Method for preparing sturgeon ovary cells secreting bile acid in vitro using isolated sturgeon ovary and bile acid
Through enzyme digestion and cell culture of ex vivo sturgeon ovarian tissue, a sturgeon ovarian cell line that can secrete bile acids in vitro was established, which solved the problem of missing bile acids in vitro in the fish ovarian cells in the prior art, and achieved efficient production High-purity bile acids are used in aquaculture and biological research, bringing economic benefits.
Patent Information
- Application Number
- CN202111515313.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-12-13
- Publication Date
- 2024-12-27
- Estimated Expiration
- 2041-12-13
AI Technical Summary
There is no report in the prior art that fish ovarian cell lines can secrete bile acids in vitro, and commercially produced bile acids are mostly extracted from pig gallbladder, which lacks efficient production methods in fish biology research and aquaculture.
Through the preparation, enzyme digestion and cell culture of ex vivo sturgeon ovary or ovarian tissue, a sturgeon ovary cell line capable of secreting bile acids in vitro was established, and cells were performed using serum-free DMEM/F12 medium and Trypsin-EDTA digestible solution. Culture to obtain a highly purified bile acid that can be passed on continuously.
The establishment of small body sturgeon ovarian cell lines can be achieved, and high-purity bile acids can be produced efficiently in vitro, used to study ovarian development mechanisms and aquaculture, bringing economic benefits, and providing new production methods for aquatic products and animal husbandry.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fish cell culture, and particularly relates to a method for preparing sturgeon ovary cells secreting bile acid in vitro by using isolated sturgeon ovaries and bile acid. Background Art
[0002] Sturgeons are ancient, economically valuable, large sub-cold-water fishes. Sturgeon meat is delicious, and sturgeon caviar is known as "egg gold". Compared with other sturgeons, the sterlet sturgeon (Acipenser ruthenus) is smaller in size and matures earlier, making it a model fish for sturgeon biology research.
[0003] Fish ovarian tissue is mainly composed of a large number of independent gonadal microenvironments, and the gonadal microenvironment cells are mainly composed of female germ cells, granulosa cells and theca cells. The gonadal microenvironment cells conduct signal transduction and communication with each other, and granulosa cells and theca cells work together under signal stimulation to secrete a large number of hormones, nutrients, cytokines and other components, providing the necessary nutrients and cell regulatory factors for the growth and development of germ cells.
[0004] Bile acid is the main active ingredient in bile. It was previously believed that it was synthesized by liver cells and often exists in the form of sodium salts, also known as bile salts. Depending on the site of generation, bile acids are divided into two categories, primary bile acids and secondary bile acids. The former is synthesized in the liver, and the latter is converted in the intestine. Depending on whether the conjugation reaction occurs in the liver, it is divided into free bile acids and conjugated bile acids. The main function of bile acid is to promote the digestion and absorption of lipids. Bile acid is considered to be a digestive aid molecule that participates in the emulsification and absorption of dietary fats and fat-soluble vitamins, and plays an important role in cholesterol homeostasis. Recent studies have found that bile acids are similar in structure to steroid hormones and have multi-organ interactive regulatory functions in metabolism, endocrine and immune functions. Currently, most commercially produced bile acids are extracted from pig bile. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing sturgeon ovary cells secreting bile acid in vitro by using isolated sturgeon ovaries and bile acid.
[0006] The present invention provides an application of an isolated sturgeon ovary or an isolated sturgeon ovarian tissue, which is as follows (a) or (b):
[0007] (a) preparing bile acid;
[0008] (b) Preparation of sturgeon ovarian cells that secrete bile acids in vitro.
[0009] The present invention also provides a method for preparing bile acid, comprising the following steps:
[0010] (1) using isolated sturgeon ovaries or isolated sturgeon ovarian tissues as raw materials, sequentially performing tissue block preparation, tissue block digestion and cell culture to obtain sturgeon ovarian cells that can secrete bile acid in vitro;
[0011] (2) culturing the sturgeon ovarian cells obtained in step (1) in vitro to obtain bile acid.
[0012] The present invention also provides a method for preparing sturgeon ovary cells that can secrete bile acid in vitro, comprising the following steps: using isolated sturgeon ovaries or isolated sturgeon ovary tissues as raw materials, sequentially performing tissue block preparation, tissue block digestion and cell culture to obtain sturgeon ovary cells that can secrete bile acid in vitro.
[0013] In any of the above methods, the size of the tissue block prepared is 1 mm 3 .
[0014] In any of the above methods, the method for preparing the tissue block is: then placing the isolated sturgeon ovary or the isolated sturgeon ovary tissue in serum-free DMEM / F12 culture medium and cutting it into 1 mm 3 The tissue blocks were then rinsed with PBS buffer.
[0015] In any of the above methods, the tissue block digestion adopts the enzymatic digestion of tissue blocks method.
[0016] In any of the above methods, the tissue block digestion is performed using 0.25% Trypsin-EDTA digestion solution.
[0017] In any of the above methods, the method for digesting the tissue block is: adding 0.25% Trypsin-EDTA digestion solution to the tissue block for digestion (specifically, the digestion time can be 10 minutes), and after the digestion is completed, adding 20% FBS-DMEM / F12 complete culture medium to terminate the digestion effect of trypsin.
[0018] In any of the above methods, the method for digesting the tissue block is: adding 2 mL of 0.25% Trypsin-EDTA digestion solution to the tissue block for digestion (specifically, the digestion time can be 10 minutes), and after the digestion is completed, adding 2 mL of 20% FBS-DMEM / F12 complete culture medium to terminate the digestion effect of trypsin.
[0019] In any of the above methods, the cell culture includes primary cell culture and cell subculture.
[0020] In any of the above methods, the cell culture adopts delayed digestion method.
[0021] The primary cell culture comprises the following steps: after the digestion of the tissue block is completed, the whole system is transferred to a centrifuge tube, centrifuged at 300g, and the supernatant is discarded; then serum-free DMEM / F12 medium is added to suspend the tissue block, and then inoculated into a cell culture bottle, and cultured inverted at 24°C for 7 hours; then, the liquid in the culture bottle is aspirated and discarded, 20% FBS-DMEM / F12 complete culture medium is added, and the culture is upright at 24°C. Primary cells, also known as P0 cells, are obtained.
[0022] Specifically, the primary cell culture comprises the following steps: after the digestion of the tissue block is completed, the entire system is transferred to a centrifuge tube, centrifuged at 300g for 10 minutes, and the supernatant is discarded; then 1 mL of serum-free DMEM / F12 medium is added to suspend the tissue block, and then inoculated to 25 cm 2 The cell culture flask was placed in a 24°C biochemical incubator and inverted for 7 hours (bottom of the flask facing upward); then, the liquid in the flask was aspirated and 2 mL of 20% FBS-DMEM / F12 complete culture medium was added, and the flask was placed in a 24°C biochemical incubator and inverted for 7 hours (bottom of the flask facing upward). Primary cells, also known as P0 cells, were obtained.
[0023] The upright culture was continued until the cells grew into a monolayer.
[0024] The culturing time of the upright culture is 7 days.
[0025] The upright culture is carried out until the cells occupy more than 80% of the bottom area of the bottle.
[0026] The upright culture is carried out until the cells occupy 90% of the bottom area of the bottle.
[0027] The cell subculture comprises the following steps: after the previous generation of cell culture is completed, the liquid in the culture bottle is discarded; 0.25% Trypsin-EDTA digestion solution is added for digestion; then, 20% FBS-DMEM / F12 complete culture solution is added to the culture bottle to prepare a cell suspension; the cell suspension is taken to a new culture bottle, 20% FBS-DMEM / F12 complete culture solution is added, and the cells are cultured at 24° C. until the cells grow into a single layer.
[0028] Specifically, the cell subculture comprises the following steps: after completing the previous generation of cell culture, aspirate and discard the liquid in the culture bottle; then, add 1 mL of 0.25% Trypsin-EDTA digestion solution to the culture bottle, shake the culture bottle and aspirate and discard the digestion solution; then, add 1 mL of 0.25% Trypsin-EDTA digestion solution to the culture bottle again, shake the culture bottle and digest for 2 minutes, aspirate and discard the digestion solution, and let it stand for 5 minutes; then, add 1 mL of 20% FBS-DMEM / F12 complete culture solution to the culture bottle, and gently blow the bottom of the culture bottle to make a cell suspension; take 0.5 mL of the cell suspension to a new culture bottle, add 2.5 mL of 20% FBS-DMEM / F12 complete culture solution to each culture bottle, and culture at 24°C until the cells grow into a monolayer.
[0029] 20% FBS-DMEM / F12 complete culture medium: It is composed of fetal bovine serum, insulin-like growth factor I, PSN antibiotic mixture and DMEM / F12 culture medium. In 20% FBS-DMEM / F12 complete culture medium, the concentration of fetal bovine serum is 20% (volume ratio), the concentration of insulin-like growth factor I is 40ng / ml, the concentration of penicillin is 50μg / mL, the concentration of streptomycin is 50μg / mL, and the concentration of neomycin is 50μg / mL.
[0030] Any of the above-mentioned cell subcultures can be subcultured for 60 consecutive generations. After 60 generations, it is called a cell line.
[0031] Any of the above cell subcultures may be subcultured for 40 consecutive generations.
[0032] Any of the above cell subcultures may be continuous subcultures for 3 to 38 generations.
[0033] The sturgeon ovary cells capable of secreting bile acid in vitro may specifically be cells that have been passed down to the 60th generation.
[0034] The sturgeon ovarian cells capable of secreting bile acid in vitro may specifically be cells that have been passed down to the 40th generation.
[0035] The sturgeon ovarian cells capable of secreting bile acid in vitro may specifically be cells of passage 3 to 38.
[0036] Sturgeon ovarian cells that can secrete bile acids in vitro can be serially passaged.
[0037] Any of the above-mentioned sturgeons is a small-bodied sturgeon.
[0038] The present invention also protects the sturgeon ovarian cells capable of secreting bile acid in vitro and prepared by any of the above methods.
[0039] The sturgeon ovary cell capable of secreting bile acid in vitro may specifically be a sturgeon ovary cell line capable of secreting bile acid in vitro.
[0040] The sturgeon ovarian cells capable of secreting bile acid in vitro can be widely used in the transfection of exogenous genes and plasmids and the study of sex-related gene functions.
[0041] The sturgeon ovary cells capable of secreting bile acid in vitro can be used for producing bile acid in vitro.
[0042] The present invention also protects the use of the sturgeon ovarian cells capable of secreting bile acid in vitro in the preparation of bile acid.
[0043] In the prior art, there is no report on the secretion of bile acid in vitro by fish ovarian cell lines. Based on previous research work, the inventors of the present invention found that the ovarian tissue cells of the sturgeon can synthesize and secrete bile acid by themselves. Furthermore, the inventors of the present invention established a cell line capable of secreting bile acid in vitro. On the one hand, the establishment of the cell line in the present invention will help to study the molecular mechanism of ovarian development and the effect of bile acid on ovarian development. On the other hand, bile acid has the function of improving the immunity of aquatic animals and promoting growth and development. The cell line of the present invention can be used to mass-produce high-purity bile acid in vitro, and its application in aquatic and animal husbandry will bring considerable economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 These are photos of primary cells and passaged cells under an optical microscope.
[0045] Figure 2 The results of the combined analysis of transcriptome and metabolome levels after ovarian cells were grown for 4 days. DETAILED DESCRIPTION
[0046] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0047] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0048] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.
[0049] MS222, the full name is ethyl m-aminobenzoate methanesulfonate.
[0050] Total bile acid kit (microplate method): Nanjing Jiancheng Bioengineering Institute, product catalog number E003-2-1.
[0051] 20% FBS-DMEM / F12 complete culture medium: It is composed of fetal bovine serum, insulin-like growth factor I, PSN antibiotic mixture and DMEM / F12 culture medium. In 20% FBS-DMEM / F12 complete culture medium, the concentration of fetal bovine serum is 20% (volume ratio), the concentration of insulin-like growth factor I is 40ng / ml, the concentration of penicillin is 50μg / mL, the concentration of streptomycin is 50μg / mL, and the concentration of neomycin is 50μg / mL. In 10% FBS-DMEM / F12 complete culture medium, the concentration of fetal bovine serum is 10% (volume ratio), and the rest is the same as 20% FBS-DMEM / F12 complete culture medium. PBS buffer: pH7.2, 0.01M PBS buffer.
[0052] Fetal bovine serum (FBS): Gibco, product catalog number 10099-141c. Insulin-like growth factor I (human IGF-1): Gibco, product catalog number PHG0078. DMEM / F12 medium (serum-free DMEM / F12 medium): Gibco, product catalog number 11330032. Penicillin-streptomycin-neomycin antibiotic mixture (PSN antibiotic mixture): Gibco, product catalog number 15640055. 0.25% trypsin-EDTA digestion solution: Gibco, product catalog number 25200072.
[0053] Example 1: Construction of bile acid secreting sturgeon ovarian cell line in vitro
[0054] 1. Obtaining ovarian tissue of sturgeon
[0055] A healthy sturgeon (weighing about 1.5 kg) was taken and anesthetized with MS222, then placed in a 75% ethanol aqueous solution for disinfection for 1 minute, and then placed on a sterile clean bench to cut open the abdomen and remove the internal ovarian tissue, then rinsed with PBS buffer to remove attached blood cells, and then placed the ovarian tissue in serum-free DMEM / F12 medium and cut into about 1 mm 3 The tissue blocks were then rinsed 3-5 times with PBS buffer (40 mL each time).
[0056] 2. Enzyme digestion of tissue blocks to initiate primary culture of ovarian cells
[0057] (1) After completing step 1, add 2 mL of 0.25% Trypsin-EDTA digestion solution to the tissue block for digestion (the digestion time is 10 minutes, and the tissue block is gently blown with a 1 mL pipette tip every 1 minute). After digestion is completed, add 2 mL of 20% FBS-DMEM / F12 complete culture medium to terminate the digestion effect of trypsin.
[0058] (2) After completing step (1), transfer the entire system to a centrifuge tube, centrifuge at 300 g for 10 minutes, and discard the supernatant; then add 1 mL of serum-free DMEM / F12 medium and suspend the tissue pieces, and then use a 1 mL pipette (with a cutter tip) to inoculate to 25 cm 2 The cell culture flask was placed in a 24°C biochemical incubator and inverted for culture (bottom of the flask facing upward) for 7 hours; then, the liquid in the culture flask was discarded, 2 mL of 20% FBS-DMEM / F12 complete culture medium was added, and the flask was placed in a 24°C biochemical incubator and cultured upright (flask mouth facing upward) for 7 days (after 3 days of culture, cells can be observed to migrate out of the tissue block, and after 7 days of culture, it can be observed that the cell area occupies 90% of the bottom area of the flask. These cells are primary cells, also known as P0 generation cells).
[0059] 3. Cell passaging and cell line establishment
[0060] Cell culture and passage method (delayed digestion method): After completing the previous step, discard the liquid in the culture bottle; then, add 1 mL of 0.25% Trypsin-EDTA digestion solution to the culture bottle, gently shake the culture bottle and discard the digestion solution; then, add 1 mL of 0.25% Trypsin-EDTA digestion solution to the culture bottle again, gently shake the culture bottle and digest for 2 minutes, discard the digestion solution, and let it stand for 5 minutes; then, add 1 mL of 20% FBS-DMEM / F12 complete culture solution to the culture bottle, and use a 1 mL pipette to gently blow the bottom of the culture bottle to make a cell suspension; take 0.5 mL of the cell suspension to a new culture bottle, add 2.5 mL of 20% FBS-DMEM / F12 complete culture solution to each culture bottle, and place it in a 24°C biochemical incubator to culture until the ovarian cells grow into a single layer again.
[0061] The cell line of the ovarian sturgeon was constructed by continuous subculturing according to the above method to the 60th generation.
[0062] The cells obtained from each passage are P1 cells, P2 cells, ..., P60 cells.
[0063] Figure 1 These are photos of primary cells and passaged cells under an optical microscope; Figure A, primary cells that have just migrated out of tissue blocks; Figure B, P3 cells; Figure C, P40 cells; scale bar = 100 μm.
[0064] 4. Detection of total bile acid content in cell-free culture system using total bile acid kit (microplate method)
[0065] The cell-free culture systems were: fetal bovine serum (FBS), 20% FBS-DMEM / F12 complete culture medium or 10% FBS-DMEM / F12 complete culture medium. Five replicates were set for each system.
[0066] The results of total bile acid content (μmol / L) in the cell-free culture system are shown in Table 1.
[0067] Table 1
[0068] FBS 20% FBS-DMEM / F12 complete culture medium 10% FBS-DMEM / F12 complete culture medium 11.683 4.167 3.431
[0069] 5. Detection of total bile acid content in cell culture supernatant
[0070] P3 cells were taken and cultured in a 24°C biochemical incubator for 5 days using 20% FBS-DMEM / F12 complete culture medium. The supernatant (denoted as p3-d5) was then collected and the total bile acid content in the culture supernatant was detected using a total bile acid kit (microplate method).
[0071] P13 cells were taken and cultured in a 24°C biochemical incubator for 11 days using 20% FBS-DMEM / F12 complete culture medium. The supernatant (denoted as p13-d11) was then collected and the total bile acid content in the culture supernatant was detected using a total bile acid kit (microplate method).
[0072] P20 cells were taken and cultured in a 24°C biochemical incubator for 3 days using 20% FBS-DMEM / F12 complete culture medium. The supernatant (denoted as p20-d3) was then collected and the total bile acid content in the culture supernatant was detected using a total bile acid kit (microplate method).
[0073] P38 cells were taken and cultured in a 24°C biochemical incubator for 15 days using 20% FBS-DMEM / F12 complete culture medium. The supernatant (denoted as p38-d15) was then collected and the total bile acid content in the culture supernatant was detected using a total bile acid kit (microplate method).
[0074] The results of total bile acid content (μmol / L) in the supernatant are shown in Table 2.
[0075] Table 2
[0076] p3-d5 p13-d11 p20-d3 p38-d15 8.415 6.781 6.863 7.353
[0077] The total bile acid content in the supernatant minus the total bile acid content in 20% FBS-DMEM / F12 complete culture medium is the total bile acid content produced by the cells (μmol / L). The results are shown in Table 3.
[0078] Table 3
[0079] P3 cells cultured for 5 days P13 cells cultured for 11 days P20 cells cultured for 3 days P38 cells cultured for 15 days 4.248 2.614 2.696 3.186
[0080] Example 2: Repeated verification
[0081] 20 sturgeons were used to carry out Example 1, and cell lines were obtained and all had the ability to secrete bile acid. The corresponding data of the parallel experiment had no significant difference with Example 1.
[0082] Example 3: Combined analysis of transcriptome and metabolome
[0083] After the ovarian cells of sturgeon were proliferated in vitro, the transcriptome and metabolome of the bile acid synthesis and secretion pathways were analyzed. The results of the combined analysis of transcriptome and metabolome levels after ovarian cells were grown for 4 days are shown in Figure 2 . Figure 2 In the figure, ovarian cells grown for 1 day after passage were used as the control group; the number of parallel samples was 6; the red boxes were significantly up-regulated genes, the dark green boxes were significantly down-regulated genes, and the green circles were significantly down-regulated metabolites. The bile acid secretion pathway was found in the KEGG enrichment pathway.
[0084] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. Use of isolated sturgeon ovaries or sturgeon ovarian cells prepared from isolated sturgeon ovarian tissues in the preparation of bile acid, wherein the sturgeon is Acipenser sternum.
2. A method for preparing bile acid, comprising the following steps: (1) Using isolated sturgeon ovaries or isolated sturgeon ovarian tissues as raw materials, sequentially performing tissue block preparation, tissue block digestion and cell culture to obtain sturgeon ovarian cells that can secrete bile acid in vitro; (2) culturing the sturgeon ovarian cells obtained in step (1) in vitro to obtain bile acid; The method for digesting the tissue block is as follows: adding 0.25% Trypsin-EDTA digestion solution to the tissue block for digestion, and after digestion, adding 20% FBS-DMEM / F12 complete culture medium to terminate the digestion effect of trypsin; The cell culture includes primary cell culture and cell subculture; The primary cell culture comprises the following steps: after the digestion of the tissue block is completed, the entire system is transferred to a centrifuge tube, centrifuged at 300 g, and the supernatant is discarded; then serum-free DMEM / F12 medium is added and the tissue block is suspended, and then inoculated into a cell culture bottle, and inverted cultured at 24° C. for 7 hours; Then, the liquid in the culture bottle was discarded, 20% FBS-DMEM / F12 complete culture medium was added, and cultured upright at 24°C; The cell subculture comprises the following steps: after the previous generation of cell culture is completed, the liquid in the culture bottle is discarded; 0.25% Trypsin-EDTA digestion solution is added for digestion for 2 minutes; Then, add 20% FBS-DMEM / F12 complete culture medium to the culture flask to prepare a cell suspension; take the cell suspension to a new culture flask, add 20% FBS-DMEM / F12 complete culture medium, and culture at 24°C until the cells grow into a monolayer; The sturgeon is a small-bodied sturgeon.