CN115024279B - Construction method of zebra fish diabetic vasculopathy model - Google Patents
Construction method of zebra fish diabetic vasculopathy model Download PDFInfo
- Publication number
- CN115024279B CN115024279B CN202210883896.7A CN202210883896A CN115024279B CN 115024279 B CN115024279 B CN 115024279B CN 202210883896 A CN202210883896 A CN 202210883896A CN 115024279 B CN115024279 B CN 115024279B
- Authority
- CN
- China
- Prior art keywords
- zebra fish
- model
- solution
- vascular
- diabetic vasculopathy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 48
- 206010012601 diabetes mellitus Diseases 0.000 title claims abstract description 44
- 238000010276 construction Methods 0.000 title claims abstract description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 32
- 239000008103 glucose Substances 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 19
- 229930006000 Sucrose Natural products 0.000 claims abstract description 19
- 239000005720 sucrose Substances 0.000 claims abstract description 19
- 239000011259 mixed solution Substances 0.000 claims abstract description 17
- 239000005715 Fructose Substances 0.000 claims abstract description 7
- 229930091371 Fructose Natural products 0.000 claims abstract description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 7
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 7
- 208000019553 vascular disease Diseases 0.000 claims abstract description 7
- 230000004720 fertilization Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 11
- 230000002792 vascular Effects 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 6
- 230000035755 proliferation Effects 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 208000013600 Diabetic vascular disease Diseases 0.000 claims description 2
- 201000009101 diabetic angiopathy Diseases 0.000 claims description 2
- 208000014139 Retinal vascular disease Diseases 0.000 claims 1
- 201000002249 diabetic peripheral angiopathy Diseases 0.000 claims 1
- 231100000216 vascular lesion Toxicity 0.000 abstract description 5
- 210000004204 blood vessel Anatomy 0.000 description 20
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 210000000601 blood cell Anatomy 0.000 description 5
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 230000017531 blood circulation Effects 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 231100000915 pathological change Toxicity 0.000 description 4
- 230000036285 pathological change Effects 0.000 description 4
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 3
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 210000001210 retinal vessel Anatomy 0.000 description 3
- 101100540419 Danio rerio kdrl gene Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 230000002207 retinal effect Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 206010033307 Overweight Diseases 0.000 description 1
- 101710183548 Pyridoxal 5'-phosphate synthase subunit PdxS Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229940127003 anti-diabetic drug Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008289 pathophysiological mechanism Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Endocrinology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Diabetes (AREA)
- Animal Husbandry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of biotechnology model construction, and particularly discloses a construction method of a zebra fish diabetes vascular lesion model, which comprises the following steps: treating the zebra fish embryo which grows for 1d-5d after fertilization in the mixed solution for 30-96 h; the mixed solution is prepared from glucose solution and sucrose solution with mass fractions of 3-4% according to the volume ratio of 1:1, or prepared from glucose solution and fructose solution with mass fractions of 3-4% according to the volume ratio of 1:1. The construction method provided by the invention can observe obvious vascular disease phenotype after 1.5 days of treatment, and the model is quite stable, and the vascular disease rate can reach 100%.
Description
Technical Field
The invention belongs to the field of biotechnology model construction, and particularly discloses a construction method of a zebra fish diabetic vasculopathy model.
Background
With population growth, aging, overweight, obesity burden increase and the like, the global diabetes incidence rate rapidly increases, wherein the incidence rate of type 2 diabetes is the vast majority, and the macrovascular and microvascular lesions caused by type 2 diabetes also increase year by year, bring great pain to the body and mind of patients and families, and bring great burden to a medical care system. Diabetes Mellitus (DM) is characterized by an increasing level of glucose (blood glucose) in the blood, either because the pancreas is unable to secrete enough insulin to regulate blood glucose or because the body is unable to use the insulin it secretes effectively. Over time, serious damage to the heart, blood vessels, eyes, kidneys and nerves can result. Over 30% of diabetics suffer from microvascular and macrovascular complications, which are the leading cause of death worldwide. However, there is currently no good therapeutic agent for the treatment of diabetic complications.
Although there are some therapies for treating DM, such as insulin treatment for T1DM and T2DM and antidiabetic drugs, such as metformin and other drugs for T2DM, the incidence of complications such as diabetic retinopathy, nephropathy or neuropathy is still rising. In order to further improve the therapeutic efficiency and identify new disease mechanisms, various animal models have been established. Rats and mice therefore represent the most common animal model, and several important pathophysiological mechanisms have been identified in these animals, but with long periods and high costs. Moreover, these model organisms do not perfectly reflect the metabolic background of diabetics. To fill this gap, new animal models and new construction methods are needed. Therefore, it is necessary to construct a model for visual dynamic observation of diabetic vascular lesions.
The method utilizes the model organism zebra fish to construct a zebra fish diabetic vasculopathy model, and the cause of the diabetic vasculopathy can be known by observing the vasculopathy process in real time. The zebra fish diabetes model has the main advantages that: the zebra fish embryo is transparent throughout the body, and can carry out in vivo imaging observation on specific tissues and organs and changes thereof; the spawning quantity is high, the growth period is short, and a large number of experimental samples can be obtained; second, zebra fish pancreas is similar in structure to other animal pancreas, can secrete a variety of hormones including insulin, and sugar metabolism regulation mechanisms are similar to other mammals, which makes zebra fish very suitable for sugar metabolism regulation research. At present, a plurality of zebra fish blood vessel mutants and vascular fluorescence transgenic zebra fish strains are constructed by using a CRISPR/cas 9-system and a gene editing technology, so that the experimental process can be effectively simplified, and the method is very suitable for high-throughput screening of medicines.
The construction method of a diabetes model is reported by using zebra fish at present: culturing in 111mmol/L glucose culture medium for 14 days, knocking out Pdx1 (pancreas and duodenum homologous frame 1 transcription factor) genes, and continuously culturing in 4% glucose culture medium for 28 days.
Disclosure of Invention
In view of the above technical problems, the present invention provides the following technical solutions:
the invention provides a method for constructing a zebra fish diabetic vasculopathy model, which comprises the following steps: treating the zebra fish embryo which grows for 1d-5d after fertilization in the mixed solution for 30-96 h;
wherein the mixed solution is prepared from glucose solution and sucrose solution with mass fractions of 3-4% according to the volume ratio of 1:1, or prepared from glucose solution and fructose solution with mass fractions of 3-4% according to the volume ratio of 1:1.
Preferably, the development time is 2.5d.
Preferably, the mass fraction of the glucose solution and the sucrose or fructose solution is 3.5%.
Preferably, the treatment time is 30 to 36 hours.
The invention also provides the application of the zebra fish diabetic vasculopathy model obtained by the construction method in vascular disease research.
Preferably, the zebra fish diabetic vasculopathy model is used for developing diabetic vasculopathy medicines.
Preferably, the diabetic vascular disease is vascular obstruction and/or vascular proliferation.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the construction method provided by the invention, the zebra fish embryo which develops for 1d to 5d after fertilization is treated for 30 to 96 hours in the mixed solution of the glucose solution with the mass fraction of 3 to 5 percent and the sucrose/fructose solution with the mass fraction of 3 to 5 percent, so that the obvious vascular disease phenotype can be observed, the model is quite stable, and the vascular disease rate can reach 100 percent.
2. The construction method provided by the invention can cause the zebra fish retina blood vessel and branch blood vessel to have obvious lesions and obstruction, and the branch blood vessel can also be seen to have proliferated blood vessel.
Drawings
FIG. 1 is a zebra fish retinal vascular condition; A. green fluorescence; B. red fluorescence;
FIG. 2 is a diagram of a diabetic branch vascular lesion in zebra fish; A. green fluorescence; B. red fluorescence;
FIG. 3 is blood glucose concentration for the high sugar group and the control group; in comparison with the control group, *** P<0.001；
FIG. 4 shows glycosylated hemoglobin concentrations of high sugar group and control group; in comparison with the control group, ** P<0.01；
fig. 5 is nitric oxide concentrations for the high sugar group and the control group; in comparison with the control group, *** P<0.001；
FIG. 6 is triglyceride concentration in the high sugar group and the control group; in comparison with the control group, *** P<0.001。
Detailed Description
The invention will be further described in detail below by way of specific examples. The equipment and reagents used in the examples and test examples were commercially available unless otherwise specified. The specific embodiments described in these examples are intended to be illustrative only and are not intended to be limiting.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, times, percentages, and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.
Example 1
Construction of a Zebra fish diabetic vasculopathy model
Transgenic fish lines Tg (flk 1: GFP; gata1: dsRed) using vascular green fluorescence-specific markers and blood cell red fluorescence-specific markers were purchased from the China zebra fish resource center and were bred and embryo collected according to the protocol of the animal protection agency and committee. After spawning, the adult zebra fish is collected, cultured in a 28.5 ℃ incubator for 2.5 days, mixed liquid (3.5% high sugar for short) with the mass fraction of 3.5% glucose and the mass fraction of 3.5% sucrose which are equal in volume is added for treatment for 1.5d, confocal photographing is carried out for recording the obstruction condition of retinal blood vessels and branch blood vessels, and meanwhile, a treatment group without glucose and sucrose is used as a control group.
The retina was seen (fig. 1, right-most column in the figure refers to the case of blood in blood vessels, which is a superimposed graph of the two left-hand figures, flk1: GFP refers to green fluorescent-labeled blood vessels, i.e., part A in the figure, gata1: dsRed refers to red fluorescent-labeled blood cells, i.e., part B in the figure), the green fluorescent-labeled vascular endothelium was blurred in the high sugar group, red fluorescent-labeled red blood cells were substantially disappeared, indicating that obvious lesions and obstruction were present in the retinal blood vessels, leading to inability of blood circulation, and disappearance of retinal blood cells.
The branch vessels are visible when observed (fig. 2, the rightmost column in the figure refers to the condition of blood in the blood vessels, which is a superposition of the two left images, flk1: GFP refers to the blood vessels marked by green fluorescence, namely the A-referenced part in the figure, gata1: dsRed refers to the blood cells marked by red fluorescence, namely the B-referenced part in the figure), the blood vessels originally with unobstructed blood flow have obvious obstruction, and the blood cells cannot normally enter the branch vessels for transportation. And many proliferated blood vessels appeared after the high sugar treatment, indicating that a significant vasculopathy phenotype appeared after 3.5% of the high sugar treatment.
To further determine whether diabetes is or is not, the present invention detects a diabetes-related index: blood glucose concentration (Glucose concentration), glycosylated hemoglobin (HbA 1 c) level, nitric Oxide (NO) content, and Triglyceride (TG) content.
Figures 3-6 show that the blood glucose concentration is significantly increased, the glycosylated hemoglobin is significantly increased, the nitric oxide content is reduced, and the triglyceride content is increased, which is consistent with the relevant indexes of human diabetes, and can be determined as diabetes.
Example 2
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: embryos are collected after spawning and cultured in an incubator at 28.5 degrees celsius for 1.0 day.
Example 3
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of 3.0% glucose and 3.0% sucrose by volume for treatment for 4d.
Example 4
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of 3.0% glucose and 4.0% sucrose by volume for treatment for 4d.
Example 5
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: the mixture is treated for 4 days by adding equal volume mixing liquid of 3.0 percent of glucose and 3.5 percent of sucrose.
Example 6
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of equal volume mixing of glucose with mass fraction of 4% and sucrose with mass fraction of 4%, and treating for 1.5d.
Example 7
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of equal volume mixing of glucose 3% by mass and fructose 4% by mass for treatment for 1.5d.
Example 8
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of equal volume mixing of glucose with mass fraction of 4% and sucrose with mass fraction of 3%, and treating for 1.5d.
Example 9
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of 3.5% glucose by mass and 3.5% sucrose by mass in equal volume, and treating for 30h.
Example 10
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of 3.5% glucose and 3.5% sucrose by mass percentage in equal volume, and treating for 96h.
Comparative example 1
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: only 3.5% glucose by mass was added for 1.5d.
Comparative example 2
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: only 3.5% sucrose by mass fraction was added for 1.5d treatment.
Comparative example 3
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: only 2% by mass of glucose was added for treatment for 4d.
Comparative example 4
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: only 5% sucrose by mass was added for treatment for 4d.
Comparative example 5
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of equal volume mixing of glucose with mass fraction of 3% and sucrose with mass fraction of 5% for treatment for 1.5d.
Comparative example 6
The construction method of the zebra fish diabetic vasculopathy model is different from that of the embodiment 1 in that: adding mixed solution of 5% glucose by mass and 3% sucrose by mass in equal volume for treatment for 1.5d.
640 embryos collected after spawning of adult zebra fish are randomly divided into groups of examples 1-10 and comparative examples 1-6, 40 each. Each group of the zebra fish diabetic vasculopathy models are respectively constructed according to the corresponding method, after the completion, the pathological changes of the retinal blood vessels and branch blood vessels and the changes of the hyperplastic blood vessels of each group of the pathological changes models are observed by referring to the method described in the embodiment 1, and the pathological changes induction rate, the pathological changes severity and the death rate of each group of the vasculopathy are counted. The results are shown in Table 1.
TABLE 1 vascular lesion induction rates, vascular lesion severity and mortality for each group
Note that: unobstructed: normal blood flow and no obstruction of blood vessels; slightly: blood flow is slow and blood vessels are slightly obstructed. And (3) moderately: blood stops, obvious obstruction of blood vessels occurs, and new blood vessels are not many; serious: blood stops, vascular obstruction is severe, and many new blood vessels appear.
The foregoing disclosure is merely illustrative of specific embodiments of the invention, but the embodiments are not limited thereto and variations within the scope of the invention will be apparent to those skilled in the art.
Claims (7)
1. The method for constructing the zebra fish diabetic vasculopathy model is characterized by comprising the following steps of: treating the zebra fish embryo which grows for 1d-5d after fertilization in the mixed solution for 30-96 h;
wherein the mixed solution is prepared from glucose solution and sucrose solution with mass fractions of 3-4% according to the volume ratio of 1:1, or prepared from glucose solution and fructose solution with mass fractions of 3-4% according to the volume ratio of 1:1;
the constructed zebra fish diabetic vasculopathy model is used for developing medicines for improving retinal vascular diseases and branch vascular diseases or obstruction or medicines for improving branch vascular proliferation.
2. The method of claim 1, wherein the zebra fish embryo develops for 2.5 days before being treated in the mixed solution.
3. The method according to claim 1, wherein the mass fraction of the glucose solution, the sucrose solution and the fructose solution is 3.5%.
4. The method of claim 1, wherein the treatment time is 30 to 36 hours.
5. Use of the zebra fish diabetic vasculopathy model obtained by the construction method of any one of claims 1 to 4 in vascular disease research.
6. The use according to claim 5, wherein the zebra fish diabetic vasculopathy model is used for developing diabetic vasculopathy drugs.
7. The use according to claim 6, wherein the diabetic vascular disorder is vascular obstruction or vascular proliferation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210883896.7A CN115024279B (en) | 2022-07-26 | 2022-07-26 | Construction method of zebra fish diabetic vasculopathy model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210883896.7A CN115024279B (en) | 2022-07-26 | 2022-07-26 | Construction method of zebra fish diabetic vasculopathy model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115024279A CN115024279A (en) | 2022-09-09 |
| CN115024279B true CN115024279B (en) | 2024-04-12 |
Family
ID=83130954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210883896.7A Active CN115024279B (en) | 2022-07-26 | 2022-07-26 | Construction method of zebra fish diabetic vasculopathy model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115024279B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118355882A (en) * | 2024-05-23 | 2024-07-19 | 南通大学附属医院 | Construction method of zebra fish diabetic vasculopathy model |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003094610A1 (en) * | 2002-05-08 | 2003-11-20 | Angiogenetics Sweden Ab | Research models for the evaluation of retinopathy or microangiopathy and uses thereof |
| CN102812921A (en) * | 2012-09-04 | 2012-12-12 | 东北师范大学 | Method for establishing type 2 diabetes animal model and application of type 2 diabetes animal model in screening of blood sugar reducing medicaments |
| KR20140136402A (en) * | 2014-08-18 | 2014-11-28 | 광주과학기술원 | Methods for zebrafish -Based Screenig Diabetes Therapeutics |
| CN107929311A (en) * | 2017-12-19 | 2018-04-20 | 电子科技大学 | A kind of method for building up of diabetes B animal model |
| CN109481428A (en) * | 2018-11-20 | 2019-03-19 | 陕西中药研究所 | A kind of construction method of diabetic cardiomyopathy animal model |
| CN110714026A (en) * | 2019-10-22 | 2020-01-21 | 闽南师范大学 | Construction method and application of type II diabetic zebra fish model |
| CN112602668A (en) * | 2020-12-17 | 2021-04-06 | 四川大学华西医院 | Rat model construction method for proliferative diabetic retinopathy |
| CN113080108A (en) * | 2021-03-19 | 2021-07-09 | 温州大学 | Method for constructing diabetic zebra fish model |
| CN113491255A (en) * | 2021-06-16 | 2021-10-12 | 温州大学 | Construction method and application of obese type II diabetic zebra fish model |
| CN113736844A (en) * | 2021-08-03 | 2021-12-03 | 温州大学 | Method for evaluating developmental toxicity of high-concentration glucose by using zebra fish |
-
2022
- 2022-07-26 CN CN202210883896.7A patent/CN115024279B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003094610A1 (en) * | 2002-05-08 | 2003-11-20 | Angiogenetics Sweden Ab | Research models for the evaluation of retinopathy or microangiopathy and uses thereof |
| CN102812921A (en) * | 2012-09-04 | 2012-12-12 | 东北师范大学 | Method for establishing type 2 diabetes animal model and application of type 2 diabetes animal model in screening of blood sugar reducing medicaments |
| KR20140136402A (en) * | 2014-08-18 | 2014-11-28 | 광주과학기술원 | Methods for zebrafish -Based Screenig Diabetes Therapeutics |
| CN107929311A (en) * | 2017-12-19 | 2018-04-20 | 电子科技大学 | A kind of method for building up of diabetes B animal model |
| CN109481428A (en) * | 2018-11-20 | 2019-03-19 | 陕西中药研究所 | A kind of construction method of diabetic cardiomyopathy animal model |
| CN110714026A (en) * | 2019-10-22 | 2020-01-21 | 闽南师范大学 | Construction method and application of type II diabetic zebra fish model |
| CN112602668A (en) * | 2020-12-17 | 2021-04-06 | 四川大学华西医院 | Rat model construction method for proliferative diabetic retinopathy |
| CN113080108A (en) * | 2021-03-19 | 2021-07-09 | 温州大学 | Method for constructing diabetic zebra fish model |
| CN113491255A (en) * | 2021-06-16 | 2021-10-12 | 温州大学 | Construction method and application of obese type II diabetic zebra fish model |
| CN113736844A (en) * | 2021-08-03 | 2021-12-03 | 温州大学 | Method for evaluating developmental toxicity of high-concentration glucose by using zebra fish |
Non-Patent Citations (3)
| Title |
|---|
| 斑马鱼模型在糖尿病研究中的应用;王雪等;中国比较医学杂志;第27卷(第8期);1-4 * |
| 葡萄糖溶液对斑马鱼胚胎发育的影响;李俊霞;;安徽农业科学(第19期);全文 * |
| 高浓度葡萄糖对斑马鱼胚胎及多巴胺神经元发育的影响;吴永梅;杨琴;李淑蓉;苏炳银;;第三军医大学学报(第15期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115024279A (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Eames et al. | Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis | |
| CN115024279B (en) | Construction method of zebra fish diabetic vasculopathy model | |
| CN110302278A (en) | A kind of glutinous rehmannia excretion body and its preparation method and application | |
| CN114214364A (en) | Method for constructing stichopus japonicus gene editing system | |
| CN113491255B (en) | Construction method and application of obese type II diabetic zebra fish model | |
| CN112618570B (en) | Preparation method of graphene quantum dots and application of graphene quantum dots in preparation of medicine for treating non-alcoholic fatty liver disease | |
| TW201137354A (en) | Myelin basic protein luciferase (MBP-luci) bioimaging model | |
| CN108967358A (en) | Construction method and application of zebra fish F L tumor model | |
| Zhang et al. | Artificial infection pathway of largemouth bass LMBV and identification of resistant and susceptible individuals | |
| Khan | Animal biotechnology | |
| CN106456674A (en) | Transgenic pig islets and uses thereof for treating diabetes | |
| CN113100178A (en) | Method for establishing drosophila melanogaster tumor invasion model | |
| CN104059887A (en) | Application of long-chain non-coding RNA Ovol2-AS | |
| WO2019227882A1 (en) | Construction of mouse model with idiopathic basal ganglia calcification pathogenic gene mutation | |
| CN116602268B (en) | Application of gene knockout mutant zebra fish in preparation of animal model for reducing pigment | |
| Eichhorn-Mulligan et al. | The genetics of leber hereditary optic neuropathy—prototype of an inherited optic neuropathy with mitochondrial dysfunction | |
| Gautam et al. | Zebrafish as a Fascinating Animal Model: A Robust Platform for in vivo Screening for Biomedical Research. | |
| RU2766689C1 (en) | Use of lidocaine for anaesthesia of model organism danio rerio under experimental conditions | |
| Da Cunha et al. | Studies on cytology and differentiation in Sciaridae IV. Nuclear and cytoplasmatic differentiation in salivary glands of Bradysia elegans (Diptera, Sciaridae) | |
| CN114868707B (en) | Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application thereof | |
| Gardner et al. | Maternal effect transferred by injection and further analysis of temperature effective period for tumorous head in Drosophila melanogaster | |
| CN108841869A (en) | construction method and application of zebra fish NK/TC L tumor model | |
| US20080069805A1 (en) | Use of stem cells to cure genetic diseases in humans cure for sickle cell anemia | |
| CN116396965B (en) | Application of AHDC1 in construction of obese animal model, method and drug screening method | |
| CN113249383B (en) | Method for constructing Serac1 gene knockout mouse model |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |