A method for reducing the immunological rejection of sea water pearl oyster xenografts

Abstract

This invention provides a method for reducing immune rejection during xenotransplantation in marine pearl oysters, belonging to the field of marine pearl farming technology. The method includes the following steps: S1. Dissolving asteroidin in a cell treatment solution to obtain an immunosuppressant solution; S2. Preparing a small mantle piece from a donor oyster and thoroughly wetting the surface of the mantle piece with mercurochrome solution; S3. Then, thoroughly wetting the surface of the mantle piece with the immunosuppressant solution; S4. Immersing the pearl nucleus from the donor oyster in the immunosuppressant solution; S5. Implanting the mantle piece and pearl nucleus into the nucleus of the recipient oyster; S6. Performing pearl cultivation. This method uses giant pearl oysters, pearl mussels, and penguin pearl oysters as donor oysters for mantle pieces and Hepu pearl oysters as recipient oysters for nucleus implantation. By applying asteroidin as an immunosuppressant in marine pearl farming, it can reduce the immune rejection reaction during xenotransplantation in marine pearl oysters, promote the formation of pearl sacs, and cultivate high-quality pearls.

Description

Technical Field

[0001] This invention relates to the field of marine pearl farming technology, specifically to a method for reducing immune rejection during xenotransplantation in marine pearl oysters. Background Technology

[0002] The Hepu pearl oyster is the main pearl oyster used in my country's marine pearl farming. Its seedling cultivation, culture, and nucleus insertion pearl cultivation techniques are very mature and easy to operate. However, the pearls produced are thin-layered and small in size, resulting in low commercial value. The large pearl oyster, pearl oyster, and penguin pearl oyster are important pearl oysters for cultivating large, high-quality pearls. The pearls they produce are of high quality, with thick nacre and bright luster, and are priced per pearl, making them extremely valuable. However, their nucleus insertion pearl cultivation techniques are very difficult, and some even have great difficulties in seedling cultivation and culture, so large-scale production has not yet been achieved.

[0003] Therefore, if small pieces of the mantle of pearl oysters such as the giant pearl oyster, the pearl oyster, and the penguin pearl oyster could be transplanted into the body of the Hepu pearl oyster for pearl cultivation, the difficulties in pearl cultivation and growth could be avoided, resulting in high-quality pearls and greatly improving the pearl farming efficiency of the Hepu pearl oyster. However, the immune rejection during this type of transplantation is very serious, and industrial-scale production has not yet been achieved. Therefore, reducing immune rejection during pearl oyster implantation is a key problem that urgently needs to be solved in the pearl farming industry. Patent application CN201811164622.2 discloses a method for reducing immune rejection during transplantation in recipient oysters by introducing the immunosuppressant mycophenolate mofetil into the recipient oyster. Patent application CN201811164623.7 discloses another method for reducing immune rejection during transplantation in recipient oysters by introducing the immunosuppressant cyclosporine A into the recipient oyster. Both methods promote pearl sac formation, thereby increasing pearl yield. However, since they are all of the same species, the rejection reaction is relatively small. If small pieces of the mantle of pearl oysters such as large pearl oysters, pearl oysters, and penguin pearl oysters are inserted into the body of Hepu pearl oysters for transplantation, the rejection reaction will be greater. The above two methods cannot solve the problems of severe immune rejection and low survival rate faced by xenotransplantation. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing a method to reduce immune rejection during xenotransplantation in marine pearl oysters. By using asteroidin as an immunosuppressant in marine pearl farming, the immune rejection reaction generated during xenotransplantation in marine pearl oysters can be reduced, the formation of pearl sacs can be promoted, and high-quality pearls can be cultivated through surrogate motherhood.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for reducing immune rejection in xenotransplantation of marine pearl oysters includes the following steps:

[0007] S1. Dissolve asteroidin in cell treatment solution to obtain an immunosuppressant solution.

[0008] S2. Prepare a small piece of the mantle membrane of the donor shellfish, and add mercurochrome solution to the surface of the small piece of mantle membrane to fully impregnate it.

[0009] S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragment obtained in step S2 to fully impregnate it.

[0010] S4. Immerse the donor pearl nucleus in an immunosuppressant solution.

[0011] S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient bead nucleus.

[0012] S6. Conduct pearl farming.

[0013] Preferably, the donor oyster is a large pearl oyster, a pearl oyster, or a penguin pearl oyster.

[0014] Preferably, the recipient oyster is the pearl oyster.

[0015] Preferably, the immunosuppressant solution contains 0.35% to 0.55% astrococcus by volume.

[0016] Preferably, the cell treatment solution contains 1%-2% polyvinylpyrrolidone, 2-4g glucose, 5-8g clam polysaccharide, 20-45g compound vitamins, 4-6g taurine, 4-8g yeast polysaccharide, 0.2-0.3g curcumin, 0.3g cefuroxime sodium, 0.002g nystatin, 1g streptomycin, 800,000 units gentamicin, 1,000,000 units penicillin, 0.04g norfloxacin, and 0.15g metronidazole, dissolved in phosphate buffer per liter.

[0017] Preferably, the phosphate buffer solution is prepared by dissolving 1.08 g of potassium dihydrogen phosphate, 1.08 g of potassium chloride, 25.22 g of sodium chloride, 0.45 g of sodium bicarbonate, and 0.15 g of disodium hydrogen phosphate in each liter of double-distilled water, adjusting the pH to 7.1, and then pasteurizing for 30 min.

[0018] Multivitamins are a combination of vitamin A, vitamin C, vitamin E and several other vitamins in a mass ratio of 5:8:5:2.

[0019] Preferably, the mercurobromine solution is mercurobromine powder dissolved in filtered seawater, with a mass concentration of 2%.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] 1. The method of reducing immune rejection in xenografting of marine pearl oysters according to the present invention involves applying the immunosuppressant astrococcus to the production of pearl oysters through xenografting. By utilizing its immunosuppressive effect on recipient oysters, the method reduces the immune rejection reaction in the early stage of nucleus implantation in marine pearl oysters, promotes the formation of immune tolerance in recipient oysters, reduces the nucleus expulsion rate and mortality rate of recipient oysters, and promotes the formation of pearl sacs, thereby improving pearl quality and yield.

[0022] 2. The method of reducing immune rejection in xenotransplantation of marine pearl oysters according to the present invention uses giant pearl oysters, pearl oysters, penguin pearl oysters, etc., as donor oysters with mantle fragments, and Hepu pearl oysters as recipient oysters for nucleus insertion, employing asteroidporine as an inhibitor to achieve pearl culturing via surrogate implantation. Staurosporine (STS) is an indole[2,3-α]carbazolium alkaloid extracted from microbial metabolites. It is a potent, ATP-competitive kinase inhibitor that effectively inhibits the activity of protein kinase C (PKC-α, PKC-δ, PKC-ζ). In addition, asteroidporine can also inhibit the activity of protein kinase A, protein kinase G, tyrosine protein kinase, and calmodulin kinase II. Attached image description:

[0023] Figure 1 The study compared the expression levels of hematopoietic immune-related genes at different time points after nucleation of *Pterocarya stenoptera* in the astrosporin-treated group and the control group; specifically, the relative expression level of protein kinase C delta type (PKC-δ) (same letters indicate no significant difference, different letters indicate significant difference).

[0024] Figure 2 The study compared the expression levels of hematopoietic immune-related genes at different time points after nucleation of *Pterocarya stenoptera* in the asteroid-treated group and the control group; specifically, the relative expression levels of allogeneic inflammatory factor-1 (AIF-1) (same letters indicate no significant difference, different letters indicate significant difference).

[0025] Figure 3 Comparison of blood cells on the surface of small patches of the mantle of pearl oysters treated with immunosuppressants (left) and control group (right) 48 hours after nucleation in Hepu pearl oysters (black arrows represent blood cells on the surface of the small patches). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1

[0028] Time: Nucleus insertion on October 22, 2019. Mortality and survival rates of the recipient shellfish were recorded on days 1, 3, 6, 10, 15, 22, and 30 post-insertion.

[0029] Location: Xincun Village, Lingshui County, Hainan Province

[0030] A method for reducing immune rejection in xenotransplantation of pearl oysters in marine waters, using the large pearl oyster as the donor and the Hepu pearl oyster as the recipient, includes the following steps:

[0031] S1. Dissolve asteroidin in cell treatment solution to prepare a 0.35% immunosuppressant solution.

[0032] S2. Prepare small pieces of the mantle membrane of the pearl oyster. Add mercurochrome solution to the surface of the small pieces of mantle membrane and soak them for 5 minutes. Use sterile absorbent cotton or sterile absorbent paper to absorb the excess mercurochrome solution from the small pieces of mantle membrane.

[0033] S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragments obtained in step S2 to fully wet them;

[0034] S4. Immerse the donor pearl nucleus in an immunosuppressant solution;

[0035] S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient's bead nucleus;

[0036] S6. Pearl breeding and cultivation shall be carried out using conventional methods.

[0037] The cell treatment solution was prepared by dissolving 1% polyvinylpyrrolidone (percentage by volume) per liter of phosphate buffer, along with 2 g of glucose, 5 g of clam polysaccharide, 20 g of complex vitamins, 4 g of taurine, 4 g of yeast polysaccharide, 0.2 g of curcumin, 0.3 g of cefuroxime sodium, 0.002 g of nystatin, 1 g of streptomycin, 800,000 units of gentamicin, 1,000,000 units of penicillin, 0.04 g of norfloxacin, and 0.015 g of metronidazole.

[0038] The above phosphate buffer solution was prepared by dissolving 1.08 g of potassium dihydrogen phosphate, 1.08 g of potassium chloride, 25.22 g of sodium chloride, 0.45 g of sodium bicarbonate, and 0.15 g of disodium hydrogen phosphate per liter of double-distilled water, adjusting the pH to 7.1, and then pasteurizing for 30 minutes. The above-mentioned compound vitamins are a mixture of vitamin A, vitamin C, vitamin E, and several other vitamins in a mass ratio of 5:8:5:2.

[0039] Experimental Design: 300 birds in each of the immunosuppressant treatment group and control group (conventional nucleation method, without asteroidin, using cell treatment solution instead of immunosuppressant solution) underwent nucleation. Nucleus patch preparation and nucleation were performed by the same team. Hemocytes and mantle patches were collected at the aforementioned time points, and the mortality and survival rates of the nucleated birds were statistically analyzed. The relative expression levels of immune-related genes PKC-δ and AIF-1 in hemocytes during the early stage of nucleation were detected using quantitative real-time PCR. Mantle patches were fixed in 4% paraformaldehyde solution for tissue sectioning and HE staining to observe hemocytes on the patch surface.

[0040] Result: As Figure 1 and Figure 2 The expression levels of PKC-δ and AIF-1 genes in the astrocytocin-treated group were significantly lower than those in the control group 24 hours after nucleation of pearl oysters in Hepu. Figure 3 The number of blood cells on the surface of the mantle fragments of pearl oysters in the immunosuppressant treatment group was significantly lower than that in the control group 48 hours after nucleation; this indicates that immunosuppressant treatment can significantly reduce the immune rejection reaction generated during nucleation.

[0041] As shown in Table 1, compared with the control group, the survival rate of recipient pearl oysters in the group treated with the immunosuppressant astrocytosporine was significantly higher in the early stage of transplantation of mantle pieces from large pearl oysters in Hepu.

[0042] Table 1 Comparison of pearl production data for each group

[0043]

[0044] Example 2

[0045] Time: Nucleus insertion on November 10, 2019. Mortality and survival rates of the recipient shellfish were recorded every 30 days.

[0046] Location: Bailong Village, Hepu County, Guangxi

[0047] A method for reducing immune rejection in xenotransplantation of pearl oysters in marine waters, using the large pearl oyster as the donor and the Hepu pearl oyster as the recipient, includes the following steps:

[0048] S1. Dissolve asteroidin in cell treatment solution to prepare a 0.4% immunosuppressant solution.

[0049] S2. Prepare small pieces of the mantle membrane of the pearl oyster. Add mercurochrome solution to the surface of the small pieces of mantle membrane and soak them for 5 minutes. Use sterile absorbent cotton or sterile absorbent paper to absorb the excess mercurochrome solution from the small pieces of mantle membrane.

[0050] S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragments obtained in step S2 to fully wet them;

[0051] S4. Immerse the donor pearl nucleus in an immunosuppressant solution;

[0052] S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient's bead nucleus;

[0053] S6. Pearl breeding and cultivation shall be carried out using conventional methods.

[0054] The cell treatment solution was prepared by dissolving 1.5% polyvinylpyrrolidone (the percentage is by volume), 2 g glucose, 5 g clam polysaccharide, 35 g compound vitamins, 4 g taurine, 4 g yeast polysaccharide, 0.2 g curcumin, 0.3 g cefuroxime sodium, 0.002 g nystatin, 1 g streptomycin, 800,000 units gentamicin, 1,000,000 units penicillin, 0.04 g norfloxacin, and 0.015 g metronidazole per liter of shellfish phosphate buffer. The shellfish-specific phosphate buffer was prepared by dissolving 1.08 g potassium dihydrogen phosphate, 1.08 g potassium chloride, 25.22 g sodium chloride, 0.45 g sodium bicarbonate, and 0.15 g disodium hydrogen phosphate per liter of double-distilled water, adjusting the pH to 7.1, and pasteurizing for 30 min. The aforementioned compound vitamin is a mixture of vitamin A, vitamin C, vitamin E, and several other vitamins in a ratio of 5:8:5:2, where the ratio is by mass.

[0055] Experimental design: 1000 embryos were implanted into each of the immunosuppressant treatment group and the control group (using standard implantation method). The embryo preparation and implantation were performed by the same team. The embryo development cycle lasted 6 months, with mortality and survival rates recorded monthly.

[0056] Results: Compared with the control group, the survival rate of receptors in the immunosuppressant group was significantly higher.

[0057] Table 2 Comparison of pearl production data for each group

[0058]

[0059] Example 3

[0060] Time: Nucleus insertion on November 11, 2019. Mortality and survival rates of the recipient shellfish were recorded every 30 days.

[0061] Location: Bailong Village, Hepu County, Guangxi

[0062] A method for reducing immune rejection in xenotransplantation of pearl oysters, using the pearl oyster as the donor and the Hepu pearl oyster as the recipient. The method includes the following steps:

[0063] S1. Dissolve asteroidin in cell treatment solution to prepare a 0.4% immunosuppressant solution.

[0064] S2. Prepare small pieces of the mantle membrane of the pearl oyster. Add mercurochrome solution to the surface of the small pieces of mantle membrane and soak them for 5 minutes. Use sterile absorbent cotton or sterile absorbent paper to absorb the excess mercurochrome solution from the small pieces of mantle membrane.

[0065] S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragments obtained in step S2 to fully wet them;

[0066] S4. Immerse the donor pearl nucleus in an immunosuppressant solution;

[0067] S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient's bead nucleus;

[0068] S6. Pearl breeding and cultivation shall be carried out using conventional methods.

[0069] The cell treatment solution was prepared by dissolving 2% polyvinylpyrrolidone (percentage by volume), 4 g glucose, 6 g clam polysaccharide, 45 g complex vitamins, 5 g taurine, 6 g yeast polysaccharide, 0.3 g curcumin, 0.3 g cefuroxime sodium, 0.002 g nystatin, 1 g streptomycin, 800,000 units gentamicin, 1,000,000 units penicillin, 0.04 g norfloxacin, and 0.015 g metronidazole per liter of phosphate buffer. The phosphate buffer solution was prepared by dissolving 1.08 g potassium dihydrogen phosphate, 1.08 g potassium chloride, 25.22 g sodium chloride, 0.45 g sodium bicarbonate, and 0.15 g disodium hydrogen phosphate per liter of double-distilled water, adjusting the pH to 7.1, and pasteurizing for 30 minutes. The complex vitamins were a blend of vitamin A, vitamin C, vitamin E, and several other vitamins in a ratio of 5:8:5:2 (mass ratio).

[0070] Experimental design: 1000 pearl oysters were implanted in both the immunosuppressant group and the control group (conventional implantation method). The same team performed both pearl fragment preparation and implantation. The pearl cultivation period lasted 6 months, and the survival rate of the pearl oysters was recorded monthly.

[0071] Results: Compared with the control group, the survival rate of receptors in the immunosuppressant group was significantly higher.

[0072] Table 3. Survival rate statistics of pearl oysters in each group.

[0073]

[0074] Example 4

[0075] Time: Nucleus insertion on November 12, 2019. Mortality and survival rates of the recipient shellfish were recorded every 30 days.

[0076] Location: Bailong Village, Hepu County, Guangxi

[0077] A method for reducing immune rejection in xenotransplantation of marine pearl oysters, using penguin pearl oysters as donor oysters and Hepu pearl oysters as recipient oysters. The method includes the following steps:

[0078] S1. Dissolve asteroidin in cell treatment solution to prepare a 0.55% immunosuppressant solution.

[0079] S2. Prepare small pieces of the mantle membrane of the pearl oyster. Add mercurochrome solution to the surface of the small pieces of mantle membrane and soak them for 5 minutes. Use sterile absorbent cotton or sterile absorbent paper to absorb the excess mercurochrome solution from the small pieces of mantle membrane.

[0080] S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragments obtained in step S2 to fully wet them;

[0081] S4. Immerse the donor pearl nucleus in an immunosuppressant solution;

[0082] S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient's bead nucleus;

[0083] S6. Pearl breeding and cultivation shall be carried out using conventional methods.

[0084] The above cell treatment solution was prepared by dissolving 2% polyvinylpyrrolidone (the percentage is by volume) per liter of phosphate buffer, along with 4 g of glucose, 8 g of clam polysaccharide, 25 g of complex vitamins, 6 g of taurine, 8 g of yeast polysaccharide, 0.3 g of curcumin, 0.3 g of cefuroxime sodium, 0.002 g of nystatin, 1 g of streptomycin, 800,000 units of gentamicin, 1,000,000 units of penicillin, 0.04 g of norfloxacin, and 0.015 g of metronidazole.

[0085] The above phosphate buffer solution was prepared by dissolving 1.08 g of potassium dihydrogen phosphate, 1.08 g of potassium chloride, 25.22 g of sodium chloride, 0.45 g of sodium bicarbonate, and 0.15 g of disodium hydrogen phosphate per liter of double-distilled water, adjusting the pH to 7.1, and then pasteurizing for 30 minutes. The above-mentioned compound vitamin is a mixture of vitamin A, vitamin C, vitamin E, and several other vitamins in a ratio of 5:8:5:2, where the ratio is by mass.

[0086] Experimental design: 1000 pearl oysters were implanted in both the asteroidin group and the control group (conventional implantation method). The same team was responsible for both pearl fragment preparation and implantation. The pearl cultivation period lasted 6 months, and the survival rate of the pearl oysters was recorded monthly.

[0087] Results: Compared with the control group, the survival rate of receptors in the immunosuppressant group was significantly higher.

[0088] Table 4. Survival rate statistics of pearl oysters in each group.

[0089]

[0090] Based on the above examples, it can be concluded that in the xenotransplantation experiment of pearl oysters in Hepu, treating the donor and recipient oysters with immunosuppressants can significantly reduce the immune rejection reaction in the early stage of implantation, promote the formation of immune tolerance in the recipient oysters, reduce the nucleus expulsion rate and mortality rate of the recipient oysters, thereby improving pearl quality and yield.

[0091] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method of reducing the immunological rejection of a sea water pearl oyster xenograft, characterized by, Includes the following steps: S1. Asteroidin is dissolved in a cell treatment solution to obtain an immunosuppressant solution; the immunosuppressant solution contains 0.35%–0.55% asteroidin by volume; the cell treatment solution is prepared by dissolving 1%–2% polyvinylpyrrolidone, 2–4g glucose, 5–8g clam polysaccharide, 20–45g compound vitamins, 4–6g taurine, 4–8g yeast polysaccharide, 0.2–0.3g curcumin, 0.3g cefuroxime sodium, 0.002g nystatin, 1g streptomycin, 800,000 units gentamicin, 1,000,000 units penicillin, 0.04g norfloxacin, and 0.15g metronidazole in phosphate buffer per liter. S2. Prepare a small piece of mantle membrane from a donor mussel, and drip a mercurochrome solution onto the surface of the mantle membrane piece to fully wet it; the mercurochrome solution is mercurochrome powder dissolved in filtered seawater, with a concentration of 2%; S3. Then, the immunosuppressant solution is dripped onto the surface of the mantle fragments obtained in step S2 to fully wet them; S4. Immerse the donor pearl nucleus in an immunosuppressant solution; S5. The mantle fragments and bead nuclei obtained in steps S3 and S4 are implanted into the recipient's bead nucleus; S6. Conduct pearl farming.

2. The method of reducing the immunological rejection of a marine pearl oyster xenograft according to claim 1, wherein, The donor oyster is a large pearl oyster, a pearl oyster, or a penguin pearl oyster.

3. The method of reducing the immunological rejection of a marine pearl oyster xenograft according to claim 1, wherein, The recipient oyster is the Hepu pearl oyster.

4. The method for reducing immune rejection in xenotransplantation of marine pearl oysters according to claim 1, characterized in that, The phosphate buffer solution was prepared by dissolving 1.08 g of potassium dihydrogen phosphate, 1.08 g of potassium chloride, 25.22 g of sodium chloride, 0.45 g of sodium bicarbonate, and 0.15 g of disodium hydrogen phosphate in each liter of double-distilled water, adjusting the pH to 7.1, and then pasteurizing for 30 min.

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