CN117050937A - Method for extracting primary cells of terminal bud base of salamander - Google Patents
Method for extracting primary cells of terminal bud base of salamander Download PDFInfo
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Abstract
The extraction method of the primary cells of the terminal buds of the salamander comprises the steps of salamander feeding, amputation, post-operation treatment, bud tissue acquisition, bud primary cell extraction and the like, and has strong operability; the method carries out corresponding postoperative treatment and feeding on the amputated salamander, samples after waiting for 3-4 weeks and generating mature buds, carries out sterilization treatment on the buds by a specific means, can effectively remove parasitic bacteria contained in the buds, realizes good separation of bud-based tissues from skin by microscopic technology and surgical technique, and finally uses trypsin to digest the bud-based tissues, and successfully extracts primary cells from the bud-based of the salamander by means of centrifugation, resuspension, filtration and the like. The extracted primary cells are planted in a culture dish, and the salamander bud-based cells are successfully cultured to be in a normal growth cycle through reasonably preparing a culture environment, so that the biological functions of division, proliferation, migration and the like are maintained.
Description
Technical Field
The application relates to the technical field of cell extraction, in particular to a primary extraction method of salamander limb terminal bud base cells.
Background
The salamander belongs to the sub-order of the salamander, the salamander family, and the genus of the salamander. The salamander belongs to the genus 6 of 23 in China, and accounts for about 30% of the world. The current 8 species of salamander and subspecies thereof are distributed in the eastern part of asia, 6 species of china (containing 2 subspecies) and 2 species of japan. The salamander is a salamander family, a salamander genus with tail amphibian, also called as Chinese fire belly salamander, and is a special species distributed in the middle and eastern part of China. The salamander has extremely strong regeneration capability, can regenerate the amputation, and can restore to the original state even if the salamander is partially excised from eyeballs, hearts and brains. Surprisingly, we could not find the trace that it was ever created and wound repair was performed, and this species did indeed heal without scars. More surprisingly, species with such a strong regeneration capacity are also cancer-free species.
The salamander amputation regeneration model is a biological model widely applied to growth and development and regeneration medicine, and the salamander amputation is completely regenerated and depends on terminal bud basal cells. At the beginning of the tissue injury regeneration process, a specific class of cells called germ cells is produced. The repair of lesions results from the different memory functions and regeneration capacity of the germ cells of different species are quite different. The germ cells are the most important mesenchymal components in the limb regeneration process and are also an undifferentiated pluripotent stem cell. The germ cells proliferate and aggregate to form a specific cell population, forming a germ. The germ cells are highly plasticized cells having regenerative and multifunctional differentiation capabilities and can differentiate into a variety of cell types including neural cells, muscle cells, bone cells, fibroblasts, etc., thereby providing multicellular support for tissue regeneration. At the same time, the germ cells have a high proliferative capacity, which also allows the germ to recover and reconstruct damaged tissue. The bud cell of the salamander has high memory and regeneration function and can realize the complete regeneration of damaged tissues.
The salamander bud cell belongs to an amphibian cell, and the culture conditions of the amphibian cell and the mammalian cell are completely different, because the culture solution is different, the nutrient substances are different, the growth environment is different, and the like. The culture report of amphibian at home and abroad is less, the current successful culture is that the African claw cell, the Australian frog toe cell and the like are carried out, the salamander bud cell belongs to the mesenchymal stem cell, the growth cycle is very slow, the differentiation is possible, and the culture difficulty is more than that of the common amphibian. If the salamander terminal bud base cells are extracted and cultured by conventional methods (such as tissue mass culture, digestion culture, suspension cell culture, organ culture, etc.), the following difficulties exist: 1. the salamander can completely regenerate the amputated limb after amputating the amputated limb in 3-5 months, and when the bud is sampled, no literature report exists at home and abroad; 2. the salamander bud base is wrapped by skin, when the bud base primary cell extraction is carried out, 1-3mm bud base is required to be peeled, and no effective method for removing bud base skin is reported at home and abroad; 3. the salamander bud base contains a large amount of parasitic bacteria, and no document is reported at home and abroad how to effectively sterilize the salamander bud base without damaging the bud base tissue; 4. the method is suitable for the conditions of cell culture and amplification of the salamander, and no document is reported at home and abroad.
Disclosure of Invention
The application provides a method for extracting primary cells of the terminal buds of salamanders, which successfully extracts the primary cells from the buds of the salamanders through the steps of salamanders feeding, amputation and post-operation treatment, bud tissue acquisition, bud primary cell extraction and the like. According to the extraction method, corresponding post-operation treatment is carried out on the salamander after amputation, and the salamander is fed, sampling is carried out after waiting for 3 to 4 weeks and no post-operation complications appear and mature buds are generated, and the salamander buds are sterilized by a specific means, so that parasitic bacteria contained in the salamander buds can be effectively removed on the premise of not damaging bud base tissues, good separation of the salamander bud base tissues and skin is realized by microscopic technology and surgical skill, primary cells are successfully extracted by means of trypsin digestion, centrifugation, resuspension, filtration and the like, and a plurality of difficulties existing in the extraction process of the salamander bud base cells in the prior art are effectively solved. The primary cells extracted by the method are planted in a culture dish, and the salamander bud-based cells are successfully cultured and are in a normal growth cycle by reasonably preparing a culture environment, so that the biological functions of division, proliferation, migration and the like are maintained, and the cells are proved to be mesenchymal cells with dryness and regeneration capability.
The technical scheme of the application is as follows:
the method for extracting the primary cells of the terminal buds of the salamander comprises the following steps:
s1, raising salamanders in the eastern direction: simulating ecological environment, raising salamander, and feeding animal feed once every 1-2 days;
s2, amputation and postoperative treatment of the salamander: a. anesthetizing the eastern salamander; b. placing the salamander under a stereo microscope, horizontally cutting two forelimbs at the middle part of the humerus of the salamander by using surgical scissors, wherein the length of the forelimbs is 1-1.5cm; c. pruning the protruding bones of the skeleton belt, pruning the protruding muscle tissues, and ensuring the surface smoothness of the amputation limb; d. transferring the amputated salamander into 0.5% sulfadiazine water solution, changing the solution after 24 hours, and normally raising the salamander according to the step S1, and waiting for the salamander to generate a bud base;
s3, obtaining the salamander bud base tissue: a. waiting for 3 to 4 weeks, and after the salamander does not have postoperative complications and generates mature bud bases, anesthetizing the salamander and sterilizing the salamander; b. placing the salamander in a stereo microscope, shearing the bud base by using an operation shear, and transferring the sheared bud base into an amphibian culture medium (A-MEM); c. after all buds are collected, sequentially soaking the buds in a pasteurized solution diluted 1000 times, iodophor and chloramphenicol diluted 10 times, and sterilizing the buds; d. placing the sterilized bud base in an amphibian balancing solution (APBS), and performing aseptic peeling operation on the bud base by means of a microscope; e. repeatedly washing with balance liquid of amphibian for several times after peeling to obtain bud-based tissue;
s4, extracting primary cells of the salamander bud group: a. digesting the granulated bud-based tissue by trypsin; b. centrifuging for 5 minutes at 1200 revolutions to obtain a bud-based primary tissue precipitate; c. re-suspending the bud-based primary tissue sediment, and filtering undigested bud-based tissue through a filter membrane to obtain bud-based primary cells.
Compared with the prior art, the method for extracting the primary cells of the terminal buds of the salamander in the eastern salamander comprises four steps of feeding, amputation and post-operation treatment of the salamander, bud-based tissue acquisition and bud-based primary cell extraction, has strong operability, can effectively solve a plurality of problems existing in the process of extracting the bud-based cells of the salamander in the prior art, and successfully extracts the primary cells from the bud-based of the salamander. In the extraction method, corresponding postoperative treatment is carried out on the amputation salamander (the amputation salamander is placed in 0.5% sulfadiazine water solution for 24 hours, the operation complications are not easily caused by external environment factors), the salamander is fed in a specific environment, after the 3-4 weeks of waiting for the occurrence of the operation complications (including infection, emaciation, hematemesis, death and the like) and mature bud bases are generated, sampling is carried out (the bud base tissues smaller than 3 weeks are too small, bud base cells are difficult to obtain, mainly because the following points are that 1, the bud base tissues are too small to be peeled, and the peeling treatment is difficult to carry out, 2, the bud base cells can be symbiotic with a large number of scale of parasitic bacteria, 3, even if the peeling treatment is carried out effectively, the bud base cells cannot be extracted due to the fact that the bud base tissues are too small and the extraction process is lost, the small paws of the salamander can be basically seen after the bud base cells are differentiated for a period of more than 4 weeks; sterilizing the salamander bud base by a specific means: soaking the bud base in 1000 times of Pasteur disinfectant, iodophor and 10 times of chloramphenicol in sequence, so as to effectively remove parasitic bacteria contained in the salamander bud base; the aseptic peeling operation is carried out in the APBS through the microscopic technology and the surgical operation skill, so that the salamander bud base tissue and the skin are well separated, the peeled bud base tissue is repeatedly washed for a plurality of times by using the APBS, the bud base tissue is convenient to carry out subsequent operation, and the bud base tissue suspension is prepared; finally, the primary cells are successfully extracted by using trypsin for digestion, centrifugation, resuspension, filtration and the like. The primary cells extracted by the method are planted in a culture dish, and the salamander bud-based cells are successfully cultured and are in a normal growth cycle by reasonably preparing a culture environment, so that the biological functions of division, proliferation, migration and the like are maintained, and the cells are proved to be mesenchymal cells with dryness and regeneration capability.
As an optimization, in the method for extracting the primary cells of the terminal buds of the salamander, in the step S1, the salamander is an adult salamander with complete metamorphosis, and the length of the salamander is required to be more than 7cm. The bud base of the salamander is smaller, and even the adult salamander is generally about 2 mm; the bud base tissue of the salamander with the length smaller than 7cm is too small, and the in-vitro extraction of bud base cells is difficult to realize, so that the adult salamander with the length larger than 7cm is suitable to be selected as an experimental object. Further, in step S1, the salamander is raised in the ecological vat before the amputation operation, and the water is changed, the residual bait and the feces are cleaned at regular time, so that the water quality is always kept clean, the bacteria breeding can be avoided, and the living environment of the salamander is ensured. Further, in the step S1, during the breeding process of the salamander, the room temperature is controlled to be 14-18 ℃, and the water temperature in the ecological vat is controlled to be 24+/-1 ℃. At this time, the growth of the salamander is facilitated.
As an optimization, in the method for extracting the primary cells of the terminal buds of the salamander, in the steps S2 and S3, the salamander is placed in a 0.1% tricaine solution for 7-10 minutes to anesthetize the salamander. At this time, the anesthesia effect is better, and the follow-up operation is convenient. The concentration of the tricine solution and the anesthetic time can be adjusted within a certain range (for example, 0.1+/-0.03% concentration and 5-20 minutes of anesthetic time), but the adjustment range is not excessively large, otherwise, the situation that the user can die or cannot fall down can occur.
As an optimization, in the method for extracting the primary cells of the terminal buds of the salamander, in the step S3, the salamander is sterilized by using an iodophor disinfectant or autoclaving water.
As an optimization, in the method for extracting the primary cells of the terminal bud bases of the salamander, in the step S3, the buds are soaked in the pasteurizing solution diluted 1000 times for 1 minute, soaked in the iodophor for 5 minutes, and soaked in the chloramphenicol diluted 10 times for 1 minute. At this time, the sterilization effect is better, and parasitic bacteria on the salamander bud base can be effectively removed in a large amount.
As an optimization, in the method for extracting the primary cells of the terminal bud base of the salamander, in the step S4, before the bud base tissue is digested, the bud base tissue is granulated by using an ophthalmic scissors. Therefore, the digestion efficiency can be improved, granulation of the bud-based tissue is realized by a physical mode, and the influence of other substances introduced into the bud-based primary cell extraction can be avoided. In addition, the salamander bud cell is much larger than human cells, the in vitro culture can reach hundreds of micrometers, and the extracted bud cell can be conveniently cultured after granulation.
Preferably, in the method for extracting the primary cells of the terminal bud of the salamander, in step S4, the bud tissues of different thicknesses are digested by trypsin with a gradient time of 15 minutes (preferably 30 minutes, 45 minutes, and 60 minutes). Therefore, the germ tissue is digested more thoroughly, and the separation efficiency of germ primary cells is improved. Gradient time digestion at 15 minutes intervals was used because the germ tissue had a certain thickness, but soaking the cells too long in trypsin affected cell viability, so that it was necessary to collect germ tissue that had been digested within a period of time, terminate digestion, and continue digestion for a period of time with undigested germ tissue remaining.
Drawings
FIG. 1 is a flow chart of the method of extraction of the primary cells of the terminal bud of the salamander of the present application;
FIG. 2 is a schematic representation of the recovery of the ampullate salamander ampullate limb in step S2 and the extraction of the germ cells in step S4 in the extraction method according to the application;
FIG. 3 is a graph comparing staining of newly developed shoot-based tissues with normal tissues (including HE staining and masson staining) of the salamander after 4 weeks of amputation;
FIG. 4 is a schematic representation of the microscopic isolation and decortication of the salamander bud;
FIG. 5 is a morphology of the salamander bud-based primary cell as it grows, divides, proliferates and migrates;
FIG. 6 is a graph of the staining of actin filaments, vimentin, CD44 of the primary cells of the salamander bud group;
figure 7 is an EIF6, histone staining pattern of the primary cells of the salamander bud group.
Detailed Description
The application is further illustrated by the following figures and examples, which are not intended to be limiting.
In order to solve a plurality of difficulties existing in the process of extracting the salamander germ cells in the prior art, including selection of germ sampling time, a microscopic peeling technology and an effective removal method of salamander germ tissue parasitic bacteria, the applicant develops a primary cell extraction method of the salamander terminal germ, successfully extracts primary cells from the salamander germ, and has important significance for the study of amphibian life and human regenerative medicine.
The extraction method of the application comprises the following aspects: 1. raising salamander; 2. performing an amputation operation of the salamander; 3. post-operation treatment of the salamander; 4. obtaining the bud base tissue of the salamander; 5. sterilizing and peeling technology of the salamander bud base; 6. obtaining the salamander bud-based tissue suspension. In particular as follows (see fig. 1 to 4).
The application relates to a method for extracting primary cells of the terminal bud base of an salamander, which comprises the following steps:
s1, raising salamanders in the eastern direction: feeding multiple adult salamanders with complete metamorphosis (length of about 7cm-10 cm) into an ecological jar (glass jar), simulating ecological environment (controlling room temperature at 16 ℃ and water temperature in the jar at 24 ℃) once daily feeding animal feed (frozen red worms can be selected), changing water regularly, cleaning residual bait and feces, and keeping water quality clean all the time (avoiding bacteria breeding);
s2, amputation and postoperative treatment of the salamander: a. placing the salamander in 0.1% tricaine solution for 8 minutes to anesthetize the salamander; b. placing the salamander under a stereo microscope, horizontally cutting two forelimbs at the middle part of the humerus of the salamander by using surgical scissors, wherein the length of the forelimbs is 1-1.5cm; c. pushing the musculature attached to the bones using the hammer forceps, trimming the protruding bones of the bone strap (any protruding bones can obstruct the wound healing process and delay regeneration; also can lead to epithelial rupture and fungal infection during limb regeneration), and trimming the protruding musculature, ensuring a smooth amputation limb surface; d. transferring the amputated salamander into 0.5% sulfadiazine water solution (the action is equivalent to that of sterilized water), changing the solution after 24 hours, and normally raising the salamander according to the step S1, waiting for the salamander to generate bud bases (generally generating bud bases 12-14 days after amputation);
s3, obtaining the salamander bud base tissue: a. waiting for 4 weeks (see figure 3, comparing the masson and HE staining of newly grown bud base tissues and normal tissues after the limb of the salamander is broken for 4 weeks, finding that the regenerated bud base tissues after the limb is broken for 4 weeks contain a large number of immature and undifferentiated bud base cells, indicating that the limb is broken for a proper sampling time after 4 weeks), after the salamander generates mature buds (the salamander can realize limb breaking regeneration through early bud, medium bud, mature bud, bud differentiation, limb breaking regeneration and other processes after the limb breaking of the salamander, as shown in figure 2), placing the salamander in a 0.1% trimethyl caine solution for 8 minutes to anesthetize the salamander, and sterilizing the salamander by using high-pressure sterilized water; b. placing salamander in a stereo microscope, shearing bud base (the surgical scissors are perpendicular to the direction of limbs) by using surgical scissors, clearly observing conical bump from residual tissues, and placing the sheared bud base into an amphibian culture medium (A-MEM); c. after all buds are collected, sequentially soaking the buds in a pasteurized solution diluted 1000 times for 1 minute, soaking in an iodophor for 5 minutes, soaking in chloramphenicol diluted 10 times for 1 minute, and performing sterilization treatment (removing parasitic bacteria); d. placing the sterilized bud in an amphibian balancing solution (APBS), and performing aseptic peeling operation on the bud by means of a microscope; e. repeatedly washing with amphibian balancing solution for 3 times after peeling to obtain bud-based tissue (see figure 4); (wherein A-MEM, pasteurization solution, iodophor, chloramphenicol, APBS, etc. are contained in a sterile petri dish)
S4, extracting primary cells of the salamander bud group: a. granulating the bud-based tissue by using an ophthalmic scissors in a physical way; b. the shoot base tissues of different thickness after granulation were subjected to gradient time digestion (30 min, 45 min, 60 min) by trypsin at 15 min intervals; c. centrifuging for 5 minutes at 1200 revolutions to obtain a bud-based primary tissue precipitate; d. the bud-based primary tissue is precipitated and resuspended by using a proper culture system (A-MEM+10% fetal bovine serum+1% penicillin/streptomycin), and undigested bud-based tissue is filtered through a filter (a microporous filter of 0.22 micrometers), thus obtaining bud-based primary cells.
After the primary bud cell is successfully extracted, the applicant finds out the culture conditions suitable for the survival and the expansion of the primary bud cell of the salamander, cultures the primary bud cell of the salamander to ensure that the primary bud cell is in a normal growth cycle and maintains the biological functions of division, proliferation, migration and the like through the attempts of various culture environments; meanwhile, the morphology of the cells is observed under a microscope, and proper proteins (actin filaments, vimentin, CD44, histone and EIF 6) homologous to human beings are screened for staining, so that the primary cells (1) extracted from the salamander bud group are successfully identified to have the following morphological characteristics: the cell size can reach hundreds of micrometers, the nucleus is large, and the genetic material in the nucleus is long; the cell morphology is different, has antenna, and is similar to nerve cell; (2) the biological behavior characteristics are as follows: slow growth, slow division speed and certain migration and proliferation capacity; (3) is a mesenchymal cell with dry and regenerative capacity.
The primary cell culture method of the salamander bud base comprises the following steps: a. planting the extracted bud-based primary cells in a collagen-coated culture dish, controlling the temperature of a culture environment to be 25 ℃, and controlling the concentration of carbon dioxide to be 2%, wherein a culture system is A-MEM+10% fetal bovine serum+1% penicillin/streptomycin; b. culturing for 24h in a Transwell chamber, lightly flushing with culture medium (A-MEM), flushing off primary cells of the salamander bud base, and replanting on a collagen-coated 24-well plate, wherein the culture environment is as in step a; c. the liquid is changed once every two days. The method can effectively separate the parasitic bacteria remained in the primary cell extraction process through Ttranswell technology and physical sedimentation; the specific principle is as follows: the salamander bud-based cells are large (the size of the salamander bud-based cells still can reach hundreds of microns after trypsin digestion), and the growth speed of the parasitic bacteria in a specific culture environment is low, so that the parasitic bacteria can be removed through physical sedimentation.
Referring to figure 5, during the culture of the primary cells of the salamander in the eastern world, by morphological observation of the cells under a microscope, it was confirmed that the primary cells of the salamander can be elongated up to several hundred microns, have large nuclei, are lengthy and have various forms (can be spindle-shaped, circular, dendritic, conical, etc.), because the cells have feelings, and have large inter-cell feelings; the primary cells of the salamander bud base have weak wall attaching capability and slow growth. In the figure, a is the state where the primary bud-based cells adhere to and grow, and the individual cells are up to several hundred micrometers in length; b is a primary bud-based cell in the dividing stage; c is the process of germ primary cell migration; d is the proliferation process of the primary bud-based cells; e is the general appearance of the form, proliferation and migration of the primary bud-based cells after expansion, and p is the fixed marker at the bottom of the culture dish.
Referring to fig. 6 and 7, actin filaments (cytoskeleton) were stained by phalloidin (phalloidin), defining a cell morphology unique to the primary bud-based cells; through immunofluorescence staining technology, respectively carrying out marker staining such as Vimentin (mesenchymal skeleton protein), CD44 (membrane protein markers of embryonic stem cells and some stem cells), histone (Histone, plays a role in regulating and controlling nuclear protein transcription, and is also an epigenetic important regulatory molecule), EIF6 (eukaryotic translation initiation factor, is expressed in cell nucleus and nucleolus, plays a role in regulating and controlling regeneration, DNA repair and the like), and the like, wherein the Vimentin staining determines whether the Vimentin is mesenchymal cells, the CD44 staining determines whether the Vimentin is embryonic stem, and the Histone and EIF6 staining determine whether partial nuclear proteins are highly conserved and regeneration related biological characteristics; wherein, vimentin staining positive, CD44 staining positive, EIF6 staining positive and Histon staining positive prove that the primary cells extracted from the salamander bud base are mesenchymal cells with dryness and regeneration capability.
The above general description of the application and the description of specific embodiments thereof in relation to the present application should not be construed as limiting the scope of the application. Those skilled in the art can add, subtract or combine the features disclosed in the foregoing general description and/or the detailed description (including examples) to form other technical solutions within the scope of the application without departing from the disclosure of the application.
Claims (8)
1. The method for extracting the primary cells of the terminal buds of the salamander is characterized by comprising the following steps of:
s1, raising salamanders in the eastern direction: simulating ecological environment, raising multiple salamanders, and feeding animal feed once every 1-2 days;
s2, amputation and postoperative treatment of the salamander: a. anesthetizing the eastern salamander; b. placing the salamander under a stereo microscope, horizontally cutting two forelimbs at the middle part of the humerus of the salamander by using surgical scissors, wherein the length of the forelimbs is 1-1.5cm; c. pruning the protruding bones of the skeleton belt, pruning the protruding muscle tissues, and ensuring the surface smoothness of the amputation limb; d. transferring the amputated salamander into 0.5% sulfadiazine water solution, changing the solution after 24 hours, and normally raising the salamander according to the step S1, and waiting for the salamander to generate a bud base;
s3, obtaining the salamander bud base tissue: a. waiting for 3 to 4 weeks, and after the salamander does not have postoperative complications and generates mature bud bases, anesthetizing the salamander and sterilizing the salamander; b. placing the salamander in a stereo microscope, shearing the bud base by using an operation shear, and transferring the sheared bud base into an amphibian culture medium; c. after all buds are collected, sequentially soaking the buds in a pasteurized solution diluted 1000 times, iodophor and chloramphenicol diluted 10 times, and sterilizing the buds; d. placing the sterilized bud base into balance liquid of the amphibian, and performing aseptic peeling operation on the bud base by means of a microscope; e. repeatedly washing with balance liquid of amphibian for several times after peeling to obtain bud-based tissue;
s4, extracting primary cells of the salamander bud group: a. digesting the bud-based tissue by trypsin; b. centrifuging for 5 minutes at 1200 revolutions to obtain a bud-based primary tissue precipitate; c. re-suspending the bud-based primary tissue sediment, and filtering undigested bud-based tissue through a filter membrane to obtain bud-based primary cells.
2. An extraction method of primary cells of the terminal bud group of the salamander of claim 1, wherein: in step S4, granulation of the shoot bud tissue is performed prior to digestion of the shoot bud tissue.
3. An extraction method of primary cells of the terminal bud group of the salamander of claim 1, wherein: in steps S2 and S3, the salamander is placed in a 0.1% solution of tricaine for 7-10 minutes to anesthetize the salamander.
4. An extraction method of primary cells of the terminal bud group of the salamander of claim 1, wherein: in step S3, the salamander is sterilized using an iodophor disinfectant or autoclaved water.
5. An extraction method of primary cells of the terminal bud group of the salamander of claim 1, wherein: in step S3, the bud base is soaked in the pasteurized solution diluted 1000 times for 1 minute, in iodophor for 5 minutes, and in chloramphenicol diluted 10 times for 1 minute.
6. An extraction method of primary cells of the terminal bud group of the salamander of claim 2, wherein: in step S4, shoot-based tissues of different thickness were digested by trypsin with a gradient time interval of 15 minutes.
7. An extraction method of primary cells of the terminal bud group of the salamander of claim 1, wherein: in step S1, the salamander is an metamorphically developed adult salamander with a length of more than 7cm.
8. An extraction method of primary cells of the terminal bud group of the salamander of claim 7, wherein: in the step S1, the salamander is raised in an ecological jar, the room temperature is controlled at 14-18 ℃, the water temperature in the ecological jar is controlled at 24+/-1 ℃, water is changed at regular time, residual bait and excrement are cleaned, and the water quality is always kept clean.
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