Method for promoting synthesis of steroids in lindel gland cells
By regulating the cholesterol concentration in the culture medium of musk gland cells and standardizing the process, the problem of balancing cell synthesis efficiency and survival stability in existing technologies has been solved, resulting in a significant increase in the synthesis of steroidal substances and improved reliability of experimental results, making it suitable for industrial production.
Patent Information
- Application Number
- CN202511471355.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2026-01-16
AI Technical Summary
The existing technology lacks a systematic study on the relationship between cholesterol concentration and steroid synthesis in the culture medium of forest musk gland cells, which makes it impossible to improve the synthesis efficiency through precise control, and it is difficult to balance cell survival stability and synthesis amount. The lack of standardized procedures leads to poor reproducibility of experimental results, making it difficult to meet the needs of industrial production.
By determining the tolerance range of cholesterol concentration through preliminary experiments, control groups, low-concentration and high-concentration treatment groups were set up, and the cholesterol concentration in the culture medium was adjusted to 10-20 mg/L. Combined with cell culture, sample processing and LC-MS detection, a standardized procedure was provided to improve the synthesis of steroid substances and cell survival stability.
It significantly increased the synthesis of steroidal substances in forest musk gland cells, especially the synthesis of pregnenolone and progesterone, which were increased by 4.12 times and 1.46 times, respectively, ensuring cell survival stability and improving the reproducibility of experimental results, making it suitable for industrial production.
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Figure CN121343874A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of cell engineering and biopharmaceutical technology, specifically a method for promoting the synthesis of steroidal substances in forest musk gland cells. Background Technology
[0002] Musk gland cells possess the ability to autonomously synthesize steroidal substances (such as cholesterol, pregnenolone, and progesterone), which have significant applications in medicine and biopharmaceuticals. Currently, in vitro culture technology for musk gland cells has gradually developed, but the following key issues remain: The lack of systematic research on the relationship between the concentration of key components in the culture medium (such as cholesterol) and the synthesis of steroidal substances makes it impossible to improve synthesis efficiency through precise control of culture medium composition. Existing culture protocols struggle to balance cell viability and steroidal substance synthesis. Excessively high cholesterol concentrations can lead to cell shrinkage, detachment, and even death; conversely, excessively low concentrations fail to effectively promote steroidal substance synthesis. The lack of standardized cell culture, sample processing, and detection procedures results in poor reproducibility of experimental results, hindering industrial-scale production. While existing technologies mentioned in the announcement mention the culture of *Muscaria lucida* gland cells or the detection of steroidal substances, they do not address the optimization and control of cholesterol concentration, nor do they propose specific solutions that balance cell viability and synthesis efficiency, thus failing to meet the needs of industrial-scale production of steroidal substances from *Muscaria lucida* gland cells.
[0003] In view of this, we propose a method to promote the synthesis of steroidal substances in forest musk gland cells. Summary of the Invention
[0004] The purpose of this invention is to provide a method for promoting the synthesis of steroidal substances in forest musk gland cells, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for promoting the synthesis of steroidal substances in forest musk gland cells, comprising the following steps: S1. Preliminary experiment to determine the cholesterol concentration tolerance range: Using basal culture medium as the base, eight cholesterol concentration gradients were set up: 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 70 mg / L. After culturing forest musk gland cells for 3 days, the cell status was observed, and 20 mg / L was determined to be the highest cholesterol concentration that the cells could tolerate. S2. Experimental group setup: A control group, a low-concentration treatment group, and a high-concentration treatment group were set up. The control group included the P1 group and basal culture medium. The low-concentration treatment group included the D10 group and basal culture medium + 10 mg / L cholesterol. The high-concentration treatment group included the D20 group and basal culture medium + 20 mg / L cholesterol. Each group was biologically replicated 3 times. S3. Cell culture: The forest musk gland cells were cultured in basal medium until they covered 70% of the bottom of the culture dish. The medium was then replaced with the corresponding experimental group medium and cultured for another 3 days. S4. Sample Collection and Processing: Collect cell samples after 3 days of culture, ensuring a cell count ≥1×10⁻⁶. 7 Cells / samples were quenched in liquid nitrogen for 30 seconds and then stored at -80 degrees Celsius. Subsequently, the cells were disrupted, centrifuged, dried, reconstituted, and sonicated to obtain the supernatant for testing. S5, LC-MS detection; Ultra-high performance liquid chromatography-mass spectrometry is used to detect the content of steroidal substances in the supernatant to be tested, thereby improving the synthesis of steroidal substances.
[0006] Optionally, in S1, the basal culture medium is provided by Beijing Yanshe Biotechnology Development Co., Ltd., and the forest musk gland cells are third-generation passaged cells with a viability ≥95%.
[0007] Optionally, in S3, the cell culture conditions are 37 degrees Celsius and 5% CO2, ensuring that the final cell count of each group reaches 1×10⁻⁶ cells during culture. 7 cell / sample or more.
[0008] Optionally, the specific operation of cell disruption in S4 includes the following steps: Add 250 μL of ultrapure water to the cell sample, vortex mix for 30 seconds, and then place it in a cell disruptor to disrupt for 15 minutes.
[0009] Optionally, the centrifugation conditions in S4 are: 4 degrees Celsius, 12000 rpm, centrifugal force 13800×g, centrifugal radius 8.6cm, and centrifugation time 15min; the specific operation of reconstitution is: add the reconstitution solution containing the isotope-labeled internal standard at a volume ratio of methanol:acetonitrile:water = 2:2:1, and vortex mix for 30s.
[0010] Optionally, in step S4, the ultrasonic treatment conditions are an ice-water bath and an ultrasonic time of 10 minutes; followed by centrifugation.
[0011] Optionally, in S5, the ultra-high performance liquid chromatograph is a Vanquish type (Thermo Fisher Scientific), and the chromatographic column is a Waters ACQUITY UPL CBEHAmide (2.1 mm × 100 mm, 1.7 μm).
[0012] Optionally, in S5, the mobile phase of the liquid chromatography is: phase A is an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B is acetonitrile; the sample pan temperature is 4 degrees Celsius, and the injection volume is 2 μL.
[0013] Optionally, the mass spectrometer in S5 is an OrbitrapExploris 120 (ThermoFisherScientific), with mass spectrometry parameters including: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 degrees Celsius, primary mass spectrometry resolution 60,000, secondary mass spectrometry resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltage 3.8 kV in positive ion mode and -3.4 kV in negative ion mode.
[0014] Compared with the prior art, the present invention provides a method for promoting the synthesis of steroidal substances in forest musk gland cells, which has the following beneficial effects: 1. This method for promoting the synthesis of steroidal substances in forest musk gland cells, by adjusting the cholesterol concentration in the culture medium to 10-20 mg / L, can upregulate the synthesis of seven steroidal substances in forest musk gland cells, among which the content of pregnenolone is increased by 4.12 times (P<0.05) and the content of progesterone is increased by 1.46 times (P<0.05), which is significantly better than the existing culture without cholesterol regulation.
[0015] 2. This method for promoting the synthesis of steroidal substances in forest musk gland cells establishes 20 mg / L as the maximum tolerated cholesterol concentration for cells. At this concentration, cell growth is only slightly slowed down without cell death, thus balancing cell survival stability and synthesis efficiency. It provides a complete standardized process from preliminary experiments, culture, sample processing to detection, with high reproducibility of experimental results, and can be directly applied to industrial production. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the process of the present invention. Detailed Implementation
[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0018] like Figure 1 As shown, the present invention provides a technical solution: a method for promoting the synthesis of steroidal substances in forest musk gland cells, comprising the following steps: S1. Preliminary experiment to determine the cholesterol concentration tolerance range: Using basal culture medium as the base, eight cholesterol concentration gradients were set up: 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 70 mg / L. After culturing forest musk gland cells for 3 days, the cell status was observed, and 20 mg / L was determined to be the highest cholesterol concentration that the cells could tolerate. The basal culture medium was provided by Beijing Yanshe Biotechnology Development Co., Ltd., and the forest musk gland cells were third-generation passaged cells with a viability of ≥95%.
[0019] S2. Experimental group setup: A control group, a low-concentration treatment group, and a high-concentration treatment group were set up. The control group included the P1 group and basal culture medium. The low-concentration treatment group included the D10 group and basal culture medium + 10 mg / L cholesterol. The high-concentration treatment group included the D20 group and basal culture medium + 20 mg / L cholesterol. Each group was biologically replicated 3 times. S3. Cell culture: The forest musk gland cells were cultured in basal medium until they covered 70% of the bottom of the culture dish. The medium was then replaced with the corresponding experimental group medium and cultured for another 3 days. The cell culture conditions were 37 degrees Celsius and 5% CO2, and the final cell count for each group was ensured to reach 1×10⁶ cells / year during the culture period. 7 cell / sample or more.
[0020] S4. Sample Collection and Processing: Collect cell samples after 3 days of culture, ensuring a cell count ≥1×10⁻⁶. 7 Cells / samples were quenched in liquid nitrogen for 30 seconds and then stored at -80 degrees Celsius. Subsequently, the cells were disrupted, centrifuged, dried, reconstituted, and sonicated to obtain the supernatant for testing. The specific steps for cell disruption are as follows: Add 250 μL of ultrapure water to the cell sample, vortex mix for 30 seconds, and then place it in a cell disruptor to disrupt for 15 minutes. The centrifugation conditions were: 4 degrees Celsius, 12000 rpm, centrifugal force 13800×g, centrifugal radius 8.6 cm, and centrifugation time 15 min; the specific operation for reconstitution was: adding the reconstitution solution containing the isotope-labeled internal standard at a volume ratio of methanol:acetonitrile:water = 2:2:1, and vortexing for 30 s. The ultrasonic treatment conditions were ice-water bath and ultrasonic time of 10 min; after ultrasonication, the cells were centrifuged again.
[0021] S5, LC-MS detection; Ultra-high performance liquid chromatography-mass spectrometry is used to detect the content of steroidal substances in the supernatant to be tested, thereby improving the synthesis of steroidal substances.
[0022] The ultra-high performance liquid chromatograph was a Vanquish (Thermo Fisher Scientific) and the chromatographic column was a Waters ACQUITY UPL CBEHAmide (2.1 mm × 100 mm, 1.7 μm).
[0023] The mobile phase for liquid chromatography was: phase A was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile; the sample pan temperature was 4 degrees Celsius, and the injection volume was 2 μL.
[0024] The mass spectrometer was an Orbitrap Exploris 120 (ThermoFisher Scientific), and the mass spectrometry parameters included: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 degrees Celsius, primary mass spectrometry resolution 60,000, secondary mass spectrometry resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltage 3.8 kV in positive ion mode and -3.4 kV in negative ion mode.
[0025] As one application of this embodiment: Experimental tissue culture: Take 8 sterile culture dishes and add 10 mL of basal culture medium containing 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 70 mg / L cholesterol, respectively; inoculate each dish with 1×10⁻⁶ ppm. 6 Musk gland cells were cultured at 37°C in a 5% CO2 incubator for 3 days. Cell status was observed daily: no cell death was observed in the 0-20 mg / L group, some cell death was observed in the 30 mg / L group, and a large number of cells died in the 40-70 mg / L group. Therefore, 20 mg / L was determined to be the highest tolerated concentration.
[0026] Experimental group culture procedures: Take 3 sterile culture dishes and inoculate each dish with 1×10 6 Musk gland cells were cultured in basal medium until they covered 70% of the dish bottom. The medium was then replaced with P1 (basal medium), D10 (10 mg / L cholesterol medium), and D20 (20 mg / L cholesterol medium), with three replicates for each group. During the culture period, the cells in P1 group covered 100% of the dish bottom in 2 days, the cells in D10 group covered 100% in 2 days, and the cells in D20 group covered 100% in 2.5 days. There was no large amount of cell shedding or death in any of the cases.
[0027] Sample processing and testing: After 3 days of culture, 3 samples were collected from each group, with each sample containing 1.2 × 10⁻⁶ cells. 7Cell / sample, quenched in liquid nitrogen for 30 seconds and stored at -80℃; sample processing was performed according to S5 procedure, with centrifugation parameters, reconstitution ratio, and sonication time strictly followed; LC-MS results: 9 steroidal substances were detected in group P1, and pregnenolone (4.12-fold) and progesterone (1.46-fold) were significantly upregulated in group D20, while the other 5 steroidal substances (such as 17α-hydroxypregnenolone) showed biological differences (Cohen's d>0.8).
[0028] In three biological replicates, the coefficient of variation of steroid content in each group was <5%, and the RSD of peak area in QC sample detection was <3%, proving that the method is stable and reliable.
[0029] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.
Claims
1. A method for promoting steroidogenesis in musk deer gland cells, the method comprising, Comprise the following steps: S1, pre-experiment determines the cholesterol concentration tolerance range; with the basal medium as the base, set 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, a total of 8 cholesterol concentration gradients, culture musk deer scent gland cells for 3 days, then observe the cell state, and determine 20 mg / L as the highest tolerance of the cell cholesterol concentration; S2, experimental group setting; set the control group, the low concentration treatment group and the high concentration treatment group, the control group includes P1 group and basal medium, the low concentration treatment group includes D10 group and basal medium+10 mg / L cholesterol, the high concentration treatment group includes D20 group and basal medium+20 mg / L cholesterol, each group is repeated 3 times biologically; S3, cell culture; the musk deer scent gland cells are cultured with the basal medium to 70% of the bottom of the culture dish, and then replaced with the corresponding experimental medium for continuous culture for 3 days; S4, sample collection and processing; collect cell sample after 3 days of culture, cell amount in cell sample ≥ 1 x 10 7 cell / sample, stored at -80 degrees Celsius after 30 seconds of liquid nitrogen quenching, and then obtain the supernatant to be detected through cell crushing, centrifugation, drying, re-dissolving, and ultrasonic treatment; S5, LC-MS detection; the content of steroid substances in the supernatant to be detected is detected by ultra-high performance liquid chromatography-mass spectrometry, so as to realize the improvement of the synthesis amount of steroid substances.
2. The method of claim 1, wherein the method is for promoting the synthesis of a steroidal substance in a forest musk deer's scent gland. In the S1, the basal medium is provided by Beijing Yanmusheng Biological Technology Development Co., Ltd., and the musk deer scent gland cells are the third generation of passaged cells with a viability of ≥95%.
3. The method of claim 1, wherein the method is for promoting the synthesis of a steroidal substance in a forest musk deer's scent gland. The S3, cell culture conditions are 37 degrees Celsius, 5% CO2, during the culture period to ensure that the final amount of each group of cells to reach 1 x 10 7 above.
4. The method of claim 1, wherein the method is for promoting the synthesis of a steroidal substance in a forest musk deer's scent gland. The specific operation of cell crushing in the S4 comprises the following steps: Add 250 μL ultrapure water to the cell sample, vortex for 30 s, and then put it into the cell crusher for 15 min.
5. The method of claim 1, wherein the method is for promoting the synthesis of a steroidal substance in a forest musk deer's scent gland. The centrifugal treatment condition in the S4 is: 4 degrees Celsius, 12000 rpm, centrifugal force 13800xg, centrifugal radius 8.6 cm, centrifugal time 15 min.
6. The method of claim 1, wherein the method is performed in the presence of a steroid. The ultrasonic treatment condition in the S4 is ice water bath, ultrasonic time 10 min; after ultrasonic, centrifuge again.
7. The method of claim 1, wherein the method is performed in the presence of a steroid. In the S5, the mobile phase of liquid chromatography is: A phase is water phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and B phase is acetonitrile; the sample disc temperature is 4 degrees Celsius, and the injection volume is 2 μL.
8. The method of claim 1, wherein the method is for promoting the synthesis of a steroidal substance in a forest musk deer's scent gland. In the S5, the mass spectrometer is OrbitrapExploris120 (ThermoFisher Scientific), and the mass spectrometry parameters include: sheath gas flow 50 Arb, auxiliary gas flow 15 Arb, capillary temperature 320 degrees Celsius, primary mass spectrometry resolution 60000, secondary mass spectrometry resolution 15000, collision energy SNCE 20 / 30 / 40, spray voltage positive ion mode 3.8 kV, negative ion mode-3.4 kV.