CN101491229A - Vitro culturing method of pearl shell mantle tissue and culture medium - Google Patents
Vitro culturing method of pearl shell mantle tissue and culture medium Download PDFInfo
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Abstract
The invention discloses an in-vitro culture method for the pearl shell mantle tissue and a special culture solution thereof. The culture medium comprises hydrolyzed pearl liquor, shell liquor, antibiotics and seawater. The method conventionally comprises the following steps: taking the material from a nacre to obtain the mantle epithelium, removing the skirt band part, cutting the tissue into tissue blocks with the size of about 1mm<3> to 2mm<3> after purification treatment, putting the tissue blocks in the protease solution for digestion, and adding the culture solution to stop the digestion. The preliminarily digested tissue blocks are respectively put into a culture dish or cling to the pearl core which is covered by adherence substance. The cultured pearl shell mantle tissue grows in good conditions; after 4 hours, the cells are emigrated from the tissue blocks; and after 2 days in-vitro pearl sacs are formed effectively. The method can be used to inject the anucleate pearl sacs at different positions in the pearl shell with the inserted pearl nucleus so as to culture the anucleate pearl. The method has low cost, a high survival rate, a high pearl formation rate, and little damage to the nacre.
Description
Technical field
The present invention relates to a kind of cultured in vitro method and medium of tissue culture technique and application, particularly pearl shell mantle tissue of aquatic invertebrate.
Background technology
Pearl is formed by the secretion of the pearl sac in pearl shell body nacre, and pearl sac is that propagation formed after the epithelial cell of pearl shell mantle was upset, so the outer embrane of pearl shell plays an important role in pearl forms.Modern artificial pearl's cultural method is that donor pearl shell mantle tissue piece is implanted no nucleus pearl of formation or nucleated pearl in the acceptor pearl shell body separately or with pearl nuclear.In the transplanting surgical procedure, epithelial cell be attached at position, tightness degree, the piece of tissue of pearl nuclear area, shape and the position of drawing materials, plant nuclear location, when planting the nuclear operation mechanical damage degree of pearly shellfish all be directly connected to the formation and the epithelial secretor state of pearl sac, often cause the generation of various defect pearls, as dirty pearl, tail pearl, plain pearl etc.In addition, outer embrane is planted the sheet method also can make the pearly shellfish damage serious, increases the lethality of planting nuclear back pearly shellfish and tells the nuclear rate, thereby seriously influence quality and the output of pearl.Because outer embrane is planted the intrinsic defective of sheet method, scholars utilize tissue culture technique and cell culture technology, extracorporeal culture nucleated pearl capsule according to the principle that pearl forms, and insert capsule again and grow cultured pearls, and attempt to set up a kind of new pearl cultivating method.
Tissue culture for pearl shell mantle has been carried out many work both at home and abroad, the pteria martensii mantle tissue that Japan's scholar's raised path between farm fields well clear (Machii) just produced Japan from phase late 1960s is cultivated and has been carried out systematic research, the mantle tissue that makes cultivation is about external survival 30d and can secrete the nacre of minitype particle, the stone peace and quiet of China Sichuan University etc. are from the fresh water pearl freshwater mussel of the initial stage eighties to China, hydriopsis cumingii, the mantle tissue of cristaria plicata and anodonta woodiana pacifica is cultivated and is carried out deep research, not only successfully set up tissue culture technique and method, and the character that outer embrane is organized in the secretion (nacre) that produces in the culture in vitro is analyzed.The common difficulty that scholars previously run into is, the outer embrane cell culture is difficult to form cell-line, follow-up work can't carry out [analogy reaches brightness, Su Tianfeng. the application of biotechnology in pearl aquaculture. aquatic science and technology, 2000, (1): 13].
The inventor thinks, present physiology, Biochemical Research weakness to the pearl shellfish, especially the immunologic mechanism to the pearl shellfish it be unclear that, the pearl of directly turning out real meaning in the laboratory is difficult, but take the method for tissue culture or cell culture, help the outer embrane epithelial cell to set up the blood circulation relation with nacre as early as possible, quicken the formation of pearl sac; Reduce the injury that cell, piece of tissue and pearl renucleation process cause nacre, be helpful not only to the bead yield rate that improves the seawater nucleated pearl, and, the meaning of particular importance in not having the production of nucleus pearl (being mainly medicinal), seawater is arranged, also is easier to obtain aborning promote.
The king of institute of oceanography, Guangxi likes that comrades such as the people studied culture in vitro nucleated pearl capsule from early 1990s, research draws a common result and is: the external pearl sac that forms with the outer embrane cell culture is used for inserting capsule grows cultured pearls and can't obtain pearl, is used for slotting capsule and grows cultured pearls and can access pearl and the mantle tissue piece is cultivated the external pearl sac that forms.Illustrate that the formed pearl sac of cell culture suspension also is not perfect pearl sac, just have complete 26S Proteasome Structure and Function and cultivate the pearl sac that forms by mantle tissue.The most important thing is to set up complete pearl sac structure at the culture in vitro pearl sac.Owing to still can't accurately separate the various types of cells in the mantle tissue at present, particularly outer epithelial cell, interior epithelial cell and phoirocyte three class cells therefore can't be in proportion at the external pearl sacs of setting up.Though many cell adhesions are consequently arranged on pearl nuclear, can't set up due connection between the cell, so simple cell culture method can not form the external pearl sac of structural integrity.On the contrary, be attached on the pearl nuclear with the mantle tissue piece and cultivate, it is consistent basically that mantle tissue is set up the process of setting up pearl sac in the process consubstantiality of external pearl sac, outer epithelial cell is moved out from the mantle tissue piece, move on surface along pearl nuclear, wraps up pearl nuclear surface gradually, and the phoirocyte in the mantle tissue is wrapped in outer surface epithelial cell more simultaneously, the final more complete pearl sac of structure that forms, this pearl sac is a pearl sac truly.
But be used for inserting the capsule experiment of growing cultured pearls with the external pearl sac that the mantle tissue piece forms and the many and high situation of pearly shellfish lethality of low, the plain pearl of bead yield rate occurs, this does not have uncorrelatedly with inserting nuclear technique, and this is because the mode that the size of otch and pearl nuclear are transported to predetermined nuclear position by otch directly has influence on the structural change of external pearl sac.If attaching is insecure on pearl nuclear, otch is too small for external pearl sac, will cause external pearl sac to be damaged, they were come off before arriving predetermined nuclear position fully, can't produce pearl, can only be plain pearl, and the final generation of the pearl of the structure pearl sac that is kept perfectly nuclear pearl.Though Wang Aimin etc. improve insertion process, but insert the capsule technology of growing cultured pearls and be applied to mariculture, just obtain 4.05% success rate, be applied to produce, also have a lot of element tasks will be (Wang Aimin, Yan Bing, Su Qiong, Deng. pteria martensii pearl sac culture in vitro and slotting capsule are grown cultured pearls. Guangxi science, 2000,7 (1): 70).
Summary of the invention
Purpose of the present invention mainly is at the not enough of above technology and the problem that reflects in previously testing, and a kind of cultured in vitro method and medium of pearl shell mantle tissue is provided.
The inventor in line with the ecological approaching thinking of nacre, hold on to and reduce cost, easy operating is convenient to promote, and improves the production technology link of output and quality, the inventor has carried out a series of useful work.
Learn from the above-mentioned background technology, because the formation of pearl realizes that by pearl sac therefore, Chinese scholars is all relatively paid close attention to the formation of pearl sac and the structure of pearl sac.On the details of pearl sac formation and structure, though the result of different researchers research is variant, but they generally believe that outer embrane plants the outer epithelial cell of sheet (tissue) and can form the epithelial cell of pearl sac through migration and propagation, the phoirocyte one common peripheral of its phoirocyte and acceptor shellfish is formed complete pearl sac around the pearl sac epithelial cell, outer embrane is planted the interior epithelial cell of sheet and is then degenerated, and does not participate in the formation of pearl sac.The groups of cells that should solve external pearl sac when the culture in vitro pearl sac is a problem, and solves the sticking problem of cell on pearl nuclear surface again.For obtaining the external pearl sac of structural integrity fast, the technology of the present invention has been selected to cultivate through postdigestive outer embrane epithelial tissue of short time; In order to solve cell better at the sticking problem on pearl nuclear surface and reduce production costs, the technology of the present invention is extracted high molecular adherent material from planting abandoning of pearl process the soft tissues such as expecting closed shell flesh, technology is simply cheap, has successfully solved the sticking problem of cell.
Method of the present invention mainly comprises the cultured in vitro base (culture fluid) and the shellfish outer embrane cultured in vitro method of shellfish mantle tissue.
The pearl oyster shellfish is waited invertebrate for hanging down, commercially available medium is cultivated research and development at vertebrate and insect cell, indiscriminately imitate general cell culture scheme and apply to the cultivation of shellfish jacket membrane epithelial cell, can not obtain desirable effect, and reagent costs an arm and a leg, ingredients is numerous and diverse, and for this reason, we improve medium.
The cultured in vitro base (culture fluid) of pearl shell mantle tissue of the present invention comprises natural sea-water, Margarita hydrolyzed solution, shellfish liquid and antibiotic, and its primary raw material and mass fraction are as follows:
Margarita hydrolyzed solution: 0.1-0.5 part;
Shellfish liquid: 2-10 part;
Antibiotic 0.01-0.03 part;
Seawater 90-100 part.
Above-described Margarita hydrolyzed solution is that the mother pearl shellfish that will clean soaks with acetic acid, and is softening, through mechanical grinding, obtains pure nacreous layer slice, the sulfuric acid heating hydrolysis is used in drying, pulverizing, levigate, calcium carbonate or milk of lime neutralization, vacuum concentration, decolorization and impurity removal by active carbon obtains pearl crude liquid.
Or the honey of nacre powder and pH2.5～6 mixed the back in stirring at room more than 5 ℃ by 0.1～5: 100, or after the stirring at room again water proof heat piecemeal and stirred 2～3 hours, obtain.
Above-described antibiotic is to contain penicillins, streptomycin class antibiotic, can be the two anti-solution of penicillin-streptomycin, or single penicillin, streptomycin, ampicillin or streptomycin sulphate.Penicillin concn is 100IU/ml, and streptomycin concentration is 0.1g/L.
Above-described shellfish liquid is shellfish tissue juice or shellfish serum, shellfish tissue juice be get in the pearl shellfish body liquid after filtration degerming obtain, shellfish serum is to get blood from pearl shell heart or trunk, through centrifugal, get supernatant and is pearl shell serum.Filtration sterilization wherein can be adopted forms such as plate filter, the airtight filtration of miillpore filter.
The pH value of the cultured in vitro base of above-described shellfish mantle tissue is 6.8～7.2, if depart from, can adjust acid-base value with hydrochloric acid or sodium hydroxide.
The cultured in vitro method of pearl shell mantle tissue of the present invention is as follows:
1, prepares culture dish
Culture dish of the present invention can use pottery or glass container, but the bottom surface of culture dish anticipate with the homemade adherent material that extracts from shellfish closed shell flesh or mantle tissue, this adherent material extracts the back dry disinfection from shellfish closed shell flesh or outer embrane, and is stand-by.
2, the cultivation of external pearl sac
Routine is drawn materials and is obtained the pearl shell mantle epithelial tissue, removes the opotism part, is cut into about 1mm after the purified treatment
3～2mm
3The piece of tissue of size is put in the protein enzyme solution (filtration sterilization, 4 ℃ store for future use) of 0.1-0.8%, adds homemade culture fluid and end digestion after digesting 5～10min under the temperature Celsius 20 ℃-25 ℃.To place 35mm * 10mm culture dish (or being attached on the pearl nuclear of smearing adherent material) through the piece of tissue of preliminary digestion respectively, 15-25 block organization piece is pasted (as adopting the culture dish of different size specifications in each culture dish bottom, the piece of tissue number that attaches should adjust with reference to this density), after putting piece of tissue, add a spot of culture fluid in culture dish, piece of tissue is moistening to get final product keeping.Build bottle cap, place 20 ℃ of incubators or gnotobasis Celsius 20 ℃-28 ℃ to cultivate.Add a subculture behind the 24h.Every other day or visual cell's upgrowth situation change liquid.The pearl shell mantle tissue 4h epithelium posterius cell of cultivating by this method is moved out from tissue and is increased rapidly, and upgrowth situation is good, the outer pearl sac of organizator effectively after 2 days.
Described protein enzyme solution is commercial enzymes such as papain, trypsase, bromelain.
The preparation of described protein enzyme solution is to weigh protease in proportion, be dissolved in the protein enzyme solution that is mixed with 0.1-0.8% in the double distilled water, mixing, filtration sterilization, 4 ℃ standby, and filtration sterilization wherein can be adopted forms such as plate filter, the airtight filtration of miillpore filter.
3, the breed of pearl sac transplanting and pearl
With the elbow suction pipe pearl sac is transplanted in the pearly shellfish body, or examine in the homologous transplantation pearly shellfish body with pearl, it is to hang in the 15-30 ℃ of seawater to support 9-12 month that the shellfish of will performing the operation after the recuperation phase is positioned over temperature range, higher survival rate and bead yield rate be can obtain, high-quality no nucleus pearl or nucleated pearl obtained.
The healthy nacre of above-described employing be meant pearl culture produce in main marine products shellfish kind, comprising: pteria martensii, separate martensii, white butterfly nacre, black martensii, penguin pearl shell.
It is above-described that the nacre of outer embrane epithelial tissue and shellfish serum is provided is the marine products shellfish of same kind, or different genera, so long as shellfish serum is just passable.If get shellfish serum with the above-mentioned pearl shell of getting the outer embrane epithelial tissue, can reduce cost greatly.
External pearl sac in the above-described injection pearly shellfish body can be expelled to the nacre of different cultivars.
Beneficial effect of the present invention is:
1, being transplanted in the pearly shellfish body is the pearl sac that forms at extracorporeal culture, rather than nacre mantle tissue piece, more be different from and traditional directly seize the method that pearl nuclear inserts on both sides by the arms with apparatus, the pearl sac of implantation more is suitable in the nacre body than piece of tissue grows.
2, survival rate and bead yield rate height: piece of tissue is through preliminary digestion, move out rapidly, be transplanted in the pearly shellfish body is the complete pearl sac of cell structure, be easy to set up the blood circulation relation with pearly shellfish, and the method that adopts the elbow suction pipe to inject, pearly shellfish is not caused damage substantially, not to the damage of pearl sac, so use technology pearl shellfish survival rate of the present invention and bead yield rate height yet.Previously the slotting capsule of pearl sac cultivated with the outer embrane epithelial tissue of the report bead yield rate of growing cultured pearls is 4.05%, observing the seawater inserted sheet according to us educates the bead yield rate of seedless pearl and has only about 25%, nuclear sea water pearls high-quality pearl is arranged about 30%, and with the technology of the present invention bead yield rate and high-quality pearl more than 50%, recuperation phase and the phase of growing cultured pearls pearl shellfish survival rate improve more than 10%.
3, cost is low: that supplies with outer embrane epithelium, shellfish tissue juice, shellfish serum and shellfish closed shell flesh can use same shellfish body, has saved the shellfish resource; In addition, because few, can in being inserted with the pearl shellfish of nucleus pearl capsule, choose diverse location and inject seedless pearl sac and cultivate no nucleus pearl, not only can gather in the crops and view and admire pearl but also can gather in the crops medicinal pearl, the income of increase pearl farming nacre damage.
4, simple to operate, reduce opportunities for contamination, reduce dirty pearl rate: the general condition of domestic plant is relatively poor, surgical procedure is difficult to accomplish sterile working, and elbow suction pipe injection method is more easy to operate than insertion, and pearl sac adheres to, operating personnel easily grasp, few to the damage of pearl shellfish, reduced infection chance, reduced dirty pearl rate.
5, social benefit and remarkable in economical benefits: the price of natural peral and high quality pearl is always expensive than gold, the present invention provides technical support for output and the quality that improves pearl, for carrying forward southern pearl culture, improve the international status of Chinese southern pearl, increase pearl farming income, drive and support that local economic construction has great importance.
Embodiment
The invention will be further described below in conjunction with example, but protection scope of the present invention is not limited to this.
One, the preparation of special culture media
Embodiment 1
Get the natural sea-water that 100ml boils, add shellfish tissue juice or shellfish serum 2-10ml, (shellfish tissue juice be with the liquid in the pteria martensii body after filtration degerming obtain, shellfish serum is to get blood from pearl shell heart or trunk, through centrifugal, get supernatant and obtains pearl shell serum), add 0.1-5% Margarita hydrolyzed solution 10ml, and add 100IU/ml penicillin 1ml, and 0.1g/L streptomycin 1ml, adjust pH is to 6.8-7.2.The preparation of Margarita hydrolyzed solution be honey with nacre powder and pH2.5～6 by 0.1～5: 100 mix the back in stirring at room more than 5 ℃, or after the stirring at room again water proof heat piecemeal and stirred 2～3 hours, obtain.
Embodiment 2
Get the natural sea-water that 100ml boils, add shellfish tissue juice or shellfish serum 2-10ml, (shellfish tissue juice be with the liquid in the pteria martensii body after filtration degerming obtain, shellfish serum is to get blood from pearl shell heart or trunk, through centrifugal, get supernatant and obtains pearl shell serum), add 0.1-5% Margarita hydrolyzed solution 10ml, and add 100IU/ml ampicillin 2ml, or 0.1g/L streptomycin sulphate 2ml, adjust pH is to 6.8-7.2.The preparation of Margarita hydrolyzed solution is that the mother pearl shellfish that will clean soaks with acetic acid, and is softening, through mechanical grinding, obtains pure nacreous layer slice, the sulfuric acid heating hydrolysis is used in drying, pulverizing, levigate, calcium carbonate or milk of lime neutralization, vacuum concentration, decolorization and impurity removal by active carbon obtains pearl crude liquid.
Two, the cultured in vitro method of pearl shell mantle tissue:
Embodiment 1
(1) weigh trypsase in proportion, be dissolved in the trypsin solution that is mixed with 0.1-0.5% in the tri-distilled water, mixing, filtration sterilization, 4 ℃ are standby; Take out the 1-2 healthy pteria martensii outer embrane epithelial tissue in age according to a conventional method, remove the opotism part, be cut into about 1mm after the purified treatment
3～2mm
3The piece of tissue of size is put and is done preliminary digestion in the trypsin solution, and temperature is 20 ℃-25 ℃ Celsius, and the time is 5～10min.Add following culture fluid and end digestion.
(2) special culture media preparation: can be fast from extraction shellfish tissue juice from the above-mentioned pteria martensii body that the outer embrane epithelial tissue is provided, shellfish serum (get blood from pearl shell heart or trunk, the centrifuging and taking supernatant is pearl shell serum); The airtight filtration of miillpore filter also can prepare shellfish serum in-20 ℃ of preservations (also can use shellfish serum not of the same race) from pteria martensii of the same race in advance.Insert 4 ℃ of thawings before the use, use as early as possible, need not the water-bath inactivation treatment before using; The preparation hydrolyzed pearl solution uses behind the filtration sterilization.
(3) will place 35mm * 10mm culture dish (or being attached on the pearl nuclear of smearing adherent material) through the piece of tissue of preliminary digestion respectively, the bottom surface of blake bottle (or pearl nuclear) anticipates with the homemade adherent material that extracts from soft tissues such as shellfish closed shell fleshes.15-25 block organization's piece (as adopting the culture dish of different size specifications, the piece of tissue number of attaching should adjust with reference to this density) is pasted in each culture dish bottom, put piece of tissue after, in culture dish, add a spot of culture fluid, piece of tissue is moistening to get final product keeping.Build bottle cap, place 20 ℃ of aseptic culture casees Celsius (or gnotobasis Celsius 20 ℃-25 ℃) to cultivate.Add a subculture behind the 24h.Every other day or visual cell's upgrowth situation change liquid.The pearl shell mantle tissue 4h epithelium posterius cell of cultivating by this method is moved out from tissue and is increased rapidly, and upgrowth situation is good, the outer pearl sac of organizator effectively after 2 days.
(4) with glass elbow suction pipe pearl sac is transplanted in the pearly shellfish body, or examine in the homologous transplantation pearly shellfish body with pearl, it is to hang in the 15-30 ℃ of seawater to support 9-12 month that the shellfish of will performing the operation after the recuperation phase is positioned over temperature range, higher survival rate and bead yield rate be can obtain, high-quality no nucleus pearl or nucleated pearl obtained.
Embodiment 2
(1) bromelain that weighs 0.15g is dissolved in and is mixed with 0.3% bromelain enzyme solutions in the tri-distilled water of 50ml, mixing, and filtration sterilization, 4 ℃ are standby; Take out the 1-2 healthy white butterfly nacre outer embrane epithelial tissue in age according to a conventional method, remove the opotism part, be cut into about 1mm after the purified treatment
3～2mm
3The piece of tissue of size is put and is done preliminary digestion in the bromelain enzyme solutions, and temperature is 20 ℃-25 ℃ Celsius, and the time is 5～10min.Add following culture fluid and end digestion.
(2) special culture media preparation: can be fast from the above-mentioned white butterfly nacre body that the outer embrane epithelial tissue is provided extraction shellfish tissue juice (EK) and shellfish serum (get blood from pearl shell heart or trunk, the centrifuging and taking supernatant is pearl shell serum); Also can prepare shellfish serum from white butterfly nacre of the same race in advance and preserve shellfish serum perhaps not of the same race in-20.Insert 4 ℃ of thawings before the use, use as early as possible, need not the water-bath inactivation treatment before using; The preparation hydrolyzed pearl solution uses behind the filtration sterilization.
(3) will place 35mm * 10mm culture dish (or being attached on the pearl nuclear of smearing adherent material) through the piece of tissue of preliminary digestion respectively, the bottom surface of blake bottle (or pearl nuclear) anticipates with the homemade adherent material that extracts from soft tissues such as shellfish closed shell fleshes.15-25 block organization's piece (as adopting the culture dish of different size specifications, the piece of tissue number of attaching should adjust with reference to this density) is pasted in each culture dish bottom, put piece of tissue after, in culture dish, add a spot of culture fluid, piece of tissue is moistening to get final product keeping.Build bottle cap, place 20 ℃ of aseptic culture casees Celsius (or gnotobasis Celsius 20 ℃-25 ℃) to cultivate.Add a subculture behind the 24h.Every other day or visual cell's upgrowth situation change liquid.The pearl shell mantle tissue 4h epithelium posterius cell of cultivating by this method is moved out from tissue and is increased rapidly, and upgrowth situation is good, the outer pearl sac of organizator effectively after 2 days.
(4) with glass elbow suction pipe pearl sac is transplanted in the pearly shellfish body, or examine in the homologous transplantation pearly shellfish body with pearl, it is to hang in the 15-30 ℃ of seawater to support 9-12 month that the shellfish of will performing the operation after the recuperation phase is positioned over temperature range, higher survival rate and bead yield rate be can obtain, high-quality no nucleus pearl or nucleated pearl obtained
Claims (9)
1, a kind of cultured in vitro method of pearl shell mantle tissue is characterized in that:
(1) prepare culture dish, adopt pottery or glass container as culture dish, anticipate with homemade adherent material from shellfish closed shell flesh or mantle tissue extraction the bottom surface of culture dish, and this adherent material extracts the back dry disinfection from shellfish closed shell flesh or outer embrane, and is stand-by;
(2) cultivation of external pearl sac, routine are drawn materials and are obtained the pearl shell mantle epithelial tissue, remove the opotism part, are cut into about 1mm after the purified treatment
3～2mm
3The piece of tissue of size, put the protein enzyme solution of 0.1-0.8%, filtration sterilization, 4 ℃ store for future use, after digesting 5～10min under the temperature Celsius 20 ℃-25 ℃, add culture fluid and end digestion, to place 35mm * 10mm culture dish through the piece of tissue of preliminary digestion respectively, or be attached on the pearl nuclear of smearing adherent material, some block organizations piece is pasted in each culture dish bottom, put piece of tissue after, in culture dish, add a spot of culture fluid, build bottle cap, place 20 ℃ of incubators to cultivate, add a subculture behind the 24h, every other day or visual cell's upgrowth situation change liquid;
(3) breed of pearl sac transplanting and pearl, with the elbow suction pipe pearl sac is transplanted in the pearly shellfish body, or examine in the homologous transplantation pearly shellfish body with pearl, it is to hang in the 15-30 ℃ of seawater to support 9-12 month that the shellfish of will performing the operation after the recuperation phase is positioned over temperature range, higher survival rate and bead yield rate be can obtain, high-quality no nucleus pearl or nucleated pearl obtained.
2, the cultured in vitro method of pearl shell mantle tissue according to claim 1 is characterized in that: the healthy nacre of described employing is meant main marine products shellfish kind in the pearl culture production.
3, the cultured in vitro method of pearl shell mantle tissue according to claim 1 is characterized in that: described the nacre of outer embrane epithelial tissue and shellfish serum is provided is the marine products shellfish of same kind or the marine products shellfish kind of different genera.
4, the cultured in vitro method of pearl shell mantle tissue according to claim 1 is characterized in that: the external pearl sac in the described injection pearly shellfish body is expelled to the nacre of identical or different kind.
5, the cultured in vitro method of pearl shell mantle tissue according to claim 1, it is characterized in that: described protein enzyme solution is the commercial enzyme of papain, trypsase or bromelain, the preparation of protein enzyme solution is to weigh protease in proportion, be dissolved in the protein enzyme solution that is mixed with 0.1-0.8% in the double distilled water, mixing, filtration sterilization, 4 ℃ standby.
6, a kind of cultured in vitro liquid of pearl shell mantle tissue as claimed in claim 1 is characterized in that: it comprises natural sea-water, Margarita hydrolyzed solution, shellfish liquid and antibiotic, and its primary raw material and mass fraction are as follows:
Margarita hydrolyzed solution: 0.1-0.5 part;
Shellfish liquid: 2-10 part;
Antibiotic: 0.01-0.03 part;
Seawater: 90-100 part.
7, the cultured in vitro liquid of pearl shell mantle tissue according to claim 6, it is characterized in that: Margarita hydrolyzed solution is that the mother-of-pearl shell that will clean soaks with acetic acid, softening, through mechanical grinding, obtain pure nacreous layer slice, drying, pulverizing, levigate, use the sulfuric acid heating hydrolysis, calcium carbonate or milk of lime neutralization, vacuum concentration, decolorization and impurity removal by active carbon obtains pearl crude liquid; Or the honey of nacre powder and pH 2.5～6 mixed the back in stirring at room more than 5 ℃ by 0.1～5: 100, or after the stirring at room again water proof heat piecemeal and stirred 2～3 hours, obtain.
8, the cultured in vitro liquid of pearl shell mantle tissue according to claim 6, it is characterized in that: described antibiotic is to contain penicillins, streptomycin class antibiotic, select the two anti-solution of penicillin-streptomycin, or single penicillin, streptomycin, ampicillin or streptomycin sulphate, penicillin concn is 100IU/ml, and streptomycin concentration is 0.1g/L.
9, the cultured in vitro liquid of pearl shell mantle tissue according to claim 6, it is characterized in that: described shellfish liquid is shellfish tissue juice or shellfish serum, shellfish tissue juice be get in the pearl shellfish body liquid after filtration degerming obtain, shellfish serum is to get blood from pearl shell heart or trunk, through centrifugal, get supernatant and be pearl shell serum.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102524121A (en) * | 2011-12-27 | 2012-07-04 | 浙江山下湖珍珠集团股份有限公司 | Pearl cell staining agent and application thereof in pearl culture |
| CN103314912A (en) * | 2013-07-03 | 2013-09-25 | 浙江海洋学院 | Method of producing pearls on the basis of pearl shell mantel cell culture |
| CN105309345A (en) * | 2015-06-07 | 2016-02-10 | 福建师范大学 | A method of culturing pearls by implanting nucleuses after in vitro co-culture of mantle free cells and the tissue engineering scaffold pearl nucleuses |
| CN105660489A (en) * | 2016-03-23 | 2016-06-15 | 绍兴文理学院 | Large-scale perfect round seedless pearl culture technology |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524121A (en) * | 2011-12-27 | 2012-07-04 | 浙江山下湖珍珠集团股份有限公司 | Pearl cell staining agent and application thereof in pearl culture |
| CN102524121B (en) * | 2011-12-27 | 2013-10-30 | 浙江山下湖珍珠集团股份有限公司 | Pearl cell staining agent and application thereof in pearl culture |
| CN103314912A (en) * | 2013-07-03 | 2013-09-25 | 浙江海洋学院 | Method of producing pearls on the basis of pearl shell mantel cell culture |
| CN105309345B (en) * | 2015-06-07 | 2017-10-31 | 福建师范大学 | Outer embrane free cell plants the method that core cultivates pearl after being co-cultured in vitro with tissue engineering bracket pearl core |
| CN105309345A (en) * | 2015-06-07 | 2016-02-10 | 福建师范大学 | A method of culturing pearls by implanting nucleuses after in vitro co-culture of mantle free cells and the tissue engineering scaffold pearl nucleuses |
| CN105660489A (en) * | 2016-03-23 | 2016-06-15 | 绍兴文理学院 | Large-scale perfect round seedless pearl culture technology |
| CN107043741A (en) * | 2017-05-26 | 2017-08-15 | 健生生物技术有限公司 | The preparation of pearl freshwater mussel outer embrane epithelial cell line and the application of the cell line and cell |
| CN107517899A (en) * | 2017-09-29 | 2017-12-29 | 北海市旺海珠宝有限公司 | The sea water pearls breeding method of seedless pattra pearl |
| CN107517900A (en) * | 2017-09-29 | 2017-12-29 | 北海市旺海珠宝有限公司 | Sea water pearls hurtless measure plants kernel method |
| CN112586414A (en) * | 2020-12-31 | 2021-04-02 | 广东尊鼎珍珠有限公司 | Cultivation method of seedless seawater pearls |
| CN112655615A (en) * | 2020-12-31 | 2021-04-16 | 广东尊鼎珍珠有限公司 | Method for cultivating seawater micro natural pearls through calcium carbonate powder stimulation |
| CN113583936A (en) * | 2021-07-20 | 2021-11-02 | 三亚热带水产研究院 | In-vitro culture solution for mantle cells of pinctada fucata as well as preparation method and application method thereof |
| CN113462634A (en) * | 2021-08-12 | 2021-10-01 | 中国水产科学研究院南海水产研究所 | In-vitro culture solution for mantle cells of pinctada maxima, preparation method and application method |
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