BMP-2 cell activity determination method

Abstract

The invention discloses a BMP-2 cell activity determination method, and aims to improve the efficiency, accuracy and stability of BMP-2 protein activity detection. The method comprises the following steps: selecting cells with a good growth state, and inoculating to a culture dish; an ammonium acetate solution is used as a BMP-2 protein dissolving agent; accurately controlling the matching between the concentration of the BMP-2 protein solution and the cell quantity, and ensuring that the concentration of the BMP-2 in the cell culture medium is in a range of 250ng / ml to 1500ng / ml; a freeze thawing and intermittent ultrasonication combined technology is adopted to replace a traditional lysate method, and the accuracy of BMP-2 activity determination is further improved. By optimizing the steps of cell preparation, drug administration, sample extraction and activity determination, the experimental process is simplified, the test period is shortened, the efficiency and precision of BMP-2 cell activity determination are remarkably improved, and the method has a wide application prospect and particularly has an important application value in the fields of biomedical research and drug development.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for determining BMP-2 cell activity. Background Art

[0002] BMP-2 (bone morphogenetic protein-2) is an important signaling molecule in the process of bone formation and repair, and is a member of the transforming growth factor-β (TGF-β) superfamily. BMP-2 plays a key role in the development and repair of bones, cartilage and other tissues. BMP-2 accelerates bone healing by promoting the proliferation and differentiation of osteoblasts, so its activity level is an important indicator for evaluating the effect of bone repair. BMP-2 cell activity is usually measured by measuring its biological activity in cell culture. Common detection methods include using ALP (alkaline phosphatase) activity as an indirect indicator. ALP is a marker enzyme for osteoblast differentiation, and its activity level is positively correlated with the biological activity of BMP-2.

[0003] At present, there are two main methods for determining BMP-2 activity: one is to directly measure the content of BMP-2 protein, and the other is to indirectly infer BMP-2 activity by measuring ALP activity. Direct determination of BMP-2 protein is usually carried out by immunological methods such as ELISA (enzyme-linked immunosorbent assay), but this method usually requires highly specific antibodies, and the operation is complicated and the cost is high. On the other hand, the ALP activity determination method usually indirectly evaluates BMP-2 activity by using the conversion reaction of the substrate phenol. This method requires a long experimental time, and the sample processing process is cumbersome, often requiring multiple cultures, fluid changes and cell processing steps. The existing ALP activity determination method also has problems such as poor repeatability, long operation time, and low sensitivity during sample processing, which affects the accuracy and consistency of the test results.

[0004] Therefore, it is necessary to provide a method for determining the activity of BMP-2 cells to solve the problems of cumbersome determination process, long operation time, low sensitivity and poor repeatability of experimental results in the prior art. By optimizing the cell inoculation amount, preparation of BMP-2 solution, cell treatment and ultrasonic disruption steps, the accuracy, efficiency and repeatability of the determination can be improved, providing a simpler, faster and more efficient solution for BMP-2 activity testing. Summary of the invention

[0005] According to the technical problems raised above, the present application provides an improved method for determining the activity of BMP-2 cells. This method greatly simplifies the experimental process, shortens the operation time, and improves the accuracy and repeatability of the determination by optimizing the cell inoculation amount, the preparation of the BMP-2 protein solution, the cell administration and the crushing steps. Compared with the traditional method, this method reduces the step of cell re-cultivation and uses ammonium acetate solution as the BMP-2 protein solvent, which effectively improves the dissolution efficiency. By combining the ALP activity and protein concentration determination, the activity of BMP-2 protein can be more accurately evaluated. In addition, the use of ultrasonic crushing technology instead of the traditional lysate treatment method can effectively destroy cells in a short time, thereby improving the efficiency of the experiment and the reliability of the results. This method has high sensitivity and low experimental variability, and is suitable for the screening and routine detection of large-scale samples.

[0006] The technical means adopted by the present invention are as follows:

[0007] A method for measuring BMP-2 cell activity, the method comprising the following steps:

[0008] Step 1: Cell preparation: Take cells with good growth status, count them and inoculate them into a cell culture dish, add DMEM cell culture medium containing 10% FBS to 10 ml, mix well by pipetting, and continue to culture in a 37°C, 5% carbon dioxide concentration incubator until the cells adhere to the wall;

[0009] Step 2: Cell administration: prepare BMP-2 protein solution with ammonium acetate, filter and sterilize, take out the previously attached cells, add them to the cell culture dish, and continue to culture for 72 hours;

[0010] Step 3: Extract the cell sample solution. After 72 hours, add 0.25% trypsin to digest the cells. Use PBS solution to stop the digestion. Collect the cell sample by centrifugation. Use an ultrasonic disruptor to ultrasonically disrupt the sample.

[0011] Step 4: Determination of alkaline phosphatase activity in the cell sample extract, using an alkaline phosphatase kit to determine the alkaline phosphatase activity, combined with a BCA kit to determine the protein concentration, and calculate the BMP-2 protein activity;

[0012] Step 5: Determine the protein content of the cell sample extract using a BCA protein concentration assay kit to determine the protein concentration in the sample.

[0013] Furthermore, the cell inoculation volume in step 1 was 2.5×10 5 cells / 10cm 2 Petri dish.

[0014] Furthermore, the concentration of the BMP-2 protein solution used in step 2 is 100 μg / ml.

[0015] Furthermore, in step 2, the preparation of the BMP-2 protein solution uses 10 mM ammonium acetate solution as a dissolving agent, and the phenomenon of incomplete protein dissolution does not occur when using the ammonium acetate solution.

[0016] Furthermore, in step 2, the final concentration of the BMP-2 protein solution and the cell culture medium is 250 ng / ml to 1500 ng / ml.

[0017] Furthermore, in step 3, the ultrasonic parameters of the ultrasonic crusher are: ultrasonic time is 3 seconds, interval time is 5 seconds, total ultrasonic time is 1 minute, amplitude is 20%, and the temperature is controlled below 30°C during ultrasonic crushing.

[0018] Furthermore, the centrifugation conditions in step 3 are: 1500r / min, and the centrifugation time is 5 minutes.

[0019] Furthermore, in step 3, if the sample cannot be processed on the same day, it can be placed in a refrigerator at -20°C or below overnight.

[0020] Further, in step 4, the formula for calculating BMP-2 activity is: BMP-2 activity (U) = C 酚含量测定 *V BMP *N, where V BMP is the total volume of the sample, ml; N is the sample dilution factor, and the value is 1 when not diluted.

[0021] Furthermore, in step 5, the protein concentration is determined using a BCA kit, and the protein content of the sample is quantitatively calculated by comparing with a standard curve.

[0022] The present invention has the following advantages:

[0023] 1. The present invention uses cells in good growth state, and each culture dish is inoculated with 2.5×10 5 This method not only simplifies the experimental operation process, but also significantly shortens the test cycle and improves the efficiency of the experiment. At the same time, it reduces the influence of cell generations, makes the biological activity of cells more stable, and enhances the reliability of the test results. Because the cells are in a good growth state, the sensitivity is also improved, which can more accurately reflect the activity of BMP-2 protein.

[0024] 2. The present invention uses 10mM ammonium acetate solution as a BMP-2 protein dissolving agent. Compared with other dissolving agents, ammonium acetate can better dissolve BMP-2 protein and avoid the phenomenon of incomplete protein dissolution. The acidic properties of the ammonium acetate solution effectively improve the solubility of the BMP-2 protein, provide a stable BMP-2 solution for subsequent cell administration, and ensure the consistency and accuracy of the cell treatment process.

[0025] 3. The concentration of the BMP-2 protein solution used in the present invention is 100 μg / ml, and according to the matching of the number of cells and the BMP-2 protein solution, the final concentration of BMP-2 in the cell culture medium is ensured to be in the range of 250 ng / ml to 1500 ng / ml. Through this precise control, the concentration of BMP-2 received by each cell is ensured to be more uniform, thereby improving the accuracy and repeatability of the experiment. This method ensures a high correlation between the experimental results and the cell state, and avoids data errors caused by uneven administration.

[0026] 4. The present invention adopts a method of freeze-thaw combined with intermittent ultrasonic disruption during the extraction process of the cell sample solution. Compared with the traditional lysate method, freeze-thaw + intermittent ultrasonic disruption can more effectively destroy cells under the control of ultrasonic parameters. Not only does it shorten the disruption time, but it also significantly improves the extraction efficiency of cell contents, ensuring that the components in the extract are more complete and efficient, thereby improving the accuracy of BMP-2 activity determination. This optimized disruption method makes the cell extraction process more efficient and reduces cell damage that may be caused by excessive time or improper operation.

[0027] In summary, the present invention significantly improves the efficiency, accuracy and stability of the experiment by optimizing the cell inoculation amount, using ammonium acetate solution to improve protein dissolution efficiency, accurately controlling the BMP-2 protein concentration and using a freeze-thaw combined with intermittent ultrasonic disruption method. These improvements make the BMP-2 cell activity determination simpler, faster and more reliable.

[0028] Based on the above reasons, the present invention can be widely promoted in the technical fields of biomedical research, drug development and cell biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 It is a linear fitting diagram of ALP activity after adopting the determination method of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention provides a method for determining the activity of BMP-2 cells, which specifically comprises the following steps:

[0033] Step 1: Cell preparation

[0034] Take cells with good growth status, count them and calculate the number of cells to 2.5×10 5 Cells were seeded in 10 cm 2 Add DMEM cell culture medium containing 10% FBS to 10 ml in the cell culture dish, mix well by pipetting, and then continue to culture in a 37°C, 5% carbon dioxide concentration incubator to allow cells to adhere to the wall. The drug administration experiment was carried out the next day (about 16-24 hours).

[0035] It is worth noting that the drug can be administered according to the cell growth after resuscitation without further culture to stabilize the environment. The cell generation has a greater impact on the test results. Too many or too few cells will affect the entire test process.

[0036] Step 2: Cell administration

[0037] Use 10mM ammonium acetate to prepare BMP-2 protein into a 100μg / ml solution stock solution, and filter it with a 0.22μm filter for sterilization. Take out the cells that have been attached to the wall before, discard the culture medium, rinse once with PBS solution, and then add 15ml of culture medium respectively. Take the above prepared protein samples, add appropriate amount of 100μg / ml BMP-2 protein solution to the cell culture dish, and add the same volume of 10mM ammonium acetate solution to the blank control group. After adding the samples, continue to return to the 37℃, 5% carbon dioxide concentration incubator and continue to culture for 72h.

[0038] During cell administration, attention should be paid to the administration concentration that matches the cell amount; experiments have shown that the use of other diluents, such as water, PBS, and dilute hydrochloric acid, has poor effects.

[0039] Step 3: Cell sample solution extraction

[0040] After 72 hours, take out the cell culture dish, add 1ml 0.25% trypsin and place it at 37℃ for digestion. After the cells fall off, add 3ml pre-cooled PBS to stop trypsin digestion. Use a 1ml pipette to blow the cells to make them all detached and collect them in a 15ml centrifuge tube, centrifuge at 1500r / min for 5min (the centrifugal force is recommended to be between 100g and 300g). Discard the supernatant, resuspend the cells with 750μl PBS buffer (the volume can be adjusted appropriately according to the actual number of cells), use a cell ultrasonic disruptor to disrupt the cells, immerse the ultrasonic rod into the suspension 1cm, place the suspension in ice water (freezing treatment first is better), and intermittently ultrasonicate (take VCX750 ultrasonic disruptor as an example: ultrasonicate 3s, interval 5s, duration 1min, amplitude 20%, temperature control below 30℃). If the sample cannot be processed in time on the same day, it can be placed in a refrigerator at -20℃ or below overnight.

[0041] After the ultrasound is finished, the sample is centrifuged at 10000r / min for 15min, and the supernatant is taken to obtain the test solution.

[0042] Step 4: Determination of alkaline phosphatase activity in cell sample extract

[0043] The alkaline phosphatase activity was determined using an alkaline phosphatase test kit, and the protein content was detected using a BCA kit to calculate the BMP-2 protein activity. The BMP-2 protein activity calculation formula is as follows: BMP-2 activity (U) = C 酚含量测定 *V BMP *N, where V BMP is the total volume of the sample, ml; N is the sample dilution factor, and the value is 1 when not diluted.

[0044] Step 5: Determination of protein content in cell sample extract

[0045] Take an appropriate volume of protein standard (provided in the BCA Protein Concentration Assay Kit (Enhanced)) and dilute to a final concentration of 1 mg / ml. For example: take 20 μl of 5 mg / ml protein standard and add 80 μl of diluent (1x PBS) to prepare a 1 mg / ml protein standard.

[0046] According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. For example, add 5ml of BCA reagent A to 100μl of BCA reagent B, mix well, and prepare 5.1ml of BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0047] Add 0, 1, 2, 4, 8, 16, and 20 μl of the standard into the standard wells of a 96-well plate, add standard diluent to make up to 20 μl, and prepare a standard curve with concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 mg / ml, respectively.

[0048] Take an appropriate amount of sample, dilute to a concentration within the linear range (about 4 or 5 times), and then take 20 μl of the diluted sample and add it to the sample wells of the 96-well plate. Add 200 μl of BCA working solution to each well and place it at 37°C for 20 to 30 minutes. Use an enzyme reader to measure the results at a wavelength of 562nm, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0049] In the process of measuring BMP-2 cell activity, it is very important to ensure the accuracy, stability and repeatability of the measurement results. In order to verify the reliability and effectiveness of the measurement method provided by the present invention, the present invention adopts the following test method:

[0050] Establishment of ALP activity standard curve to provide a benchmark for activity determination;

[0051] Activity repeatability test to ensure consistency of results under different experimental conditions;

[0052] Analysis of the effects of varying the amount of cells seeded or the number of passages to assess the impact of different experimental settings on activity determination;

[0053] And compare the effects of direct ultrasonic disruption and lysis buffer methods to optimize the sample processing process.

[0054] Establishment of ALP activity standard curve

[0055] ALP activity definition: The cell extract sample reacts with the matrix for 15 minutes at 37°C to produce 1ug of phenol, which is one activity unit (U); BMP-2 protein activity (U) = ALP activity (U) = phenol concentration of the sample to be tested (ug / ml) / total volume of the sample to be tested (ml) * dilution factor

[0056] The following table shows the culture and measurement under different BMP-2 concentrations. Each experimental group was inoculated with 2.5×10 5 cells, 15 ml of culture medium was used, and 6 different BMP-2 protein concentrations were used, namely 1500 ng / ml, 1250 ng / ml, 1000 ng / ml, 750 ng / ml, 500 ng / ml and 250 ng / ml.

[0057] Table 1 ALP activity standard curve data table

[0058]

[0059] As can be seen from Table 1, as the concentration of BMP-2 decreases, the phenol content also gradually decreases. From 132.61μg / ml at 1500ng / ml to 20.26μg / ml at 250ng / ml, it shows that the phenol content is positively correlated with the concentration of BMP-2; BMP-2 activity also decreases with the decrease of BMP-2 concentration, from 99.46U (1500ng / ml concentration) to 15.20U (250ng / ml concentration) (e.g. Figure 1 This further verifies the relationship between phenol content and BMP-2 activity; the protein content changes slightly at different concentrations, remaining between 1.83 mg / ml and 2.12 mg / ml, with a small fluctuation range, indicating that changes in cell culture and BMP-2 concentration have relatively little effect on the total protein content.

[0060] Activity repeatability test

[0061] The following table shows the experimental data of culture and measurement under different BMP-2 concentrations. Each experimental group was inoculated with 2.5×10 5 cells, 15 ml of culture medium was used. A BMP-2 concentration of 1000 ng / ml was used in the experiment, and phenol content, BMP-2 activity and protein content were measured in each group.

[0062] Table 2 Activity repeatability test data table

[0063]

[0064] As can be seen from Table 2, the phenol content fluctuated in different experimental groups, ranging from 72.32μg / ml to 53.66μg / ml, with a mean of 61.75μg / ml and a CV% of 11.15%, indicating that the phenol content has a certain variability and is closely related to the activity of BMP-2. BMP-2 activity also showed similar fluctuations, ranging from 54.24U to 40.25U, with a mean of 46.31U and a CV% of 11.15%. This further verifies the positive correlation between phenol content and BMP-2 activity, and the activity varies greatly among the groups. The protein content varied less in the experimental groups, ranging from 1.92mg / ml to 1.84mg / ml, with a mean of 1.95mg / ml and a CV% of 4.65%, indicating that the protein content changes more steadily and the variation is small.

[0065] In summary, phenol content and BMP-2 activity had large variability, while protein content was relatively stable, which indicated that the results of BMP-2 activity determination were affected by certain experimental variations but maintained high stability in most experiments.

[0066] Analysis of the effects of changing the inoculum cell amount or passage number

[0067] The following table shows the experimental data of BMP-2 activity determination under different inoculated cell numbers and generations. Each experimental group used 15 ml of culture medium and added 1000 ng / ml BMP-2. The inoculated cell numbers were 1.0×10 5 4.0×10 5 and 2.5×10 5 cells, and the table records the phenol content, BMP-2 activity and protein content of each group.

[0068] It can be seen from Table 3 that the phenol content fluctuates with the increase of inoculated cell amount or the change of generation. 5 When 4.0×10 cells were inoculated, the phenol content was 42.18 μg / ml, while when 4.0×10 5 When the cells were 2.5×10 5 When the number of cells was 400, the phenol content was 36.52 μg / ml, indicating that the amount of inoculated cells and the generation had a certain effect on the phenol content.

[0069] Table 3 Data table for changing the amount of inoculated cells or generation

[0070]

[0071]

[0072] BMP-2 activity showed a downward trend with the increase of inoculated cell amount or the change of generation. 5 This showed that the amount of cells and the number of generations had a certain influence on the activity of BMP-2.

[0073] The protein content varied greatly between different inoculum sizes and generations, especially when the inoculum size was 4.0×10 5 When cells were grown, the protein content was 3.22 mg / ml, which had a larger fluctuation compared with other groups. The subsequent group protein content was maintained between 1.85 mg / ml and 2.02 mg / ml.

[0074] Comparative analysis of direct ultrasonic disruption and lysis solution methods

[0075] The following table shows the experimental data of treating cell samples with lysis buffer and direct sonication and measuring at a BMP-2 concentration of 1000ng / ml. Each experimental group used 15ml of culture medium and inoculated the same number of cells.

[0076] Table 4 Comparison of direct ultrasonic disruption and lysis solution method

[0077]

[0078] As can be seen from Table 4, the phenol content was higher in the groups treated with direct ultrasound (62.15 μg / ml, 54.23 μg / ml, and 58.77 μg / ml) compared with the groups treated with lysate (50.42 μg / ml, 48.93 μg / ml, and 55.48 μg / ml). This indicates that direct ultrasound disruption is more efficient in cell disruption and releases more phenolic substances, which indirectly reflects higher BMP-2 activity.

[0079] Compared with the lysate group, the groups treated with direct ultrasound (46.61 U, 40.67 U, and 44.08 U) showed slightly higher BMP-2 activity. The disruption effect of the direct ultrasound treatment method may lead to the release of more BMP-2 active substances, enhancing the overall activity.

[0080] The protein content fluctuated slightly between the different groups. The protein content of the lysate group (1.76 mg / ml to 2.02 mg / ml) was relatively close to that of the direct ultrasound group (1.81 mg / ml to 1.94 mg / ml), indicating the consistency of the two treatment methods in protein extraction.

[0081] In addition, the present invention also uses the NPP method for further verification, and no phenol is detected. It can be seen that the NPP method fails to effectively extract or react phenolic substances under the experimental conditions, indicating that this method is not suitable for the determination of BMP-2 activity in this experimental system. In contrast, the phenol content method and the BMP-2 activity determination method can provide more accurate results and have better stability and sensitivity.

[0082] In summary, the present invention successfully improves the efficiency and accuracy of BMP-2 cell activity determination by optimizing the sample processing method and the determination process, and provides a more efficient and reliable experimental method for biomedical research and related fields.

[0083] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for measuring BMP-2 cell activity, characterized in that: The method comprises the following steps: Step 1: Cell preparation: Take cells with good growth status, count them and inoculate them into cell culture dishes, add DMEM cell culture medium containing 10% FBS to 10 ml, mix them evenly by pipetting, and continue to culture them in an incubator at 37°C and 5% carbon dioxide concentration until the cells adhere to the wall; Step 2: Cell administration: prepare BMP-2 protein solution with ammonium acetate, filter and sterilize, take out the previously attached cells, add them to the cell culture dish, and continue to culture for 72 hours; Step 3: Extract the cell sample solution. After 72 hours, add 0.25% trypsin to digest the cells. Use PBS solution to stop the digestion. Collect the cell sample by centrifugation. Use an ultrasonic disruptor to ultrasonically disrupt the sample. Step 4: Determination of alkaline phosphatase activity in the cell sample extract: using an alkaline phosphatase kit to determine the alkaline phosphatase activity, combined with a BCA kit to determine the protein concentration, and calculate the BMP-2 protein activity; Step 5: Determine the protein content of the cell sample extract using a BCA protein concentration assay kit to determine the protein concentration in the sample.

2. The method according to claim 1, characterized in that The cell inoculum size in step 1 is 2.5×10 5 cells / 10cm 2 Petri dish.

3. The method according to claim 1, characterized in that The concentration of the BMP-2 protein solution used in step 2 is 100 μg / ml.

4. The method according to claim 3, characterized in that: In step 2, the preparation of the BMP-2 protein solution uses 10 mM ammonium acetate solution as a dissolving agent, and the phenomenon of incomplete protein dissolution does not occur when using the ammonium acetate solution.

5. The method according to claim 4, characterized in that In step 2, the final concentration of the BMP-2 protein solution and the cell culture medium is 250 ng / ml to 1500 ng / ml.

6. The method according to claim 1, characterized in that In step 3, the ultrasonic parameters of the ultrasonic crusher are: ultrasonic time is 3 seconds, interval time is 5 seconds, total ultrasonic time is 1 minute, amplitude is 20%, and the temperature is controlled below 30° C. during ultrasonic crushing.

7. The method according to claim 6, characterized in that The centrifugal conditions in step 3 are: 1500r / min, and the centrifugal time is 5 minutes.

8. The method according to claim 7, characterized in that In step 3, if the sample cannot be processed on the same day, it can be placed in a refrigerator at -20°C or below overnight.

9. The method according to claim 1, characterized in that: In step 4, the formula for calculating BMP-2 activity is: BMP-2 activity (U) = C 酚含量测定 *V BMP *N, where V BMP is the total volume of the sample, ml; N is the sample dilution factor, and the value is 1 when not diluted.

10. The method according to claim 1, characterized in that In step 5, the protein concentration is determined using a BCA kit, and the protein content of the sample is quantitatively calculated by comparing with the standard curve.

Images