 5% CO 2 Culture in incubator for 24 hours.

[0038] Step 2, induction stage: replace the culture medium in the cell culture plate with the induction medium, which is a high-glucose DMEM medium containing 2-4% horse serum, and induce the culture for 2-3 days; as a reference, 100 μL per well, incubate at 37°C, 5% CO2 incubator for 2-3 days;

[0039] Step 3, drug action and detection stage: replace the culture medium in the cell culture plate with a drug-added medium, which is a medium-sugar DMEM medium supplemented with 2-4% horse serum (HS), and then add different dilutions of human insulin reference and human insulin and analog test products to the induced cell culture plate, and place at 37°C, 5% CO 2 The cells were incubated for 20 to 30 hours. During the incubation period, the supernatant in different wells was aspirated at different times to detect the glucose concentration. As a reference, 10 to 20 μl of the supernatant from the 96-well cell culture plate was aspirated and the glucose concentration was detected in sequence using a Johnson & Johnson blood glucose meter (model: ONETOUCH Verio Flex).

[0040] As a reference, the glucose detection values ​​(i.e., the glucose detection values ​​obtained by directly detecting the culture medium with a Johnson & Johnson blood glucose meter) in the high-glucose DMEM culture medium (i.e., DMEM-H), medium-glucose DMEM culture medium (i.e., DMEM-M) or low-glucose DMEM culture medium (i.e., DMEM-L) in this protocol are 22.5-24, 17.5-18.5, and 4-5.5, respectively, indicating equal glucose concentrations.

[0041] Step 4, result analysis stage: According to the glucose concentration value and dilution concentration of the human insulin and analogs test samples and human insulin reference samples obtained in the above test, four-parameter fitting is performed to calculate the in vitro biological activity of the test samples. The relative activity calculation formula of the test samples is as follows:

[0042] Relative activity of test sample = reference sample IC50 / test sample IC50×100%

[0043] Experimental Example 1: Effects of acclimatization, induction medium, and induction time on detection efficiency

[0044] Referring to the steps in the above embodiments, combinations of acclimation, different induction culture media, different induction times, and different drug-added culture media were selected to set up treatment groups (the design of the treatment groups is detailed in Table 1), and the glucose detection values ​​in the culture media after different treatment times with different gradient insulin concentrations were detected. The effects of the selection of various parameters on the insulin detection efficiency of ordinary people were detected, and the glucose detection values ​​obtained by using a blood glucose meter to detect the supernatant of the drug-added culture media were detailed in Table 1.

[0045] Table 1 Results of glucose detection in the supernatant of the culture medium with different treatment combinations

[0046]

[0047]

[0048] Note: “-” in the table means exceeding the minimum detection line of the blood glucose meter.

[0049] Experimental data show that compared with C2C12 cells that have not been trained with insulin, C2C12 cells trained with 1 μg / mL insulin are more sensitive to insulin under the same induction and drug-added culture medium treatment conditions, effectively shortening the time required for the same blood sugar-lowering effect, thereby effectively improving the activity detection time of insulin and analogs.

[0050] Specifically, for example, in treatment 2 and treatment 10, after 3 days of induction culture using the induction medium 2% HS+DMEM-H, the untamed treatment 2 treated the myotubes with 58ng / ml insulin for 24h and reduced the sugar to 7 (glucose detection value, the same below), while the tamed treatment 10 treated the myotubes with 58ng / ml insulin for 12h and reduced the sugar to 7.8 (equivalent to the sugar reduction level in treatment 2), and reduced the sugar to 4.4 after 24h (the sugar reduction effect was significantly better than the sugar reduction level in treatment 2). It can be seen that the use of 1μg / mL insulin to tamed C2C12 cells in advance effectively improved the sensitivity of the induced myotubes to insulin, thereby shortening the detection time and improving the detection efficiency. And the same conclusion can be drawn under various treatment conditions of this scheme (such as treatment 1 and treatment 9, treatment 3 and treatment 13, treatment 4 and treatment 14), indicating that the taming treatment of this scheme has high operability, good repeatability, improved detection accuracy, and is conducive to widespread promotion and use.

[0051] In addition, under the same acclimation treatment, by comparing treatment 5 and treatment 6, treatment 7 and treatment 8, treatment 9 and treatment 10, treatment 11 and treatment 12, treatment 13 and treatment 14, it can be seen that when the induction time and induction medium remain unchanged, the higher the HS concentration in the drug-added medium, the faster the glucose consumption rate.

[0052] By comparing treatment 5 and treatment 7, and treatment 6 and treatment 8, it can be seen that when the induction time and the drug-added culture medium remain unchanged, the higher the HS concentration in the induction culture medium, the faster the glucose consumption rate, thereby shortening the detection time and improving the detection efficiency.

[0053] By comparing treatment 5, treatment 9 and treatment 13, and by comparing treatment 6, treatment 10 and treatment 14, it can be seen that when the HS concentration in the induction medium is the same as 2% and the drug-added medium remains unchanged, the longer the induction time, the faster the glucose consumption rate, thereby shortening the detection time and improving the detection efficiency.

[0054] By comparing treatment 7 and treatment 11, and treatment 8 and treatment 12, it can be seen that when the HS content in the induction medium is the same at 4% and the drug-added medium remains unchanged, the longer the induction time, the slower the glucose consumption rate, which is not conducive to rapid detection. The inventors analyzed that the reason is that the excessively high HS concentration in the induction medium leads to an excessively high degree of differentiation of the induced cells, which in turn slows down the glucose consumption rate of the cells and reduces the detection speed.

[0055] It can be seen that under the same acclimation treatment, after 2 days of induction with the induction medium 4% HS+DMEM-H, the drug addition medium is 4% HS+DMEM-M, which can further shorten the detection time and obtain faster detection efficiency and higher detection accuracy.

[0056] Experimental Example 2: Effects of different drug-added culture media on test results

[0057] According to the steps in the above embodiment, the cells were first acclimated with a high-glucose DMEM medium containing 1 μg / mL human insulin and 10% fetal bovine serum (FBS), and then induced with an induction medium 4% HS+DMEM-H for 2 to 3 days. In "Step 3, drug action and detection phase", myotubes were cultured with a high-glucose DMEM medium (i.e., DMEM-H), a medium-glucose DMEM medium (i.e., DMEM-M) or a low-glucose DMEM medium (i.e., DMEM-L) supplemented with different sera, and the glucose detection values ​​in the culture medium after different gradient concentrations of ordinary human insulin were detected at different treatment times. The results are shown in Table 2.

[0058] Table 2 Glucose detection results under different drug-added culture medium treatments

[0059]

[0060] Note: “-” in the table means exceeding the minimum detection limit of the blood glucose meter;

[0061] Experimental data show that when high-glucose DMEM medium (i.e., DMEM-H), medium-glucose DMEM medium (i.e., DMEM-M) or low-glucose DMEM medium (i.e., DMEM-L) is used to detect the biological activity of insulin, the change pattern of glucose content in some high-glucose or medium-glucose culture media is more suitable for detecting insulin activity, thereby obtaining a more accurate insulin activity detection effect; however, if the sugar content in the drug-added culture medium is too low (such as DMEM-L), the glucose content will be greatly reduced after the action of low-concentration insulin (i.e., the glucose content decreases too quickly), making it impossible to accurately detect insulin activity. Specifically, in treatment 20 (2% HS+DMEM-L), due to its low initial glucose concentration (the glucose value detected by the blood glucose meter was 5.3), the blood glucose in the culture medium dropped rapidly even if the insulin dosage was low. For example, after the myotube cells were acted on with 5.8 ng / ml insulin for 12 hours (generally speaking, long-acting or medium-acting insulin can be injected at a fixed time every day, so under ideal conditions, reducing it to a normal level in 24 hours can better simulate the actual insulin application scenario), the glucose detection value in the culture medium was 1.2, which is close to the lower detection limit of the blood glucose meter, which reduces the accuracy of the test result.

[0062] It can be seen from this that the drug-added culture medium in this scheme is preferably a high-glucose DMEM culture medium (DMEM-H) supplemented with 2-4% horse serum or a medium-glucose DMEM culture medium (DMEM-M) supplemented with 2-4% horse serum.

[0063] Experimental Example 3: Effects of cell density and drug concentration on detection results

[0064] Referring to the steps in the embodiment, the concentration is selected to be 5000-9000 cm -1 C2C12 cells were used to detect the in vitro activity of insulin and its analogs. The cells were first acclimated with high-glucose DMEM medium containing 1 μg / mL human insulin and 10% fetal bovine serum (FBS), then induced with induction medium 4% HS+DMEM-H for 2 to 3 days, and then cultured in drug-added medium 4% HS+DMEM-M. The glucose detection values ​​in the culture medium after different concentrations of human insulin and ergoinsulin were recorded for different periods of time. The results are shown in Table 3.

[0065] Table 3 Glucose detection results under different cell density treatments

[0066]

[0067]

[0068] Note: “ / ” in the table means not tested.

[0069] Experimental data show that the cell density is 5000 cells cm-1 (i.e. 5000 cm -1 ), the glucose content in the culture medium decreases too slowly, resulting in a prolonged detection cycle, which is not conducive to rapid detection of insulin activity. -1 When , the glucose content in the culture medium decreases considerably and rapidly, so it is more suitable for rapid detection of insulin activity.

[0070] Experimental Example 4: Predicting the IC50 of various insulins and calculating their activity

[0071] This protocol provides an application of a method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12, which specifically includes the following steps:

[0072] Step 1, cell acclimation and plating stage: acclimate the cells with high-glucose DMEM medium containing 1 μg / mL human insulin and 10% fetal bovine serum (FBS), centrifuge the cells in the logarithmic growth phase at 1000 rpm·min-1 for 10 min, resuspend the cells with DMEM complete medium to obtain cell fluid, adjust the cell density in the cell fluid to 7000-9000 cells·mL-1, and then inoculate the cell fluid into a 96-well cell culture plate and culture it for later use; as a reference, inoculate 96-well cell culture plates with 100 μL per well and place them in a 37°C, 5% CO2 incubator for 24 h.

[0073] Step 2, induction stage: replace the culture medium in the cell culture plate with the induction medium, which is a high-glucose DMEM medium containing 2-4% horse serum, and induce the culture for 2-3 days; as a reference, 100 μL per well, incubate at 37°C, 5% CO2 incubator for 2-3 days;

[0074] Step 3, drug action and detection stage: replace the culture medium in the cell culture plate with a drug-added culture medium, which is a high-glucose DMEM culture medium supplemented with 2-4% horse serum or a medium-glucose DMEM culture medium supplemented with 2% HS and 0.5% FBS, and then add human insulin reference substances and human insulin and analog test substances of different dilution concentrations to the cell culture plate induced as described above, and incubate at 37°C, 5% CO2 for 20-30 hours; during the cell incubation process, aspirate the supernatant in different wells at different times to detect the glucose concentration value; as a reference, aspirate 10-20ul of the supernatant from the 96-well cell culture plate, and use a Johnson & Johnson blood glucose meter to detect the glucose concentration values ​​in turn.

[0075] Step 4, result analysis stage: According to the glucose concentration value and dilution concentration of the human insulin and analogs test samples and human insulin reference samples obtained in the above test, four-parameter fitting is performed to calculate the in vitro biological activity of the test samples. The relative activity calculation formula of the test samples is as follows:

[0076] Relative activity of test sample = reference sample IC50 / test sample IC50×100%

[0077] As a reference, the human insulin and analog test samples are any one of human insulin, insulin aspart, insulin degludec, and insulin ecol. The glucose detection values ​​and IC50 values ​​of the four insulins are shown in Table 4. Figures 1 to 4 .

[0078] Table 4 Activity test results of four kinds of insulin

[0079]

[0080] Experimental data show that this scheme can effectively and quickly detect the in vitro activity of insulin and analogs. And in the process of optimizing the detection method of this scheme. It was found that factors such as the glucose concentration in the culture medium, whether it was domesticated before induction, the glucose concentration and horse serum concentration in the induction culture medium, and the glucose concentration and serum composition in the drug-added culture medium during the detection stage all significantly affect the detection sensitivity and detection cycle, thereby affecting the accuracy of the detection results. This scheme effectively shortens the in vitro activity detection cycle of insulin by optimizing the parameters of each stage. Compared with the existing insulin detection, which has low detection efficiency and accuracy due to the difficulty of cell culture operation, long detection cycle, and poor repeatability, it is of great significance and can be extended to various insulin in vitro activities.

[0081] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12, characterized in that: C2C12 cells were first acclimated with high-glucose DMEM medium supplemented with human insulin, and then induced to differentiate into myotubes. Then, human insulin reference substances and human insulin and analogues test substances with different concentration gradients were added to incubate the myotubes. After the incubation, the glucose consumption and utilization rate were detected. Parameters were fitted based on the dose-effect relationship between the human insulin and analogues test substances and the human insulin reference substance and the dilution concentration, and the in vitro biological activity of the human insulin and analogues test substances was calculated.

2. The method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12 according to claim 1, characterized in that: The human insulin and analogs test samples include ordinary human insulin, aspart insulin, degludec insulin, and elabor insulin.

3. The in vitro activity determination method of human insulin and its analogs based on mouse myoblast C2C12 according to claim 1 or 2, characterized in that: The steps include: Step 1, cell acclimation and plating stage: acclimate the cells with high-glucose DMEM medium containing normal human insulin and 10% fetal bovine serum FBS, centrifuge the cells in the logarithmic growth phase, resuspend the cells with DMEM complete medium to obtain cell fluid, adjust the cell density in the cell fluid, inoculate the cell fluid into the cell culture plate, and culture for later use; Step 2, induction stage: replace the culture medium in the cell culture plate with the induction medium, and keep it for later use after induction culture; Step 3, drug action and detection stage: replace the culture medium in the cell culture plate with a drug-added culture medium, then add human insulin reference products and human insulin and analog test products of different dilution concentrations into the cell culture plate induced as described above, and during the cell incubation process, aspirate the supernatant in different wells at different times to detect the glucose concentration value; Step 4, result analysis stage: according to the glucose concentration values ​​of human insulin and analogs test samples and human insulin reference samples obtained in the above tests and the dose-effect relationship between the dilution concentration, four-parameter fitting is performed to calculate the in vitro biological activity of the test samples.

4. The method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12 according to claim 3, characterized in that: In step 1, the concentration of human insulin in the high-glucose DMEM medium is 1 μg / mL when acclimating the cells.

5. The in vitro activity determination method of human insulin and its analogs based on mouse myoblast C2C12 according to claim 3, characterized in that: In step 1, adjust the cell density in the cell solution to 7000-9000 cells / mL -1 .

6. The method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12 according to claim 3, characterized in that: In step 2, the induction medium is a high-glucose DMEM medium containing 2-4% horse serum.

7. The method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12 according to claim 6, characterized in that: In step 2, the induction time is 2 to 3 days.

8. The method for determining the in vitro activity of human insulin and its analogs based on mouse myoblasts C2C12 according to claim 3, characterized in that: In step 3, the drug-added culture medium is a medium-sugar DMEM culture medium supplemented with 2-4% horse serum.

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