CN111328920A - Feed for constructing non-alcoholic steatohepatitis model of non-human primate and using method thereof - Google Patents
Feed for constructing non-alcoholic steatohepatitis model of non-human primate and using method thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
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- A23K10/22—Animal feeding-stuffs from material of animal origin from fish
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- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate and a using method thereof, wherein the feed comprises the following raw materials in parts by weight: 22-28% of soybean meal; 2.5 to 3.5 percent of Peru fish meal; 2-3% of feed grade calcium hydrophosphate; 0.2-0.4% of salt; 0.1-0.3% of compound multivitamins; high-stability VC accounts for 0.1-0.3%; 0.4-0.6% of cholesterol; 20-25% of single crystal fructose; 15-20% of animal fat; 0.2-0.4% of methionine; 1-2% of premixed mineral; 30-35% of first-grade flour, obviously improves the single index scores in the non-alcoholic fatty liver disease scoring system standard and the fibrosis scoring standard, and can establish non-alcoholic fatty liver injury after being fed for 3 months, thereby becoming a non-alcoholic fatty liver animal model.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate and a using method thereof.
Background
As a high incidence country of liver diseases, china imposes a serious economic and social burden on the country and society of patients with liver diseases. The data show that the total medicine scale of the liver diseases in 2016 is over 500 hundred million yuan. Nonalcoholic steatohepatitis (NASH), an extremely advanced form of nonalcoholic fatty liver disease, is defined as the appearance of a steatosis phenomenon accompanied by inflammation and hepatocellular injury. NASH can lead to late stage liver fibrosis, cirrhosis, liver failure, and the development of liver tumors. NAFLD has doubled its incidence over the last 20 years and is now the most common liver disease in china and western countries. Currently, there is no drug for treating NASH and liver cirrhosis, mainly due to the lack of a new drug research in an animal model that can accurately and effectively mimic the mechanism of NASH in humans. The gap in drug treatment of the disease has led to end-stage patients seeking liver transplantation only. The fat tissue metabolism and distribution of non-human primates, including rhesus monkey and cynomolgus monkey, are very similar to those of human beings, and are the best model for researching metabolic system diseases. In addition, the abdominal fat cell metabolism of the cynomolgus monkey is very similar to the fat cell metabolism of a human body, and is a reliable model for researching human fat metabolic diseases. This is incomparable and replaceable by other rodents, canines, etc.
Disclosure of Invention
The invention provides a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate and a using method thereof.
The scheme of the invention is as follows:
the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate is characterized by comprising the following raw materials in parts by weight:
the preferable technical scheme comprises the following raw materials in parts by weight:
the invention also provides a use method of the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate, which comprises the following steps:
1) non-human primate screening: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists;
2) preparing a feed: stirring and mixing 22-28% by weight of soybean meal, 2.5-6% by weight of Peru fish meal, 2-3% by weight of feed grade calcium hydrophosphate, 0.2-0.4% by weight of salt, 0.1-0.3% by weight of compound multivitamins, 0.1-0.3% by weight of high-stability VC, 0.4-0.6% by weight of cholesterol, 20-25% by weight of single crystal fructose, 15-20% by weight of animal fat, 0.2-0.4% by weight of methionine, 1-2% by weight of premixed mineral and 30-35% by weight of primary flour to obtain a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate;
3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 40-70%, the indoor temperature is 18-26 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months;
4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively;
5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system;
6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection;
7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
Preferably, the animal of step 1) is a cynomolgus monkey.
As a preferred technical scheme, animals continuously fed for three months are anesthetized with ketamine in the step 4).
As a preferred technical scheme, in the step 5), the non-alcoholic fatty liver disease scoring system standard detection:
1) observing the HE stained sections under microscope magnification;
2) then carrying out fatty degeneration, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection on the liver respectively;
3) in the step 2), each item of steatosis, hepatocyte injury/ballooning lesion and lobular inflammation detection meets the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system reaches the scoring index; any one of steatosis, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection in the step 2) cannot meet the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system cannot meet the scoring index.
According to a preferable technical scheme, the fatty degeneration area in the fatty degeneration detection is more than or equal to 5%, the fatty degeneration detection is qualified, the fatty degeneration area is less than 5%, the fatty degeneration detection is unqualified, and the fatty degeneration area is the ratio of the surface area involved in the fatty degeneration in the medium-low power detection; the condition that the liver cells with balloon-like lesions appear in the liver cell damage/balloon-like lesions or are positioned at the boundary of the liver cell damage and the balloon-like lesions is met, the liver cell damage/balloon-like lesions are qualified, the liver cell damage/balloon-like lesions are not found, and the liver cell damage/balloon-like lesions are unqualified; the lobular inflammation of the liver appears in focus, the lobular inflammation of the liver is qualified, the lobular inflammation of the liver does not appear in focus, and the lobular inflammation of the liver is unqualified.
As a preferred technical solution, the liver fibrosis scoring standard is:
1) magnifying the sirius red stained section by 200 times under a microscope;
2) showing no fibrotic features, the liver fibrosis scoring standard is not qualified;
3) the liver fibrosis characteristics appear, and the liver fibrosis scoring standard is qualified.
Preferably, the fibrosis features include peri-sinus or peri-portal fibrosis, peri-sinus and peri-portal/portal fibrosis, bridging fibrosis and cirrhosis.
Due to the adoption of the technical scheme, the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate and the use method thereof comprise the following steps of 1) screening the non-human primate: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists; 2) preparing a feed: stirring and mixing 22-28% by weight of soybean meal, 2.5-6% by weight of Peru fish meal, 2-3% by weight of feed grade calcium hydrophosphate, 0.2-0.4% by weight of salt, 0.1-0.3% by weight of compound multivitamins, 0.1-0.3% by weight of high-stability VC, 0.4-0.6% by weight of cholesterol, 20-25% by weight of single crystal fructose, 15-20% by weight of animal fat, 0.2-0.4% by weight of methionine, 1-2% by weight of premixed mineral and 30-35% by weight of primary flour to obtain a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate; 3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 40-70%, the indoor temperature is 18-26 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months; 4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively; 5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system; 6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection; 7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
The invention has the advantages that:
after the feed is fed for three months, the scores of single indexes in the non-alcoholic fatty liver disease scoring system standard and the fibrosis scoring standard are obviously improved, and the non-alcoholic fatty liver injury can be established after the feed is fed for 3 months.
Drawings
FIG. 1 is a photograph of an HE stained section of a non-alcoholic fatty liver of an animal model cynomolgus monkey at a magnification of 100 times under a microscope;
FIG. 2 is a 400-fold microscopic photograph of a HE stained section of a cynomolgus monkey nonalcoholic fatty liver disease in an animal model, with steatosis (score 3), ballooning lesion (score 2), lobular inflammation of the liver (score 3), and NAS integration score 8, wherein the top arrow indicates ballooning lesion, the middle arrow indicates lobular inflammation of the liver, and the bottom arrow indicates steatosis;
FIG. 3 is a graph of the periantral or periportal fibrosis under a standard HE staining section microscope for fibrosis scoring (score 1; fibrosis occurring periantral or periportal; photomicrographs: sirius red chromosome; × 10 fold and × 20 fold);
FIG. 4 is a perisinus and portal vein/portal vein peripheral map under a standard HE staining sectioning microscope for fibrosis scoring (fibrosis score 2; perisinus and portal vein/portal vein periphery; photomicrograph: sirius red chromosome; × 20 fold);
FIG. 5 is a graph of bridging fibrosis under the standard HE staining section microscope for fibrosis scoring (fibrosis score 3, bridging fibrosis; photomicrograph: sirius red chromosome; × 10 fold);
FIG. 6 is a photograph of cirrhosis under a microscope of standard HE stained sections for fibrosis scoring (fibrosis score 4, cirrhosis; photomicrograph: sirius red chromosome; × 10 fold);
FIG. 7 is a statistical chart of the steatosis score of HE stained sections of 214 animals in example 3;
FIG. 8 is a statistical chart of balloon lesion scoring of HE stained sections of 214 animals in example 3;
FIG. 9 is a statistical chart of small liver inflammation of HE stained sections of 214 animals in example 3;
FIG. 10 is a statistical graph of NAS scores of 214 animals in example 3;
FIG. 11 is a statistical chart of the scores of fibrosis in 214 animals in example 3;
FIG. 12 is a statistical chart of the combined scores for animal HAS and animal fibrosis of 214 cases in example 3;
Detailed Description
In order to make up for the defects, the invention provides a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate and a using method thereof so as to solve the problems in the background technology.
The feed for constructing the non-alcoholic steatohepatitis model of the non-human primate is characterized by comprising the following raw materials in parts by weight:
the preferable technical scheme comprises the following raw materials in parts by weight:
the invention also provides a use method of the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate, which comprises the following steps:
1) non-human primate screening: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists;
2) preparing a feed: stirring and mixing 22-28% by weight of soybean meal, 2.5-6% by weight of Peru fish meal, 2-3% by weight of feed grade calcium hydrophosphate, 0.2-0.4% by weight of salt, 0.1-0.3% by weight of compound multivitamins, 0.1-0.3% by weight of high-stability VC, 0.4-0.6% by weight of cholesterol, 20-25% by weight of single crystal fructose, 15-20% by weight of animal fat, 0.2-0.4% by weight of methionine, 1-2% by weight of premixed mineral and 30-35% by weight of primary flour to obtain a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate;
3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 40-70%, the indoor temperature is 18-26 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months;
4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively;
5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system;
6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection;
7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
Preferably, the animal of step 1) is a cynomolgus monkey.
As a preferred technical scheme, animals continuously fed for three months are anesthetized with ketamine in the step 4).
As a preferred technical scheme, in the step 5), the non-alcoholic fatty liver disease scoring system standard detection:
1) observing the HE stained sections under microscope magnification;
2) then carrying out fatty degeneration, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection on the liver respectively;
3) in the step 2), each item of steatosis, hepatocyte injury/ballooning lesion and lobular inflammation detection meets the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system reaches the scoring index; any one of steatosis, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection in the step 2) cannot meet the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system cannot meet the scoring index.
According to a preferable technical scheme, the fatty degeneration area in the fatty degeneration detection is more than or equal to 5%, the fatty degeneration detection is qualified, the fatty degeneration area is less than 5%, the fatty degeneration detection is unqualified, and the fatty degeneration area is the ratio of the surface area involved in the fatty degeneration in the medium-low power detection; the condition that the liver cells with balloon-like lesions appear in the liver cell damage/balloon-like lesions or are positioned at the boundary of the liver cell damage and the balloon-like lesions is met, the liver cell damage/balloon-like lesions are qualified, the liver cell damage/balloon-like lesions are not found, and the liver cell damage/balloon-like lesions are unqualified; the lobular inflammation of the liver appears in focus, the lobular inflammation of the liver is qualified, the lobular inflammation of the liver does not appear in focus, and the lobular inflammation of the liver is unqualified.
As a preferred technical solution, the liver fibrosis scoring standard is:
1) magnifying the sirius red stained section by 200 times under a microscope;
2) showing no fibrotic features, the liver fibrosis scoring standard is not qualified;
3) the liver fibrosis characteristics appear, and the liver fibrosis scoring standard is qualified.
Preferably, the fibrosis features include peri-sinus or peri-portal fibrosis, peri-sinus and peri-portal/portal fibrosis, bridging fibrosis and cirrhosis.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1:
1) non-human primate screening: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists;
2) preparing a feed: stirring and mixing 22% by weight of soybean meal, 2.5% by weight of Peru fish meal, 2% by weight of feed grade calcium hydrophosphate, 0.2% by weight of salt, 0.1% by weight of compound multivitamins, 0.1% by weight of high-stability VC, 0.4% by weight of cholesterol, 20% by weight of single crystal fructose, 15% by weight of animal fat, 0.2% by weight of methionine, 1% by weight of premixed mineral and 30% by weight of primary flour to obtain the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate;
3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 40%, the indoor temperature is 18 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months;
4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively;
5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system;
6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection;
7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
The animal of (a) is a cynomolgus monkey.
In said step 4) animals continuously fed for three months are anesthetized with ketamine.
The standard detection of the non-alcoholic fatty liver disease scoring system in the step 5):
1) observing the HE stained sections under microscope magnification;
2) then carrying out fatty degeneration, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection on the liver respectively;
3) in the step 2), each item of steatosis, hepatocyte injury/ballooning lesion and lobular inflammation detection meets the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system reaches the scoring index; any one of steatosis, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection in the step 2) cannot meet the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system cannot meet the scoring index.
According to a preferable technical scheme, the fatty degeneration area in the fatty degeneration detection is more than or equal to 5%, the fatty degeneration detection is qualified, the fatty degeneration area is less than 5%, the fatty degeneration detection is unqualified, and the fatty degeneration area is the ratio of the surface area involved in the fatty degeneration in the medium-low power detection; the condition that the liver cells with balloon-like lesions appear in the liver cell damage/balloon-like lesions or are positioned at the boundary of the liver cell damage and the balloon-like lesions is met, the liver cell damage/balloon-like lesions are qualified, the liver cell damage/balloon-like lesions are not found, and the liver cell damage/balloon-like lesions are unqualified; the lobular inflammation of the liver appears in focus, the lobular inflammation of the liver is qualified, the lobular inflammation of the liver does not appear in focus, and the lobular inflammation of the liver is unqualified.
The liver fibrosis scoring standard is as follows:
1) magnifying the sirius red stained section by 200 times under a microscope;
2) showing no fibrotic features, the liver fibrosis scoring standard is not qualified;
3) the liver fibrosis characteristics appear, and the liver fibrosis scoring standard is qualified.
The fibrotic features include peri-sinus or periportal fibrosis, peri-sinus and periportal/portal fibrosis, bridging fibrosis and cirrhosis.
Example 2:
1) non-human primate screening: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists;
2) preparing a feed: stirring and mixing 28 weight percent of soybean meal, 6 weight percent of Peru fish meal, 3 weight percent of feed grade calcium hydrophosphate, 0.4 weight percent of salt, 0.3 weight percent of compound multivitamins, 0.3 weight percent of high-stability VC, 0.6 weight percent of cholesterol, 25 weight percent of single crystal fructose, 20 weight percent of animal fat, 0.4 weight percent of methionine, 2 weight percent of premixed mineral and 35 weight percent of first grade flour to obtain the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate;
3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 70%, the indoor temperature is 26 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months;
4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively;
5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system;
6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection;
7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
The animals in the step 1) are cynomolgus monkeys.
In said step 4) animals continuously fed for three months are anesthetized with ketamine.
The standard detection of the non-alcoholic fatty liver disease scoring system in the step 5):
1) observing the HE stained sections under microscope magnification;
2) then carrying out fatty degeneration, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection on the liver respectively;
3) in the step 2), each item of steatosis, hepatocyte injury/ballooning lesion and lobular inflammation detection meets the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system reaches the scoring index; any one of steatosis, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection in the step 2) cannot meet the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system cannot meet the scoring index.
The fatty degeneration area in the fatty degeneration detection is more than or equal to 5 percent, the fatty degeneration detection is qualified, the fatty degeneration area is less than 5 percent, the fatty degeneration detection is unqualified, and the fatty degeneration area is the ratio of the surface area involved in the fatty degeneration in the medium-low power detection; the condition that the liver cells with balloon-like lesions appear in the liver cell damage/balloon-like lesions or are positioned at the boundary of the liver cell damage and the balloon-like lesions is met, the liver cell damage/balloon-like lesions are qualified, the liver cell damage/balloon-like lesions are not found, and the liver cell damage/balloon-like lesions are unqualified; the lobular inflammation of the liver appears in focus, the lobular inflammation of the liver is qualified, the lobular inflammation of the liver does not appear in focus, and the lobular inflammation of the liver is unqualified.
The liver fibrosis scoring standard is as follows:
1) magnifying the sirius red stained section by 200 times under a microscope;
2) showing no fibrotic features, the liver fibrosis scoring standard is not qualified;
3) the liver fibrosis characteristics appear, and the liver fibrosis scoring standard is qualified.
The fibrotic features include peri-sinus or periportal fibrosis, peri-sinus and periportal/portal fibrosis, bridging fibrosis and cirrhosis.
Example 3:
HE stained sections were used for NAS (NAFLD Activity Score non-alcoholic fatty liver disease Score) scoring, standard animal model HE stained sections are shown in fig. 1 and 2, histopathological lesions in fig. 2: steatosis (score 3), balloon-like lesion (score 2), liver lobular inflammation (score 3), NAS composite score 8, top arrow in the figure indicates balloon-like lesion, middle arrow in the figure indicates liver lobular inflammation, bottom arrow in the figure indicates steatosis;
preparing a feed: stirring and mixing 25 weight percent of soybean meal, 3 weight percent of Peru fish meal, 2.5 weight percent of feed grade calcium hydrophosphate, 0.3 weight percent of salt, 0.2 weight percent of compound multivitamins, 0.2 weight percent of high-stability VC, 0.5 weight percent of cholesterol, 20 weight percent of single crystal fructose, 15 weight percent of animal fat, 0.3 weight percent of methionine, 1 weight percent of premixed mineral and 32 weight percent of primary flour to obtain the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate.
Firstly, screening standards of experimental animals selected according to the item, animal species (cynomolgus monkey), number (214), size and body weight (7.5 KG-14 KG), age (11-18)
Variety: number of cynomolgus animals: 214 is only
The animal condition of the selected item is as follows:
1 weight: not less than 7.5kg
Age 2: not less than 10 years old
3, health condition: normal liver and kidney function
Reference index:
alanine Aminotransferase (ALT) less than or equal to 40(U/L)
Alanine Aminotransferase (AST) less than or equal to 40(U/L)
Myo-hepatic (CREA) less than or equal to 100(umol/L)
UREA (UREA) less than or equal to 8(mmol/L)
4 selected animals were not fed other inducers.
Secondly, the experimental method steps (firstly, secondly, finally, whether environmental factors such as temperature, humidity, illumination and the like, feeding amount and feeding time are involved in the process)
The selected animals were fed with the feed prepared in this example under the following conditions.
Feeding conditions are as follows: feeding in a single cage;
controlling the indoor humidity to be 40% -70%;
controlling the temperature in the room: 18-26 ℃;
the illumination condition is as follows: 12 hours light +12 hours dark.
Feeding mode: 50g feed/mouse in the morning;
150g of fruits per fruit at noon;
100g feed/mouse in the afternoon.
Thirdly, experimental contents to be collected and indexes for determining experimental results
After three months of continuously fed cynomolgus monkeys are anesthetized by ketamine, liver puncture is carried out under the guidance of B ultrasonic to collect biopsy tissues, and HE staining and sirius red staining are carried out on the collected liver tissues.
Example 3 three-month-old cynomolgus monkeys were fed the following non-alcoholic fatty liver disease scoring system (NAS)
H & E staining, magnification × 200:
1. fatty degeneration scoring
0: 5% -refers to the surface area ratio involved in the presence of steatosis in the mid-low power examination; an area of steatosis of < 5% was scored as 0 points to avoid over-sampling in the case of little steatosis.
1 minute: the fat deformation accounts for 5-33%;
and 2, dividing: the percentage of fat deformation is 33-66%;
3, the ratio of fat deformation is more than 67 percent;
2. grading of hepatocyte injury/balloon-like lesions
0 minute: is not found;
1 minute: small-small means that the number is very rare, but there are cases where the hepatocytes of the balloon-like lesion are determined or are at the diagnostic border;
and 2, dividing: many cells/permanent balloon-like lesions-most cells with permanent balloon-like lesions will appear Mallory glass-like, but Mallory glass-like lesions are not scored separately here.
3. Grading of hepatic lobule inflammation
0 minute: without focal point
1 minute: zoom at 200 × <2 foci
And 2, dividing: has 2 focuses under 200 times of magnification
And 3, dividing: >4 foci under 200 x magnification
Example 3 feeding of cynomolgus monkeys for three months, sirius red stained sections for liver fibrosis scoring massen trichrome and sirius red staining, method multiple × 200
The standard of fiberization scoring is as follows
0 minute: not shown;
1 minute: periantral or periportal fibrosis (see fig. 3);
1A: mild, zone 3, mild perisinus fibrosis;
1B: moderate, zone 3, dense perisinus fibrosis;
1C: portal/periportal-this classification is to accommodate cases of portal/periportal fibrosis without peri-cellular/peri-sinus fibrosis;
and 2, dividing: perisinus and portal/periportal (see fig. 4);
and 3, dividing: bridging fibrosis (see fig. 5);
and 4, dividing: cirrhosis (see fig. 6);
according to the NAS score, 0-2 in total can be considered as not being nonalcoholic steatohepatitis NASH;
a score of 3-4 in total that is essentially diagnosed as, or at the diagnostic border or positive for, non-alcoholic steatohepatitis NASH;
the total score of 5-8 can be diagnosed as non-alcoholic steatohepatitis NASH.
In conclusion, animals with NAS scores totaling 0-2 or fibrosis score 0 as a result of histopathological section scoring were considered not to be non-alcoholic steatohepatitis (NASH) models; at least 3 points of NAS score (at least 1 point of steatosis, at least 1 point of ballooning change and at least 1 point of liver lobular inflammation) and at least 1 point of liver fibrosis score (total score is 4 points or more), and the model can be diagnosed as a non-alcoholic steatohepatitis (NASH) model.
The following statistical data were obtained by histopathological staining and sectioning of 214 animal samples in example 3
Steatosis scores are shown in fig. 7, balloon-like lesions scores are shown in fig. 8, small inflammation scores are shown in fig. 9, NAS score statistics are shown in fig. 10, and fibrosis scores are shown in fig. 11; HAS and fibrosis composite scores are shown in fig. 12;
example 3 evaluation of the results of the experiment
As can be seen from the figure, the scores of the individual indexes of the NAS scores (including steatosis, balloon-like lesion and liver lobular inflammation) and the fibrosis scores are obviously improved after the feed prepared in example 3 is prepared for three months, wherein the animal number of the NAS scores above 3 is improved by 23.3%, the animal number of the fibrosis scores above 1 is improved by 46.2%, and the animal number of the NASH model is improved by 50%, so that the nonalcoholic fatty liver injury can be established after the feed is fed with the fructose formula for 3 months.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. The feed for constructing the non-alcoholic steatohepatitis model of the non-human primate is characterized by comprising the following raw materials in parts by weight:
2. the feed for constructing the non-alcoholic steatohepatitis model of the non-human primate according to claim 1, which is characterized by comprising the following raw materials in parts by weight:
3. the use of the feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 1 or 2, comprising the steps of:
1) non-human primate screening: selecting animals, selecting the number of the animals to be cultured, wherein the weight of the animals is more than or equal to 7.5kg, the age is more than or equal to 10 years, the liver function of the animals is normal, and no history of feeding with other inducers exists;
2) preparing a feed: stirring and mixing 22-28% by weight of soybean meal, 2.5-6% by weight of Peru fish meal, 2-3% by weight of feed grade calcium hydrophosphate, 0.2-0.4% by weight of salt, 0.1-0.3% by weight of compound multivitamins, 0.1-0.3% by weight of high-stability VC, 0.4-0.6% by weight of cholesterol, 20-25% by weight of single crystal fructose, 15-20% by weight of animal fat, 0.2-0.4% by weight of methionine, 1-2% by weight of premixed mineral and 30-35% by weight of primary flour to obtain a feed for constructing a non-alcoholic steatohepatitis model of a non-human primate;
3) culturing: feeding qualified animals indoors in a single cage, wherein the indoor humidity is 40-70%, the indoor temperature is 18-26 ℃, the indoor lighting condition is 12h light illumination and 12h darkness, and the feeding mode is that 50g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the morning, 150g of fruits in the noon and 100g of feed for constructing the non-alcoholic steatohepatitis model of the non-human primate in the afternoon are continuously fed for three months;
4) anaesthetizing animals continuously fed for three months, performing liver puncture under the guidance of B-ultrasonic to collect biopsy tissues, and performing HE (high-intensity ultrasound) staining and sirius red staining on the collected liver tissues respectively;
5) the obtained HE stained section is used for standard detection of a non-alcoholic fatty liver disease scoring system;
6) the obtained sirius red staining section is used for liver fibrosis scoring standard detection;
7) non-human primates which can not reach the scoring indexes through the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of liver fibrosis scoring cannot be used as non-alcoholic steatohepatitis models; the non-human primate reaching the scoring standard by the standard detection of a non-alcoholic fatty liver disease scoring system and the standard detection of fibrosis scoring is a non-alcoholic steatohepatitis model.
4. The use method of the feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 3, wherein the method comprises: the animals in the step 1) are cynomolgus monkeys.
5. The use method of the feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 3, wherein the method comprises: in said step 4) animals continuously fed for three months are anesthetized with ketamine.
6. The method of claim 3, wherein the non-alcoholic fatty liver disease scoring system standard test in step 5) comprises:
1) observing the HE stained sections under microscope magnification;
2) then carrying out fatty degeneration, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection on the liver respectively;
3) in the step 2), each item of steatosis, hepatocyte injury/ballooning lesion and lobular inflammation detection meets the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system reaches the scoring index; any one of steatosis, hepatocyte injury/balloon-like lesion and liver lobular inflammation detection in the step 2) cannot meet the scoring requirement, and the standard detection of the non-alcoholic fatty liver disease scoring system cannot meet the scoring index.
7. The method of using a feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 6, wherein the method comprises: the fatty degeneration area in the fatty degeneration detection is more than or equal to 5 percent, the fatty degeneration detection is qualified, the fatty degeneration area is less than 5 percent, the fatty degeneration detection is unqualified, and the fatty degeneration area is the ratio of the surface area involved in the fatty degeneration in the medium-low power detection; the condition that the liver cells with balloon-like lesions appear in the liver cell damage/balloon-like lesions or are positioned at the boundary of the liver cell damage and the balloon-like lesions is met, the liver cell damage/balloon-like lesions are qualified, the liver cell damage/balloon-like lesions are not found, and the liver cell damage/balloon-like lesions are unqualified; the lobular inflammation of the liver appears in focus, the lobular inflammation of the liver is qualified, the lobular inflammation of the liver does not appear in focus, and the lobular inflammation of the liver is unqualified.
8. The method of using a feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 3, wherein the liver fibrosis score criteria is:
1) magnifying the sirius red stained section by 200 times under a microscope;
2) showing no fibrotic features, the liver fibrosis scoring standard is not qualified;
3) the liver fibrosis characteristics appear, and the liver fibrosis scoring standard is qualified.
9. The method of using a feed for constructing a non-human primate non-alcoholic steatohepatitis model according to claim 8, wherein the method comprises: the fibrotic features include peri-sinus or periportal fibrosis, peri-sinus and periportal/portal fibrosis, bridging fibrosis and cirrhosis.
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