CN117904036A - Method for producing snake oil by culturing fat cells of snake - Google Patents
Method for producing snake oil by culturing fat cells of snake Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a method for producing snake oil by culturing fat cells of a snake, the method comprising digesting a fat sample of a living organism of the snake to obtain a single cell mixture; culturing the single cell mixture, and purifying to obtain fat precursor cells through differential adherence culture; continuously subculturing the fat precursor cells to obtain immortalized fat precursor cells; performing adipogenic differentiation culture on the immortalized fat precursor cells to obtain a large number of mature fat cells; centrifuging to collect mature adipocytes, and obtaining snake oil component. The invention only needs a small amount of snake fat tissue, can obtain immortalized fat precursor cells and a large amount of mature fat cells through culture, has high safety and good stability, is convenient for industrial production, can partially replace snake oil produced by the traditional technology, and is applied to the fields of skin care products, medical scientific research and the like.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a method for producing snake oil by culturing fat cells of a snake.
Background
The main chemical components of the snake oil are various fatty acids. On the one hand, the skin care composition has the effects of promoting skin metabolism, enhancing cell activity, resisting oxidation and skin aging, increasing luster and the like, and has stronger affinity and good permeability to a skin mucosa system, so that the skin care composition is often used for preparing advanced cosmetics in the aspects of beauty and skin care; on the other hand, the external preparation has the functions of reducing vascular permeability of scalds, resisting inflammation, relieving pain, reducing blood fat, reducing blood sugar, inhibiting bacteria and the like, and is often combined with other medicines to prepare the compound external preparation for treating various skin diseases.
Snake resources have a long history in Chinese medicine and folk medicine as medicinal materials, and after the fasting instruction of wild animals is issued in 2020, the utilization of snake resources is limited to medicinal and ornamental purposes, but at present, the method for preparing snake oil on the market usually comprises the steps of directly extracting fat tissues from snake bodies, and extracting the snake oil from snake fat by adopting an acid hydrolysis method or a heating method. Although the method can directly extract the required medicinal materials, the extraction method is complex, the efficiency is low and the cost is high.
In addition, as the extraction process of the snake oil faces a series of problems of animal ethics, sanitation, infectious diseases and the like, the research capability and the treatment level of the snake oil are difficult to be improved. Many studies are now made of the use of cell culture to produce various animal tissues, such as cell culture meat, and the technology is mature. Therefore, it is urgent to establish a technology for extracting snake oil by using cell culture instead of direct extraction, and it is expected to provide a new direction and make a new contribution to scientific research.
At present, no literature reports on the culture of snake cells and the utilization of the snake cells, how to culture the snake cells and whether the snake oil can be produced by culturing the snake fat cells are yet to be further explored.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a method for producing snake oil by culturing snake fat cells, which not only provides a feasible method for culturing snake fat cells on a large scale, but also successfully obtains snake oil by culturing snake fat cells.
In a first aspect the present invention provides a method of obtaining immortalised fat precursor cells of a snake comprising the steps of:
s1: digesting and treating a fat sample of a living snake to obtain a single-cell mixture;
S2: culturing the single cell mixture, and purifying to obtain fat precursor cells through differential adherence culture;
S3: and continuously subculturing the fat precursor cells to obtain the immortalized fat precursor cells.
In some embodiments, the step S1 digestion process is preceded by: a small sample of fat is taken from a living snake and sterilized and/or cleaned.
In some embodiments, the disinfecting solution used for disinfection is an ethanol solution, such as 75% ethanol solution, and the cleaning solution used for cleaning is physiological saline containing antibiotics; the disinfection and cleaning time is 3-5 minutes respectively. The sterilization of the fat sample is beneficial to removing bacteria and viruses, and the sample is prevented from losing proliferation and differentiation capacity and even death due to infection; the washing of the sample is advantageous for removing impurities such as blood, non-adipose tissue such as mucous membrane, etc.
In some embodiments, in step S1, the digestion process employs digestive enzymes including dispase, which is a mixed enzyme of type I collagenase and neutral protease II, and pancreatin substitutes.
In some embodiments, in the step S1, the final concentration of the type I collagenase in the digestive enzyme is 0.1% -0.5% (w/v), the final concentration of the neutral protease II is 0.2% -0.5% (w/v), and the final concentration of the pancreatin substitute is 1% -5% (w/v). Above this concentration, there is a greater damage to the activity of the stem cells, which is detrimental to maintaining the integrity of the fat precursor cells; below this concentration, digestion of the tissue is not favored.
In some embodiments, in the step S1, the digestion time is 0.5 to 2 hours, and the digestion temperature is 30 to 40 ℃.
In some embodiments, in step S2, the medium for culturing the single cell mixture comprises: low sugar medium, FBS, glutamine and recombinant epidermal growth factor. The use of low sugar culture medium in early cell culture stage helps to limit the premature differentiation of adipocytes and maintain the stability of cell quality, and recombinant epidermal growth factor helps to help the rapid establishment of cell lines.
In some embodiments, in step S2, the low sugar medium is selected from the group consisting of minimal medium DMEM containing 1g/L glucose.
In some embodiments, the medium for culturing the single cell mixture comprises: 75-85% (v/v) DMEM, 15-20% (v/v) fetal bovine serum FBS, 0.5-2% (v/v) 100 Xglutamine and 0.01-0.02% (w/v) human recombinant epidermal growth factor. After glutamine and human recombinant epidermal growth factor are added, the self-renewal and potential differentiation capacity of the fat precursor cells of the snakes in the culture medium are enhanced.
In some embodiments, in the step S2, the culture temperature of the single cell mixture is 31-34 ℃, and the time of the adherent culture is 1-6 hours. The culture temperature (such as 31-34 ℃) lower than 37 ℃ is closer to the natural body temperature of the snakes under the non-hibernation condition, and is more suitable for the growth and differentiation of the cells of the snakes; the adherent culture time is selected to be 1-6 hours, so that adipocytes and other non-adipocytes in a slowly adherent differentiated state can be rapidly and well removed.
In some embodiments, in the step S3, the immortalized fat precursor cells are spontaneously generated by subculture, and 0.5-2% CEE (chicken Embryo Extract/chick embryo extract) may be added for induction to improve the immortalization efficiency, for example, more than 25 passages. Avoiding the risks of high cost and influence on the quality of the final product which can be related to the traditional methods of introducing SV40, TERT and other vectors.
In some embodiments, in step S3, the medium for serial subculture comprises: 75-85% (v/v) DMEM, 15-20% (v/v) fetal bovine serum FBS, 0.5-2% (v/v) 100 Xglutamine, 0.01-0.02% (w/w) human recombinant epidermal growth factor, and 0.5-2% (v/v) CEE.
In a second aspect, the invention provides a method of obtaining fat cells from a plurality of snakes, comprising the steps of:
s1: digesting and treating a fat sample of a living snake to obtain a single-cell mixture;
S2: culturing the single cell mixture, and purifying to obtain fat precursor cells through differential adherence culture;
s3: continuously subculturing the fat precursor cells to obtain immortalized fat precursor cells;
S4: and performing adipogenic differentiation culture on the immortalized fat precursor cells to obtain a large number of mature fat cells.
The steps S1-S3 are identical to steps S1-S3 in any of the embodiments of the first aspect of the invention.
In some embodiments, in step S4, the medium of the adipogenic differentiation culture contains glucose, NMN (beta-nicotinamide mononucleotide), Y-27632 (RhoA/ROCK inhibitor), insulin, and phosphatidylcholine as additives. The addition of glucose, NMN, Y-27632, insulin and phosphatidylcholine helps to promote the progression of differentiation of adipose precursor cells into mature adipocytes containing a large amount of lipid components, wherein Y-27632 also helps to prevent aging and fibrotic differentiation of the cells.
In some embodiments, in step S4, the medium for adipogenic differentiation culture comprises: 75-85% (v/v) of DMEM medium, 15-20% (v/v) of fetal bovine serum, 0.5-2% (v/v) of 100 Xglutamine, 35-60 mg/mL of glucose, 200-400 mg/L of NMN, 5-10 mu M Y-27632, 1.5-3 mu g/mL of insulin and 1.5-3 mg/mL of phosphatidylcholine.
In some embodiments, the step S4 further comprises the step of culturing the immortalized fat precursor cells on a large scale prior to the adipogenic differentiation culture.
In some embodiments, the medium for large scale culture comprises high sugar medium, fetal bovine serum, glutamine, recombinant epidermal growth factor, glucose, NMN, and Y-27632 (RhoA/ROCK inhibitor) medium. Fetal bovine serum, high sugar (glucose) and NMN help to provide the energy and nad+ required for rapid growth and proliferation of fat precursor cells; y-27632 helps to prevent cell senescence and fibroblast differentiation.
In some embodiments, the high sugar medium is selected from the group consisting of basal medium DMEM containing 4.5g/L glucose.
In some embodiments, the medium for large scale culture comprises: 75-85% (v/v) of DMEM medium, 15-20% (v/v) of fetal bovine serum, 0.5-2% (v/v) of glutamine, 0.01-0.02% (w/w) of recombinant epidermal growth factor, 35-60 mg/mL of glucose, 200-400 mg/L of NMN and 5-10 mu M Y-27632 (RhoA/ROCK inhibitor).
In a third aspect, the present invention provides a method for producing snake oil by culturing fat cells of a snake, comprising the steps of:
s1: digesting and treating a fat sample of a living snake to obtain a single-cell mixture;
S2: culturing the single cell mixture, and purifying to obtain fat precursor cells through differential adherence culture;
s3: continuously subculturing the fat precursor cells to obtain immortalized fat precursor cells;
S4: performing adipogenic differentiation culture on the immortalized fat precursor cells to obtain a large number of mature fat cells;
S5: centrifuging to collect mature adipocytes, and obtaining snake oil component from the collected adipocytes.
The steps S1-S4 are identical to steps S1-S4 in any of the embodiments of the second aspect of the invention.
In some embodiments, in step S5, the mature adipocytes are collected by low-speed centrifugation; the low-speed centrifugation method allows rapid collection of cells and avoids rupture of mature adipocyte membranes.
In some embodiments, in step S5, the collected mature adipocytes are subjected to low temperature ultracentrifugation to obtain a snake oil fraction. The cell rupture and the separation of the oil component and the water phase are realized by the ultracentrifugation, the damage to certain active components of the fat cells caused by the traditional high-temperature heating method of the snake fat can be avoided, and all the active components in the snake fat can be effectively protected.
In a fourth aspect, the invention provides a culture medium for continuous subculture of fat precursor cells of a snake: 75-85 (v/v)% DMEM, 15-20% (v/v) fetal bovine serum FBS, 0.5-2% (w/w) 100 Xglutamine and 0.01-0.02% (w/w) human recombinant epidermal growth factor, and 0.5-2% (v/v) CEE.
In a fifth aspect, the invention provides a culture medium for adipogenic differentiation of immortalized fat precursor cells of a snake: 75-85% (v/v) DMEM medium, 15-20% (v/v) fetal bovine serum FBS, 0.5-2% (v/v) 100 Xglutamine, 35-60 mg/mL glucose, 200-400 mg/L NMN, 5-10 mu M Y-27632 (RhoA/ROCK inhibitor), 1.5-3 mu g/mL insulin and 1.5-3 mg/mL phosphatidylcholine.
In a sixth aspect, the invention provides a medium for large scale culture of immortalized fat precursor cells of a snake: 75-85% (v/v) DMEM medium, 15-20% (v/v) fetal bovine serum FBS, 0.5-2% (v/v) 100 Xglutamine, 0.01-0.02% (w/w) recombinant epidermal growth factor, 35-60 mg/mL glucose, 200-400 mg/L NMN and 5-10 mu M Y-27632 (RhoA/ROCK inhibitor).
Compared with the prior art, the invention has the beneficial effects that at least:
(1) The invention provides a method for producing snake oil by culturing fat cells of a snake for the first time, which comprises the steps of obtaining immortalized fat precursor cells of the snake, obtaining a large number of mature fat cells by adipogenic differentiation culture, obtaining the snake oil from the mature fat cells, and providing a complete solution.
(2) According to the invention, only a small amount of snake adipose tissue is required to be obtained, and the adipose precursor cells with high purity, high activity and strong proliferation and self-renewal capacity can be obtained through subculture, and a large amount of mature adipocytes can be obtained through differentiation, wherein the subculture is carried out for more than 40 generations.
(3) The invention provides the culture medium at each stage of cell culture, and has high safety and good stability; the cell culture medium for early and medium stage culture is favorable for maintaining the fat precursor cells in an undifferentiated state of high-speed growth, and the differentiation culture medium for later stage can effectively promote the differentiation and maturation of the fat cells.
(4) The snake oil obtained by the invention is from the fat cells of the snake, has the same components as the components obtained from the fat cells of the snake body, can obtain a large amount of the fat cells of the snake body and the snake oil components, is convenient for industrial production, can partially replace the snake oil produced by the traditional technology, and is applied to the fields of skin care products, medical scientific research and the like.
Drawings
FIG. 1 is a photograph of a snake used in example 1;
FIG. 2 is a photomicrograph of fat precursor cells obtained by the differential cell attachment method of example 1;
FIG. 3 is a photomicrograph of fat precursor cells during mass growth expansion in example 1;
FIG. 4 is a photomicrograph of mature adipocytes after differentiation;
FIG. 5 shows adipocytes harvested by low-speed centrifugation as in example 1, with a white tube bottom;
FIG. 6 is a snake oil obtained by ultracentrifugation of example 1;
FIG. 7 is a photomicrograph of fat precursor cells during mass growth expansion in example 2;
FIG. 8 is a photomicrograph of the differentiated mature adipocytes of example 2.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. Examples of which are illustrated in the accompanying drawings. It should be understood that the specific examples described in the following embodiments of the present invention are intended to be illustrative of the specific embodiments of the present invention and are not to be construed as limiting the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass the range or value as being close to the range.
EXAMPLE 1 isolation and culture of yellow chain snake adipocytes and production of snake oil
1. Reagent formulation and method of formulation
1.1 The preparation method of the digestive enzyme system comprises the following steps: 75mg of neutral protease II powder (Solarbio, china) was dissolved in 25mL of PBS to prepare a neutral protease II solution having a final concentration of 3mg/mL, and 50mg of type I collagenase powder (Gibco, USA) was added to a final concentration of 0.2% (w/v), and the mixed enzyme system was placed in a refrigerator at 4℃for use. 0.5g of pancreatin substitute (Gibco, USA) was dissolved in 25mL PBS to a final concentration of 2% (w/v) and placed in a refrigerator at 4℃for use.
1.2 The preparation method of the cell culture medium comprises the following steps: 100mL of fetal bovine serum (Gibco, USA) was added to 390mL of DMEM (Gibco, USA) at a final concentration of 20% (v/v), 5mL of 100 XGlutamine (Gibco, USA) at a final concentration of 1% (v/v), 50mg of human recombinant epidermal growth factor (Gibco-BRL, USA) at a final concentration of 100ng/mL and CEE at a final concentration of 5mL of 1%. After mixing, filtration was carried out with sterile filter membranes and the filtrate was stored at 4 ℃.
2. Isolation of fat precursor cells
Obtaining fat tissue of the snake, and separating and culturing fat precursor cells by adopting a differential adherence culture method, wherein the specific steps are as follows:
S1: selecting living yellow-chain snakes (1.1 m long) as shown in figure 1, anaesthetizing, cleaning with 75% ethanol for about 10 seconds, obtaining about 5g of abdominal adipose tissue from an abdominal opening (1 cm long wound, suturing afterwards) under aseptic conditions, and soaking in 75% ethanol for 3 minutes; washing with physiological saline containing 2% double antibody for 2 times for 3 min to obtain adipose tissue;
S2: the adipose tissues were minced into small pieces of 2mm 3 with sterilized scissors, and the tissue pieces were digested with a mixed enzyme of 0.2% (w/v) type I collagenase, and 0.1% (w/v) neutral proteinase II for 1 hour at 37℃in a constant temperature water bath. Mixing every 10 minutes, adding pancreatin substitute with a final concentration of 2% after 1 hour, and continuing to digest in 37 ℃ water bath for 0.5 hour; after centrifugation at 1000rpm for 5 minutes, the pellet was resuspended in PBS and filtered through a 70 μm cell screen to give a single cell mixture, which was transferred into a flask for culture at a culture temperature of 32℃and, depending on the cell attachment rate, the suspended cells were removed and the culture medium was replaced after 3 hours, the attached cells were as shown in FIG. 2;
3. Large-scale culture and amplification of fat precursor cells
The method comprises the following specific steps:
S1: immortalizing fat precursor cells, carrying out in-vitro subculture on the fat precursor cells of the snakes to about 25 generations, finding that the growth of a plurality of parts of cells is slowed down and the growth of a few parts of cells is accelerated, and continuously culturing the cells with accelerated growth to obtain the spontaneous immortalized fat precursor cells.
S2: immortalized fat precursor cells were cultured using a new cell culture medium comprising DMEM (Gibco, USA) supplemented with fetal bovine serum (20%), glutamine (1%), recombinant epidermal growth factor (0.01%), glucose (45 mg/mL), Y-27632 (5 μm) and NMN (β -nicotinamide mononucleotide) (300 mg/L). The culture was continued for 20 days (32 ℃ C.) and the cells were expanded from 0.5X10 4 to 1X 10 10 cells, as shown in FIG. 3.
4. Adipogenic differentiation of fat precursor cells
The expanded fat precursor cells were replaced with a lipid-forming differentiated cell medium comprising fetal bovine serum (20%), glutamine (1%), recombinant epidermal growth factor (0.01%), glucose (45 mg/mL), NMN (β -nicotinamide mononucleotide) (300 mg/mL), Y-27632 (5. Mu.M), insulin (2. Mu.g/mL) and phosphatidylcholine (2 mg/mL). The culture was continued for 7 days (culture temperature of 32 ℃ C.) as shown in FIG. 4.
5. Separation of snake oil
The method comprises the following specific steps:
s1, collecting the cultured cells by using pancreatin, and separating the cells by using low-speed centrifugation (800 rpm/min) to obtain fat cells, as shown in FIG. 5.
S2, obtaining the snake oil by low-temperature ultracentrifugation, wherein the temperature is 4 ℃, the centrifugation speed is 20000 rpm/min, and the result of the snake oil is shown in figure 6.
EXAMPLE 2 isolation culture of Red chain snake adipocytes and production of snake oil
Similar to example 1, the difference is that:
1. reagent formulation and method of formulation
1.1 The preparation method of the digestive enzyme system comprises the following steps: 50mg of neutral protease II powder (Solarbio, china) was dissolved in 25mL of PBS to prepare a neutral protease II solution having a final concentration of 0.2% (w/v), 125mg of type I collagenase powder (Gibco, USA) was then added to have a final concentration of 0.5% (w/v), and the mixed enzyme system was placed in a refrigerator at 4℃for use. 1.25g of pancreatin substitute (Gibco, USA) was dissolved in 25mL of PBS to a final concentration of 5% (w/v) and placed in a refrigerator at 4℃for use.
1.2 The preparation method of the cell culture medium comprises the following steps: 15mL of fetal bovine serum (Gibco, USA) was added to 85mL of DMEM (Gibco, USA) at a final concentration of 15% (v/v), 100 Xglutamine (Gibco, USA) at a final concentration of 0.5% (v/v), human recombinant epidermal growth factor at a final concentration of 0.01% and CEE at a final concentration of 1.5%. After mixing, filtration was carried out with sterile filter membranes and the filtrate was stored at 4 ℃.
2. Isolation of fat precursor cells
S1: selecting living red chain snakes (about 0.9 m long), anaesthetizing, cleaning with 75% ethanol for about 10 seconds, obtaining about 4g of abdominal adipose tissue from an abdominal opening (about 1cm long wound, and suturing afterwards) under a sterile condition, and soaking in 75% ethanol for 2 minutes; washing with physiological saline containing 2% double antibody for 2 times for 2 min to obtain adipose tissue;
S2: the adipose tissue was cut into small pieces of 2mm 3 with sterilized scissors, and the pieces of tissue were digested with a mixed enzyme of 0.5% type I collagenase and 0.2% neutral proteinase II for 1 hour at 37℃in a constant temperature water bath. Mixing every 10 minutes, adding pancreatin substitute with a final concentration of 5% after 1 hour, and continuing to digest in 37 ℃ water bath for 0.5 hour; after centrifugation at 1000rpm for 5 minutes, the pellet was resuspended in PBS and filtered through a 70 μm cell screen to give a single cell mixture, which was transferred into a flask for culture at a culture temperature of 32℃and, depending on the cell attachment rate, the suspended cells were removed after 4 hours and the culture medium was changed.
Cells expanded in vitro and cells after adipogenic differentiation are shown in figures 7 and 8.
Finally, the above embodiments are only for illustrating the technical solution of the present invention, and do not limit the present invention. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (8)
1. A method for producing snake oil by culturing fat cells of a snake, comprising the steps of:
step S1: digesting and treating a fat sample of a living snake to obtain a single-cell mixture;
Step S2: culturing the single cell mixture, and purifying to obtain fat precursor cells through differential adherence culture;
Step S3: continuously subculturing the fat precursor cells to obtain immortalized fat precursor cells;
Step S4: performing adipogenic differentiation culture on the immortalized fat precursor cells to obtain a large number of mature fat cells;
Step S5: centrifuging to collect mature adipocytes, and obtaining snake oil component from the collected adipocytes.
2. A method for producing snake oil by culturing snake fat cells according to claim 1, wherein in step S1, the digestion treatment is performed with digestive enzymes including dispase, which is a mixed enzyme of type I collagenase and neutral proteinase II, and pancreatin substitute; the final concentration of the type I collagenase in the digestive enzyme is 0.1% -0.5% (w/v), the final concentration of the neutral protease II is 0.2% -0.5% (w/v), and the final concentration of the pancreatin substitute is 1% -5% (w/v).
3. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein in the step S1, the digestion time is 0.5-2 hours, and the digestion temperature is 30-40 ℃.
4. A method for producing snake oil by culturing snake fat cells according to claim 1, wherein in step S2, the medium for culturing the single cell mixture comprises: low sugar medium, FBS, glutamine and recombinant epidermal growth factor; the culture temperature of the single cell mixture is 31-34 ℃, and the adherent culture time is 1-6 hours.
5. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein in the step S3, the immortalized fat precursor cells are spontaneously generated by subculture or added with 0.5-2% (v/v) chick embryo extract; the medium for serial subculture comprises: 80-85% (v/v) DMEM, 15-20% (v/v) fetal bovine serum, 0.5-2% (w/v) 100 Xglutamine, 0.01-0.02% (w/v) human recombinant epidermal growth factor, and 0.5-2% (v/v) chick embryo extract.
6. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein the culture medium for the adipogenic differentiation culture comprises glucose, NMN, rhoA/ROCK inhibitor, insulin and phosphatidylcholine in step S4.
7. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein said step S4 is performed before the adipogenic differentiation culture, and further comprising the step of culturing immortalized fat precursor cells on a large scale; the medium for large scale cultivation comprises a high sugar medium and a medium for NMN.
8. A method for producing snake oil by culturing snake fat cells according to claim 1, wherein in step S5, mature fat cells are collected by low-speed centrifugation; collecting mature adipocytes, and performing low-temperature ultracentrifugation to obtain snake oil components.
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