Application of hemocyanin in DNA damage repair and cell proliferation
By using hemocyanin preparations to promote the proliferation and DNA repair of hepatopancreatic cells in shrimp, the problem of hepatopancreatic damage caused by external environmental factors in shrimp breeding was solved, which significantly reduced the tissue damage of shrimp and improved growth in fruit fly cell culture and proliferation ability.
Patent Information
- Application Number
- CN202411173429.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-26
- Publication Date
- 2025-01-10
AI Technical Summary
The hepatopancreatic damage caused by external environmental factors during shrimp farming is serious. The existing technology has failed to effectively reduce or repair this damage, resulting in death and economic losses of shrimp.
Hemocyanin is used as a preparation to promote cell growth and DNA damage repair. By adding it to the feed of shrimp or as an injection, it promotes the proliferation of shrimp hepatopancreatic cells and DNA repair and reduces tissue damage.
It significantly reduces the damage to the hepatopancreas of shrimp, improves the resistance of shrimp, and promotes cell growth and proliferation in Drosophila cell culture medium, demonstrating the effectiveness of hemocyanin in DNA damage repair and cell proliferation.
Smart Images
Figure CN119280384A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to an application of hemocyanin. Background Art
[0002] During shrimp farming, they are often subjected to the stress of pathogens (such as white spot syndrome virus, Taura virus, parasites, etc.), environmental factors (temperature, dissolved oxygen, salinity, etc.) and environmental pollutants (microplastics, nitrites, nonylphenol, heavy metals, etc.), which cause cell fragmentation, dissolution, and apoptosis, resulting in tissue damage to shrimp gills, stomachs, muscles, intestines, hepatopancreas, etc., leading to tissue dysfunction and ultimately causing shrimp death. Especially the impact of environmental factors on the hepatopancreas of shrimp, histopathological analysis shows that when the external environment changes dramatically, hepatopancreas tissues are infiltrated with blood cells, hepatopancreas tubular epithelial cells fall off and enter the lumen, and eventually the hepatopancreas is completely atrophied and necrotic, causing shrimp to die from hepatopancreatic dysfunction, causing economic losses of more than $7 billion per year to the shrimp farming industry worldwide. Therefore, in the current context, how to reduce or even repair hepatopancreas damage caused by the outside world is extremely urgent.
[0003] Hemocyanin is an oxygen-carrying protein that bears the heavy responsibility of oxygen transportation in the body. Its evolution is affected by the habitat. In the course of evolution that has lasted for hundreds of millions of years, due to the adaptability of the hemocyanin gene, it has evolved a variety of functions, such as agglutination activity, antibacterial activity, antiviral activity, etc., to adapt to the body's energy needs and to cope with changes in various biological and non-biological environmental factors. However, there is no report in the prior art that hemocyanin promotes cell proliferation and reduces tissue damage. For this reason, actively carrying out research in this area will help to find potential targets for preventing tissue damage, which has important theoretical significance and potential application value for achieving healthy shrimp farming. Summary of the invention
[0004] The purpose of the present invention is to provide a new method for reducing or even repairing the hepatopancreas damage of shrimp caused by external factors.
[0005] The following technical solutions are adopted:
[0006] An application of hemocyanin for preparing a preparation for promoting cell growth.
[0007] Preferably, the invention is used for preparing a preparation for promoting cell cycle progression.
[0008] Preferably, the invention is used for preparing a preparation for preventing or repairing DNA damage.
[0009] Preferably, it is used for preparing a preparation for reducing organ damage.
[0010] Preferably, the organ comprises the hepatopancreas.
[0011] Application of hemocyanin in DNA damage repair and cell proliferation, application of hemocyanin in DNA damage repair and proliferation of crustacean cells, reducing tissue damage.
[0012] A preparation obtained by using the above hemocyanin.
[0013] Preferably, the preparation comprises one or more of a medicine, a feed, and a cell culture medium.
[0014] Preferably, the preparation comprises one or more of a medicine and a feed for crustaceans; the medicine comprises an injection.
[0015] Preferably, the crustacean includes prawns; and the ratio of the added amount of the hemocyanin to the body weight of the crustacean is 25-200 μg / 6g.
[0016] The invention provides a protein hemocyanin, which can promote the repair of DNA damage and cell proliferation in Litopenaeus vannamei cells and reduce the tissue damage of shrimp. The added dosage is 25-200 μg / 6g.
[0017] Furthermore, the above products include injections, which can be used for shrimps and crabs, especially for promoting the proliferation of hepatopancreatic cells and blood cells of shrimps, and reducing tissue damage.
[0018] Preferably, the cell culture medium comprises a culture medium for culturing Drosophila cells; the ratio of the amount of hemocyanin per milliliter of the cell culture medium to the number of Drosophila cells comprises 20 to 60 μg / 10 6 indivual.
[0019] The application of hemocyanin in DNA damage repair and cell proliferation. Hemocyanin can promote cell growth and proliferation as an additive to Drosophila S2 cell culture racks.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The invention is based on hemocyanin which promotes cell DNA damage repair and proliferation and reduces tissue damage. It can be added to feed as an additive to improve the ability of shrimp to resist tissue damage. It can also be added to cell culture medium as an additive to improve the growth and proliferation of fruit fly S2 cells.
[0022] As an oxygen-carrying protein, hemocyanin can be used as an additive to prepare a medicament for promoting cell growth. The hemocyanin of the present invention can repair DNA damage, promote cell cycle, and thus enhance cell proliferation ability, and play a role in reducing tissue damage. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1A Analysis diagram of the interference results of hemocyanin; Figure 1 B Analysis of the effect of interfering hemocyanin on hepatopancreatic damage.
[0024] Figure 2 A heat map analysis of the effects of interfering with hemocyanin on genes related to DNA damage; Figure 2 B Analysis of the effect of interfering hemocyanin on DNA damage; Figure 2 C Statistical analysis of the effect of hemocyanin interference on DNA damage; Figure 2 D is the statistical analysis result of the tail distance of DNA damage caused by interfering hemocyanin.
[0025] Figure 3 A is a diagram analyzing the effects of interfering with hemocyanin and replenishing hemocyanin on DNA damage; Figure 3 B. Statistical analysis of the effects of interfering with hemocyanin and replenishing hemocyanin on the induced DNA damage; Figure 3 C is the statistical analysis result of interfering with hemocyanin and replenishing the tail distance of DNA damage caused by hemocyanin.
[0026] Figure 4 A Analysis of the effect of interfering with hemocyanin on the proliferation of shrimp hepatopancreatic cells; Figure 4 B Analysis of the effect of interfering hemocyanin on the hepatopancreatic cell cycle; Figure 4 C Analysis of the effect of hemocyanin interference followed by hemocyanin replenishment on the hepatopancreatic cell cycle.
[0027] Figure 5 Analysis of the effects of blood interference and hemocyanin supplementation on hepatopancreas damage in shrimp.
[0028] Figure 6 A Analysis of the effect of interfering with hemocyanin on the proliferation of shrimp blood cells; Figure 6 B is the analysis of the effect of hemocyanin interference on the expression of genes related to the blood cell cycle.
[0029] Figure 7 A Analysis of the effect of hemocyanin on the growth and proliferation of Drosophila S2 cells; Figure 7 Statistical analysis of the effect of B hemocyanin on the growth and proliferation of Drosophila S2 cells. DETAILED DESCRIPTION
[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.
[0031] The hemocyanin (HMC) used in the present invention can be extracted from plasma, such as the plasma of Penaeus vannamei, or can be directly purchased from commercial products, such as those purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., product number CAS: 9013-72-3.
[0032] Example 1: Effect of interfering with hemocyanin on hepatopancreas damage in shrimp
[0033] (1) Inject dsHMC to interfere with hemocyanin, and collect hepatopancreas tissues at 48 h. Use RNAfast2000 total RNA extraction kit (Shanghai Feijie) and strictly follow the instructions to extract shrimp hepatopancreas RNA. Then use the reverse transcription kit One-step gDNA Removal and cDNA Synthseis SuperMix (Beijing Quanshijin) Instructions: The total RNA of the liver and pancreas prepared in the above step was reverse transcribed into cDNA. Finally, the interference of hemocyanin was detected by fluorescence quantitative PCR technology. The results are as follows: Figure 1 As shown in A, the hemocyanin gene was significantly down-regulated.
[0034] (2) The shrimp were treated according to the conditions of Example 1(1), and the hepatopancreas tissues of 5 Penaeus vannamei were randomly selected at 24 and 48 hours and sent to Wuhan Bios Biotechnology for pathological section analysis. Figure 1 As shown in B, compared with the control group, the hepatopancreas infiltration of blood cells, hepatic tubule atrophy and necrosis in the shrimp hepatopancreas of the hemocyanin interference group were more serious.
[0035] Example 2: Effects of interfering with hemocyanin on the expression level of DNA damage repair genes and DNA damage in the hepatopancreas of shrimp
[0036] (1) Shrimp were treated according to the conditions of Example 1(1) to obtain total RNA samples of the hepatopancreas, which were then sent to Shanghai Meiji Biotechnology for transcriptome sequencing. The results showed that compared with the control group, the genes involved in DNA damage repair were significantly reduced after interference with hemocyanin, such as Figure 2 As shown in A.
[0037] (2) Shrimp were treated according to Example 1 (1), and hepatopancreatic cells were collected and transferred into a 1.5 mL sterile centrifuge tube. 500 μL of pre-cooled sterile phosphate buffered saline (PBS) (0.01 M, pH 7.4) was added. The hepatopancreas was gently squeezed with a grinding rod to allow its own pancreatic enzymes to separate the hepatopancreatic cells. Then, the hepatopancreatic cells were washed three times with 500 μL of pre-cooled sterile PBS and filtered through a flow tube. The hepatopancreatic cells in PBS were obtained for comet electrophoresis experiments in strict accordance with the instructions of the comet electrophoresis kit (Biyuntian, catalog number: C2041S). The results showed that interference with hemocyanin led to increased DNA damage, such as Figure 2 As shown in BD.
[0038] Example 3: Effects of hemocyanin supplementation after interference with hemocyanin on DNA damage in the hepatopancreas of shrimp
[0039] Hemocyanin was interfered by injection of dsHMC, and hemocyanin was replenished after 48 hours. The control group was replenished with bovine serum albumin (BSA), and then 5 shrimps were randomly selected to obtain hepatopancreatic tissue samples. Hepatopancreatic cells were obtained according to the steps in Implementation Case 2 (2), and then comet electrophoresis experiments were performed in strict accordance with the instructions of the comet electrophoresis kit (Biyuntian, catalog number: C2041S). The results showed that the replenishment of hemocyanin after interference with hemocyanin could significantly inhibit DNA damage ( Figure 3 A) Reduce DNA damage rate ( Figure 3 B) Inhibition of DAN damage tail distance ( Figure 3 C).
[0040] Example 4: Effects of interfering with hemocyanin on the proliferation and cell cycle of shrimp hepatopancreatic cells
[0041] (1) Inject dsHMC to interfere with hemocyanin, and inject shrimp with EdU in the cell proliferation detection kit (Cat. No.: C0071S, Biyuntian) to mark the proliferating cells. After 48 hours, randomly select 5 shrimps to obtain hepatopancreatic tissue and blood cell samples. According to the steps in Implementation Case 2 (2), hepatopancreatic cells were obtained, DNA was stained with Hocest33342, and then observed and analyzed by laser confocal microscopy. The results are shown in Figure 2. Figure 4 As shown in A, interference with hemocyanin significantly inhibited the proliferation of shrimp hepatopancreatic cells.
[0042] (2) After interfering with hemocyanin, hepatopancreatic cells were obtained according to the steps of Example 2 (2), DNA was stained with Hocest33342, and then analyzed by flow cytometry. The results showed that after interfering with hemocyanin, the cell cycle was inhibited, such as Figure 4 As shown in B.
[0043] Example 5: Effects of interference and replenishment of hemocyanin on the hepatopancreatic cell cycle of shrimp
[0044] After interfering with hemocyanin, hemocyanin was replenished, and then hepatopancreatic cells were obtained according to the steps of Example 2 (2). DNA was stained with Hocest33342 and then analyzed by flow cytometry. The results showed that after interfering with hemocyanin, hemocyanin was replenished (25-200 μg / shrimp, shrimp weighing 6 g) significantly promoted the cell cycle process, such as Figure 4 As shown in C.
[0045] Example 6: Analysis of shrimp hepatopancreas damage based on interference and supplementation of hemocyanin
[0046] The shrimp were treated in the same way as in Example 5. Five shrimps were randomly selected at 48 hours to collect hepatopancreas tissues and sent to Wuhan Bios Biotech for pathological section analysis. Figure 5 As shown, compared with the control group, the interference and supplementation of hemocyanin group significantly inhibited the pathological characteristics of shrimp hepatopancreas duct atrophy, necrosis and so on.
[0047] Example 7: Effects of interfering with hemocyanin on shrimp hemocyte proliferation and expression of proliferation-related genes
[0048] 48 hours after the injection of dsHMC, shrimp blood cells were collected and RNA was extracted from shrimp blood cells using RNAfast2000 total RNA extraction kit (Shanghai Feijie) in strict accordance with the instructions. One-step gDNARemoval and cDNASynthseis SuperMix (Beijing Quanshijin) Instructions The total RNA of blood cells prepared in the above step was reverse transcribed into cDNA. Finally, the expression of cell cycle-related genes (CTSL, GAPDH, β-actin, ANT2, HPCAS, CyclA, CyclB, CDK2, SPDY) was measured by fluorescence quantitative PCR. The results showed that after 48 hours of injection of dsHMC, shrimp significantly inhibited blood cell proliferation, such as Figure 6 As shown in A, cell proliferation-related genes were significantly downregulated, such as Figure 6 As shown in B.
[0049] Example 8: Effects of hemocyanin on the growth and proliferation of Drosophila S2 cells
[0050] Spread the activated Drosophila S2 cells in a 6-well cell plate, 1 ml per well, about 10 6 The cells were treated with 20 and 60 μg of hemocyanin, and the control group was added with the same dose of bovine serum albumin (BSA). At the same time, the proliferating cells were labeled with EdU in the cell proliferation detection kit (Cat. No. C0071S, Bio-Tech), and the cells were collected 24 hours later. The Drosophila S2 cells were treated according to the instructions of the cell proliferation detection kit and analyzed by flow cytometry. The results showed that the addition of hemocyanin promoted the growth and proliferation of Drosophila S2 cells ( Figure 7 A), and statistical analysis showed that the effect was significant ( Figure 7 B).
[0051] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.
Claims
1. An application of hemocyanin, characterized in that: Used to prepare preparations that promote cell growth.
2. The use of hemocyanin according to claim 1, characterized in that: Used to prepare preparations that promote cell cycle progression.
3. The use of hemocyanin according to claim 1, characterized in that: Used to prepare preparations for preventing or repairing DNA damage.
4. The use of hemocyanin according to claim 1, characterized in that: Used to prepare preparations for reducing organ damage.
5. The use of hemocyanin according to claim 4, characterized in that: The organs include the hepatopancreas.
6. A preparation obtained by using the hemocyanin according to claim 1.
7. The preparation according to claim 6, characterized in that The preparation includes one or more of a medicine, a feed, and a cell culture medium.
8. The preparation according to claim 7, characterized in that The preparation comprises one or more of a medicine and a feed for crustaceans; the medicine comprises an injection.
9. The preparation according to claim 7, characterized in that The crustacean includes prawns; the ratio of the added amount of the hemocyanin to the weight of the crustacean includes 25-200 μg / 6g.
10. The preparation according to claim 7, characterized in that: The cell culture medium includes a culture medium for culturing Drosophila cells; the ratio of the amount of hemocyanin per milliliter of the cell culture medium to the number of Drosophila cells is 20 to 60 μg / 10 6 indivual.