CN115413625A - Rheumatoid coronary heart disease animal model and construction method and application thereof - Google Patents
Rheumatoid coronary heart disease animal model and construction method and application thereof Download PDFInfo
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- A01K67/02—Breeding vertebrates
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- 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
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Abstract
The invention discloses an animal model of rheumatoid coronary heart disease and a construction method and application thereof, wherein the construction method comprises the following steps: (1) Administering a bovine type II collagen complete Freund's adjuvant emulsion and a bovine type II collagen incomplete Freund's adjuvant emulsion to the experimental animal; (2) administering a high fat diet to the experimental animal of step (1); (3) Separating the third and fourth ribs of the experimental animal in the step (2), cutting off the third and fourth ribs of the experimental animal, finding out the left auricle, ligating the left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a line, and observing the waveform change of an electrocardiograph of the experimental animal; (4) And loosening the ligation of the left anterior descending coronary artery after 30 minutes, recanalizing the left anterior descending coronary artery, and performing coronary artery blood flow reperfusion for 2 hours to obtain the rheumatoid coronary heart disease animal model. The molding method is simple, and the stability of each relevant index of the model is good.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a rheumatoid coronary heart disease animal model and a construction method and application thereof.
Background
Rheumatoid Arthritis (RA) is a systemic autoimmune chronic inflammatory response disease with chronic synovial inflammation in the joints, joint deformity and loss of function, inflammation affecting large, medium and small blood vessels in the extraarticular joints, and huge damage to the heart vessels. Multiple factors in the development of RA can cause intimal damage, which accumulates in the decline of arterial elastic function, predisposes the patient to atherosclerosis and significantly increases the risk of myocardial infarction and stroke. A large number of studies have shown that the inflammatory processes of rheumatoid arthritis and atheromatous plaque are very close, and have common inflammatory factor effects such as IL-17, IL-6, IL-1 and TNF-alpha, which may be important inflammatory components for the induction of cardiovascular-related diseases.
The incidence and the fatality rate of patients with rheumatoid arthritis and Coronary Heart Disease (CHD) are obviously higher than those of common people, and the disease course development is latent. Foreign research studies show that the CHD incidence rate of patients with rheumatoid arthritis is increased from 0.8% to 1.4% in 2004-2014, and the incidence rate of cardiovascular adverse events of patients with rheumatoid arthritis and coronary heart disease is 2.6% higher than that of patients with common coronary heart disease. An investigation and research in China shows that the incidence rate of coronary heart disease combined with the rheumatic heart disease is the highest in all co-diseases, and the fact that the rheumatoid arthritis patients are more susceptible to the coronary heart disease than common people is suggested.
However, a pathological process model capable of effectively simulating the rheumatoid arthritis combined with the coronary heart disease in vivo or in vitro is lacked at present, and the research on the pathogenesis of the rheumatoid coronary heart disease and the screening of safe and effective therapeutic drugs are key scientific problems. Therefore, the establishment of a model of the rheumatoid coronary heart disease which meets the clinical pathological characteristics has certain necessity.
Disclosure of Invention
Aiming at the problems, the invention aims to provide the rheumatoid coronary heart disease animal model with high success rate, effectiveness, reliability and strong repeatability, and the construction method and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses a method for constructing an animal model of rheumatoid coronary heart disease, which comprises the following steps:
(1) The method comprises the following steps of (1) administering a bovine type II complete Freund's adjuvant emulsion and a bovine type II incomplete Freund's adjuvant emulsion to experimental animals, wherein the bovine type II complete Freund's adjuvant emulsion is prepared by the following steps: dissolving the bovine type II collagen in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with complete Freund's adjuvant by using a high-speed homogenizer to prepare a bovine type II collagen complete Freund's adjuvant emulsion; the preparation process of the incomplete Freund's adjuvant emulsion of bovine type II collagen comprises the following steps: dissolving bovine type II collagen in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with incomplete Freund adjuvant by using a high-speed homogenizer to prepare a bovine type II collagen incomplete Freund adjuvant emulsion;
(2) Administering a high fat diet to the experimental animal of step (1);
(3) Separating the third and fourth ribs of the experimental animal in the step (2), cutting off the third and fourth ribs of the experimental animal, finding out the left auricle, ligating the left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a line, and observing the waveform change of an electrocardiograph of the experimental animal;
(4) And (3) loosening the ligation of the left anterior descending coronary artery after 30 minutes, restoring the left anterior descending coronary artery, and performing coronary artery blood flow reperfusion on the experimental animal in the step (3) for 2 hours to obtain the rheumatoid coronary heart disease animal model.
Further, in step (1), the specific procedures for administering the bovine type II complete Freund's adjuvant emulsion and the bovine type II incomplete Freund's adjuvant emulsion to the experimental animals are as follows: the prepared complete Freund adjuvant emulsion of bovine type II collagen is injected into 4 points of the left toe and the back of an experimental animal in the skin for initial immunization; one week later, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen is injected into the left toe part of the experimental animal in an intradermal way and injected into 4 points on the back in an intradermal way to strengthen the immunity, wherein the 4 points on the back refer to the positions of the left and right scapulae, the middle part of the back and the position of the back, which is 1cm away from the tail root part.
Further, in the step (1), the concentration of the bovine type II collagen in the bovine type II collagen solution is 2mg/ml; the rotating speed of the high-speed homogenizer is 30000r/min, and the volume ratio of the bovine II type collagen solution to the complete Freund adjuvant is 1; the volume ratio of the bovine type II collagen solution to the incomplete Freund adjuvant is 1.
Further, in the step (1), the injection amount of the bovine type II complete Freund's adjuvant emulsion in the toe area of the left foot of the animal is 0.1 mL/animal, and the injection amount of each point in 4 points on the back is 0.02 mL/animal; the injection amount of the bovine type II incomplete Freund's adjuvant emulsion in the left toe of the experimental animal is 0.1 mL/body, and the injection amount of each point in 4 points on the back is 0.02 mL/body.
Further, in the step (2), the specific process of administering the high fat diet to the experimental animal of the step (1) is as follows: and (3) feeding atherosclerosis high-fat feed every day from day 0 to day 21.
Further, the formula of the atherosclerosis high-fat feed in the step (2) comprises the following components in percentage by mass: casein 22.2222%, L-cystine 0.3333%, corn starch 23.5556%, maltodextrin 107.8889%, sucrose 12.5556%, cellulose BW 200.5556%, soybean oil 2.7778%, cocoa butter 17.2222%, compound mineral substance 1.1111%, calcium hydrogen phosphate 1.4444%, calcium carbonate 0.6111%, potassium citrate 1.8333%, compound vitamin V10001.1111%, choline bitartrate 0.2222%, cholesterol 1.2500%, sodium cholate 0.5%; wherein the casein is 80 mesh.
Further, the experimental animal is a rat.
The invention also discloses an animal model of the rheumatoid coronary heart disease, which is constructed by the construction method of the animal model of the rheumatoid coronary heart disease.
The invention also discloses application of the animal model of the rheumatoid coronary heart disease in the research of rheumatoid arthritis and coronary heart disease.
The invention has the beneficial effects that:
(1) The modeling method is simple, the stability of each relevant index of the model weight is good, the clinical pathological characteristics of the rheumatoid coronary heart disease are met, and a reliable method is provided for the construction of the rheumatoid coronary heart disease animal model.
(2) The rheumatoid coronary heart disease model constructed based on the invention can be used for simulating the disease characteristics and pathological process of clinical rheumatoid arthritis combined heart disease, discussing the pathogenesis and molecular target of the rheumatic coronary heart disease and screening the treatment drugs of the rheumatic coronary heart disease.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a graph showing the comparison of the low density lipoprotein (LDL-C) content in serum of rats in each group, data are expressed as mean. + -. Standard deviation, * P＜0.05， ** p <0.01 (compared to normal);
FIG. 2 is a graph showing the comparison of the Lactate Dehydrogenase (LDH) content in the serum of rats in each group, the data are expressed as mean. + -. Standard deviation, * P＜0.05， ** p <0.01 (compared to normal);
FIG. 3 is a graph comparing myocardial infarction areas of rats in each group, data are expressed as mean. + -. Standard deviation, ** p <0.01 (compared to normal);
fig. 4 is a graph comparing the HE staining results of rat hearts of each group at a magnification of 100 times, wherein a is a normal group; B. myocardial ischemia reperfusion group; C. a group of high fat diets; D. collagen arthritis group; E. collagen arthritis + myocardial ischemia reperfusion group; F. collagen arthritis + high fat diet group; G. myocardial ischemia reperfusion + high fat diet group; H. collagen arthritis + high fat diet + myocardial ischemia reperfusion group;
FIG. 5 is a graph comparing the results of Masson staining of the hearts of rats in each group at a magnification of 100, wherein A. The normal group; B. myocardial ischemia reperfusion group; C. a group of high fat diets; D. collagen arthritis group; E. collagen arthritis + myocardial ischemia reperfusion group; F. collagen arthritis + high fat diet group; G. myocardial ischemia reperfusion + high fat diet group; H. collagen arthritis + high fat diet + myocardial ischemia reperfusion group.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1: construction of animal model of rheumatoid coronary heart disease
1. Animals were performed:
SPF grade healthy SD rats were purchased from slacquard laboratory animals ltd, han, hunan. The study was approved by the ethical committee of southern Anhui medical college and was performed strictly in accordance with the relevant processing guidelines of the International laboratory animal protection certification and evaluation institute. The experimental animals are raised in an SPF environment at the temperature of 22-25 ℃ and the humidity of 50 percent, and are alternately kept for 12 hours day and night.
2. The implementation method comprises the following steps:
64 SD rats were randomly divided into 8 groups of 8 animals each:
(1) Normal group: each group had 8 animals, and after one week of acclimatization, on days 0 to 21, rats were fed with sufficient amount of normal clean-grade feed daily. The same amount of physiological saline was injected intradermally into the same spot in the left toe and back of the rat as the group not subjected to collagen arthritis molding on day 0 and day 7. On day 22, 2% pentobarbital (30 mg/kg) was intraperitoneally injected to anesthetize the rats, the limbs of the rats were fixed, the right and middle trachea was cut, the tracheal catheter was inserted, and mechanical ventilation was performed by a small animal ventilator. The left sternal edge 3 and 4 ribs are thoracotomy, but not ligatured by thread. Electrocardiogram and ST segment are observed, blood is obtained from abdominal artery after 2.5h, heart and liver are cut off, and the right hind limb of the rat is cut off and placed in 4% paraformaldehyde for subsequent experiments.
(2) Myocardial ischemia reperfusion group: the rats are fed with common clean-grade feed every day after being adaptively fed for one week by 8 rats in each group. Rats were injected intradermally with the same amount of physiological saline at the left toe and back of the rat as the group without collagen arthritis molding on day 0 and day 7. A model building method of a myocardial ischemia reperfusion model comprises the steps of injecting 2% pentobarbital (30 mg/kg) into an abdominal cavity on the 22 th day to anaesthetize a rat, fixing the limbs of the rat, cutting a middle trachea, inserting a tracheal catheter, and connecting the rat with a small animal respirator for mechanical ventilation. Separating the third rib and the fourth rib of the experimental animal, cutting off the third rib and the fourth rib of the experimental animal, finding out the left auricle, and ligating the left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a thread; and (3) recovering left anterior descending perfusion after 30min of ligation, observing electrocardiograms before ligation, 30min of ligation and after circulation recovery, taking ST segment significant elevation as a myocardial ischemia mark, taking ST segment slow fall back as a reperfusion mark, obtaining blood from abdominal arteries after 2h of coronary artery blood flow reperfusion, shearing hearts and livers, and meanwhile, cutting right hind limbs of rats and placing the rats in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(3) High fat diet group: each group had 8. After one week of adaptive feeding, the purchased rats were fed with sufficient atherosclerosis high-fat diet daily from day 0 to day 21. Rats were injected intradermally with the same amount of physiological saline at the left toe and back of the rat as the group without collagen arthritis molding on day 0 and day 7. On day 22, 2% pentobarbital (30 mg/kg) was intraperitoneally injected to anesthetize the rats, the limbs of the rats were fixed, the right and middle trachea was cut, the tracheal catheter was inserted, and mechanical ventilation was performed by a small animal ventilator. The third and fourth ribs of the experimental animal were separated and cut off, but no threading ligation was performed. Electrocardiogram and ST segment are observed, blood is obtained from abdominal artery after 2.5h, heart and liver are cut off, and the right hind limb of the rat is cut off and placed in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(4) Collagen arthritis group: each group had 8. One week after the purchased rats were acclimatized and fed with sufficient amount of ordinary clean-grade feed per day from day 0 to day 21. The molding method of the collagen arthritis comprises the following steps: dissolving bovine type II collagen (2 mg/ml) in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with a complete Freund adjuvant by using a high-speed homogenizer (30000 r/min) according to the ratio of 1; bovine type II collagen (2 mg/ml) was ice-bathed and dissolved in glacial acetic acid to obtain a bovine type II collagen solution, which was then homogenized with a high speed homogenizer (30000 r/min) at a speed of 1:1 and incomplete Freund's adjuvant to prepare the bovine II type incomplete Freund's adjuvant emulsion. On day 0, the prepared bovine type II collagen complete Freund's adjuvant emulsion is taken and injected intradermally (0.02 mL per spot) into the left toe part (0.1 mL injection amount) and the back 4 points (the left and right scapula parts, the middle back part and the back part 1cm away from the tail root part) of the animal for initial immunization. On day 7, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen was injected into the toe and back of a rat at multiple points and the same amount (0.1 mL of the injected dose in the toe and 0.02mL of the injected dose in each point in the back) for boosting, and collagen arthritis model evaluation was performed on day 21. And ensuring that the arthritis index score of the rat after injection immunization is more than or equal to 4 points, and then the molding is successful. On day 22, 2% pentobarbital (30 mg/kg) was intraperitoneally injected to anesthetize the rats, the limbs of the rats were fixed, the right and middle trachea was cut, the tracheal catheter was inserted, and mechanical ventilation was performed by a small animal ventilator. The third and fourth ribs of the experimental animal were separated and cut off, but no threading ligation was performed. Electrocardiogram and ST segment are observed, blood is obtained from abdominal artery after 2.5h, heart and liver are cut off, and the right hind limb of the rat is cut off and placed in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(5) Collagenous arthritis + myocardial ischemia reperfusion group: each group had 8. After one week of adaptive feeding, the purchased rats were therefore a composite model, and were fed daily with sufficient amounts of normal clean-grade feed for rats from day 0 to day 21. The molding method of the collagen arthritis comprises the following steps: dissolving bovine type II collagen (2 mg/ml) in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with a complete Freund adjuvant by using a high-speed homogenizer (30000 r/min) according to the proportion of 1; bovine type II collagen (2 mg/ml) was ice-bathed and dissolved in glacial acetic acid to obtain a bovine type II collagen solution, which was then homogenized with a high speed homogenizer (30000 r/min) at a speed of 1:1 and incomplete Freund's adjuvant to prepare the bovine II type incomplete Freund's adjuvant emulsion. On day 0, the prepared complete Freund's adjuvant emulsion of bovine type II collagen was injected intradermally (0.02 mL per spot) into the left toe (0.1 mL injection) and the back 4 spots (1 cm from the left and right scapulae, the middle back and the back to the root of the tail) of the animals for initial immunization. On day 7, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen was injected into the toe and back of a rat at multiple points in equal amounts (0.1 mL of the injected amount in the toe and 0.02mL of the injected amount in each point in the back) for boosting, and collagen arthritis model evaluation was performed on day 21. And ensuring that the arthritis index score of the rat after the injection immunization is more than or equal to 4 points, and then successfully modeling. A model building method of a myocardial ischemia reperfusion model comprises the steps of injecting 2% pentobarbital (30 mg/kg) into an abdominal cavity on the 22 th day to anaesthetize a rat, fixing the limbs of the rat, cutting a middle trachea, inserting a tracheal catheter, and connecting the rat with a small animal respirator for mechanical ventilation. Separating a third rib and a fourth rib of the experimental animal, cutting off the third rib and the fourth rib of the experimental animal, finding a left auricle, and ligating a left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a thread; and (3) recovering the perfusion of the left anterior descending branch after 30min of ligation, observing that the electrocardiogram is obviously raised by using an ST segment before ligation, 30min of ligation and after circulation recovery as a myocardial ischemia mark, slowly dropping the ST segment as a reperfusion mark, performing coronary artery blood flow reperfusion for 2h, obtaining blood from the abdominal artery, clipping the heart and the liver, simultaneously cutting the hind limb (right hind limb) of a rat which is not injected with the complete Freund's adjuvant intradermally, and placing the hind limb (right hind limb) of the rat in 4 percent paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(6) Collagen arthritis + high fat diet group: each group had 8. After one week of adaptive feeding, the purchased rats were therefore a composite model, and were fed with sufficient atherosclerosis and high fat diet daily from day 0 to day 21. The molding method of the collagen arthritis comprises the following steps: dissolving bovine type II collagen (2 mg/ml) in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with a complete Freund adjuvant by using a high-speed homogenizer (30000 r/min) according to the proportion of 1; bovine type II collagen (2 mg/ml) was ice-bathed and dissolved in glacial acetic acid to obtain a bovine type II collagen solution, which was then homogenized with a high speed homogenizer (30000 r/min) at a speed of 1:1 and incomplete Freund's adjuvant to prepare the bovine type II collagen incomplete Freund's adjuvant emulsion. On day 0, the prepared complete Freund's adjuvant emulsion of bovine type II collagen was injected intradermally (0.02 mL per spot) into the left toe (0.1 mL injection) and the back 4 spots (1 cm from the left and right scapulae, the middle back and the back to the root of the tail) of the animals for initial immunization. On day 7, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen was injected into the toe and back of a rat at multiple points and the same amount (0.1 mL of the injected dose in the toe and 0.02mL of the injected dose in each point in the back) for boosting, and collagen arthritis model evaluation was performed on day 21. And ensuring that the arthritis index score of the rat after injection immunization is more than or equal to 4 points, and then the molding is successful. On day 22, 2% pentobarbital (30 mg/kg) was intraperitoneally injected to anesthetize the rats, the limbs of the rats were fixed, the right trachea was cut, the tracheal catheter was inserted, and mechanical ventilation was performed by a small animal ventilator. The third and fourth ribs of the experimental animal were separated and cut off without ligature. Electrocardiogram and ST segment are observed, blood is obtained from abdominal artery after 2.5h, heart and liver are cut off, and the right hind limb of the rat is cut off and placed in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(7) Myocardial ischemia reperfusion + high fat diet group: each group had 8. After one week of adaptive feeding, the purchased rats were therefore a composite model, and were fed with sufficient atherosclerosis and high fat diet daily from day 0 to day 21. The same amount of physiological saline was injected intradermally into the same spot in the left toe and back of the rat as the group not subjected to collagen arthritis molding on day 0 and day 7. A model building method of a myocardial ischemia reperfusion model comprises the steps of injecting 2% pentobarbital (30 mg/kg) into an abdominal cavity on the 22 th day to anaesthetize a rat, fixing limbs of the rat, cutting a right middle trachea, inserting a tracheal catheter, and connecting a small animal respirator for mechanical ventilation. Separating the third rib and the fourth rib of the experimental animal, cutting off the third rib and the fourth rib of the experimental animal, finding out the left auricle, and ligating the left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a thread; and (3) recovering left anterior descending perfusion after 30min ligation, observing that the electrocardiogram is obviously raised by ST segment before ligation, 30min ligation and after circulation recovery to be used as a myocardial ischemia mark, the ST segment slowly falls back to be used as a reperfusion mark, obtaining blood from abdominal cavity artery after 2h of coronary artery blood flow reperfusion, clipping heart and liver, and meanwhile, intercepting hind limb of rat friend who is not injected with complete Freund's adjuvant intradermally and placing the hind limb in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
(8) Collagen arthritis + high fat diet + myocardial ischemia reperfusion group: each group had 8. After one week of adaptive feeding of purchased rats, the model is therefore a composite model, so that from day 0 to day 21, sufficient atherosclerosis high-fat diet is fed daily, collagen arthritis modeling method: dissolving bovine type II collagen (2 mg/ml) in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with a complete Freund adjuvant by using a high-speed homogenizer (30000 r/min) according to the proportion of 1; bovine type II collagen (2 mg/ml) was ice-bathed and dissolved in glacial acetic acid to obtain a bovine type II collagen solution, which was then homogenized with a high speed homogenizer (30000 r/min) at a speed of 1:1 and incomplete Freund's adjuvant to prepare the bovine type II collagen incomplete Freund's adjuvant emulsion. On day 0, the prepared bovine type II collagen complete Freund's adjuvant emulsion is taken and injected intradermally (0.02 mL per spot) into the left toe part (0.1 mL injection amount) and the back 4 points (the left and right scapula parts, the middle back part and the back part 1cm away from the tail root part) of the animal for initial immunization. On day 7, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen was injected into the toe and back of a rat at multiple points and the same amount (0.1 mL of the injected dose in the toe and 0.02mL of the injected dose in each point in the back) for boosting, and collagen arthritis model evaluation was performed on day 21. And ensuring that the arthritis index score of the rat after the injection immunization is more than or equal to 4 points, and then successfully modeling. A model building method of a myocardial ischemia reperfusion model comprises the steps of injecting 2% pentobarbital (30 mg/kg) into an abdominal cavity on the 22 th day to anaesthetize a rat, fixing limbs of the rat, cutting a right middle trachea, inserting a tracheal catheter, and connecting a small animal respirator for mechanical ventilation. Separating a third rib and a fourth rib of the experimental animal, cutting off the third rib and the fourth rib of the experimental animal, finding a left auricle, and ligating a left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a thread; and (3) recovering left anterior descending perfusion after 30min of ligation, observing electrocardiogram before ligation, 30min of ligation and after circulation recovery, taking ST segment significant elevation as a myocardial ischemia mark, taking ST segment slow fallback as a reperfusion mark, obtaining blood from abdominal cavity artery after 2h of coronary artery blood flow reperfusion, cutting heart and liver, and meanwhile, cutting hind limbs of rat friends who are not injected with complete Freund's adjuvant intradermally and placing the hind limbs in 4% paraformaldehyde for subsequent experiments. Rats with model making failure are rejected, and the same batch of rats is selected for supplementation.
The formula of the atherosclerosis high-fat feed comprises the following components in percentage by mass: casein 22.2222%, L-cystine 0.3333%, corn starch 23.5556%, maltodextrin 107.8889%, sucrose 12.5556%, cellulose BW 200.5556%, soybean oil 2.7778%, cocoa butter 17.2222%, compound mineral substance 1.1111%, calcium hydrogen phosphate 1.4444%, calcium carbonate 0.6111%, potassium citrate 1.8333%, compound vitamin V10001.1111%, choline bitartrate 0.2222%, cholesterol 1.2500%, sodium cholate 0.5%; wherein the casein is 80 mesh.
3. The monitoring index and method are as follows:
3.1 measurement of LDL-C levels in rat sera:
an enzyme-labeled analyzer is adopted to detect the HDL-C content in the serum of the rat, and the detection is carried out by an enzyme colorimetric method according to the instruction of a kit.
3.2 detection of LDH levels in rat sera:
an enzyme-labeled analyzer is adopted to detect the LDH content in rat serum, and the detection is carried out by an enzyme colorimetric method according to the kit instruction.
3.3 calculating myocardial infarction area of each group of rats:
after reperfusion, taking rat hearts of each group, washing blood stain by using normal saline, cutting heart tissues into myocardial slices of 0.1-0.2 cm, dyeing the myocardial cross section by using nitrotetrazolium blue chloride (NBT), incubating for 15min at 37 ℃, and calculating the myocardial cross section infarct area and the whole left ventricle myocardial cross section area by using image analysis software, wherein the myocardial infarct area is represented by the percentage of the cross section infarct part in the left ventricle infarct area.
3.4 myocardial histopathological observations:
fixing myocardial tissue with 4% paraformaldehyde for 12h, washing, dehydrating, clearing, embedding, slicing, and staining according to HE and Masson staining procedure. The sections were placed under an optical microscope to observe histopathological changes in the myocardium of each group.
4. The experimental results are as follows:
4.1 Effect of combined treatment of high fat diet, complete Freund's adjuvant emulsion of bovine type II collagen and incomplete Freund's adjuvant emulsion of bovine type II collagen injection and ligation of left anterior descending branch of coronary artery on the degree of swelling of secondary lateral joint in AA rat:
after the cattle complete Freund type II collagen adjuvant emulsion and the cattle incomplete Freund type II collagen adjuvant emulsion are injected into rats, d 1-d 3 show acute local inflammation; then gradually reducing, namely acute inflammation remission (d 7-d 12); delayed hypersensitivity ensues, manifested by swelling of the contralateral and forelimb paw, inflammatory nodules and erythema on the ear and tail with weight loss. In the experiment, after the immunized rats are injected with complete Freund's collagen adjuvant, the swelling degrees of secondary side joints of d14, d17 and d21 are obviously increased compared with those of a normal group, which is shown as secondary side paw swelling, and indicates that arthritis molding is successful (Table 1).
TABLE 1 Effect of collagen complete Freund's adjuvant on Secondary swelling degree of the lateral articulation in rats
* p <0.05, p <0.01vs. p <0.001vs. normal group.
4.2 effects of high fat diet, collagen complete Freund's adjuvant injection and ligation of left anterior descending coronary artery on LDL-C levels:
the effect of combined treatment of hyperlipidemia, collagen arthritis and myocardial ischemia on serum LDL-C in rats is shown in FIG. 1. Compared with the normal group, the serum LDL-C level of rats is increased in the high-fat diet group, the collagen arthritis and high-fat diet group, the myocardial ischemia reperfusion and high-fat diet group, and the collagen arthritis and high-fat diet and myocardial ischemia reperfusion group. Experimental results show that the high-fat diet can increase the LDL-C content in rat serum, and the high-fat diet, complete Freund's adjuvant emulsion of cattle type II collagen and incomplete Freund's adjuvant emulsion of cattle type II collagen injection and ligation coronary artery left anterior descending combined treatment can stably increase the LDL-C content in rat serum.
4.3 Effect of combination of high fat diet, complete Freund's adjuvant emulsion of bovine type II collagen and incomplete Freund's adjuvant emulsion of bovine type II collagen injection and ligation of left anterior descending coronary artery on LDH level
The effect of combined treatment of hyperlipidemia, collagen arthritis and myocardial ischemia on rat serum LDH is shown in figure 2. Compared with the normal group, the levels of LDH in the serum of rats in the myocardial ischemia reperfusion group, the collagen arthritis and myocardial ischemia reperfusion group, the myocardial ischemia reperfusion group and high fat diet group, and the collagen arthritis and high fat diet and myocardial ischemia reperfusion group are increased. The experimental result shows that the ligation of the left anterior descending branch of the coronary artery can increase the LDH content in the serum of the rat, and the combination of high-fat diet, complete Freund's adjuvant emulsion of bovine type II collagen, incomplete Freund's adjuvant emulsion injection of bovine type II collagen and the ligation of the left anterior descending branch of the coronary artery can stably increase the LDH content in the serum of the rat.
4.4 comparison of myocardial infarction area of rats in each group:
the heart structure of the rats in the normal group is complete and has no ischemic injury. Compared with the normal group, the myocardial ischemia reperfusion group, the collagen arthritis + myocardial ischemia reperfusion group, the myocardial ischemia reperfusion + high fat diet group, the collagen arthritis + high fat diet + myocardial ischemia reperfusion group increased the myocardial infarction area of the rats, as shown in fig. 3.
4.5 the combined treatment of high fat diet, complete Freund's adjuvant emulsion of bovine type II collagen and incomplete Freund's adjuvant emulsion of bovine type II collagen injection and left anterior descending of ligature coronary artery has the effects on the pathological change degree of heart:
according to the result of HE staining, the normal group heart tissue has compact and clear structure and no obvious pathological change; the heart tissues of the high-fat diet group and the collagen arthritis model group have slight inflammatory cell infiltration; the collagen arthritis group, the myocardial ischemia reperfusion group, the high fat diet and myocardial ischemia reperfusion group and the collagen arthritis group have obvious hypertrophy of myocardial cells, more fibroblasts are visible among muscle bundles, the arrangement of myocardial fiber layers is irregular, and a fracture layer is formed, wherein the pathological change is most obvious in the high fat diet, myocardial ischemia reperfusion and collagen arthritis group, as shown in figure 4.Masson staining results show that the myocardial cells of the rats in the normal group are red, blue is not seen, and no obvious collagen deposition exists; the heart tissues of the high-fat diet group and the collagen arthritis model group have slight inflammatory infiltration; the pathological changes of the groups of collagen arthritis, myocardial ischemia reperfusion, high fat diet and myocardial ischemia reperfusion and the groups of high fat diet, myocardial ischemia reperfusion and collagen arthritis are most obvious, as shown in figure 5, the rat hearts have more fibroblasts, the fibrous connective tissue is hyperplastic, the myocardial interstitium can be seen as large blue, a large amount of myocardial collagen is deposited in the myocardial interstitium.
The method of the invention comprises the steps of injecting bovine type II collagen complete Freund's adjuvant emulsion into a male mouse, simultaneously administering a high-fat diet and feeding the male mouse in a sufficient amount for 3 weeks, and then ligating coronary artery left anterior descending branch for combined treatment, wherein the collagen arthritis, the high-fat diet and myocardial ischemia reperfusion groups have the typical characteristics similar to rheumatoid coronary heart diseases such as increased serum LDL-C and LDH levels, increased heart infarction area, pathological changes of different degrees and the like, thereby successfully establishing a model consistent with the clinical characteristics of the rheumatic coronary heart diseases. The molding method is simple, and the stability of indexes such as LDL-C level, LDH level, heart pathology and the like in the model is good, which shows that the molding method is stable, effective, reliable and strong in repeatability.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. The construction method of the animal model of the rheumatoid coronary heart disease is characterized by comprising the following steps:
(1) The method comprises the following steps of (1) administering a bovine type II complete Freund's adjuvant emulsion and a bovine type II incomplete Freund's adjuvant emulsion to experimental animals, wherein the bovine type II complete Freund's adjuvant emulsion is prepared by the following steps: dissolving bovine type II collagen in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution and the complete Freund adjuvant by using a high-speed homogenizer to prepare a bovine type II collagen complete Freund adjuvant emulsion; the preparation process of the incomplete Freund's adjuvant emulsion of bovine type II collagen comprises the following steps: dissolving bovine type II collagen in glacial acetic acid after ice bath to obtain a bovine type II collagen solution, and emulsifying the bovine type II collagen solution with incomplete Freund adjuvant by using a high-speed homogenizer to prepare a bovine type II collagen incomplete Freund adjuvant emulsion;
(2) Administering a high fat diet to the experimental animal of step (1);
(3) Separating the third and fourth ribs of the experimental animal in the step (2), cutting off the third and fourth ribs of the experimental animal, finding out the left auricle, ligating the left anterior descending coronary artery at a position 2-4 mm below the left auricle by using a line, and observing the waveform change of an electrocardiograph of the experimental animal;
(4) And (4) loosening the ligation of the left anterior descending coronary artery after 30 minutes, dredging the left anterior descending coronary artery again, and performing coronary artery blood flow reperfusion on the experimental animal in the step (3) for 2 hours to obtain the rheumatoid coronary heart disease animal model.
2. The method for constructing an animal model of rheumatoid coronary heart disease according to claim 1, wherein the specific procedures for administering the complete Freund's adjuvant emulsion of bovine type II and the incomplete Freund's adjuvant emulsion of bovine type II to the experimental animal in step (1) are as follows: the prepared complete Freund adjuvant emulsion of bovine type II collagen is injected into 4 points of the left toe and the back of an experimental animal in an intradermal way for initial immunization; one week later, the prepared incomplete Freund's adjuvant emulsion of bovine type II collagen is injected into the left toe part of the experimental animal in an intradermal way and injected into 4 points on the back in an intradermal way to strengthen the immunity, wherein the 4 points on the back refer to the positions of the left and right scapulae, the middle part of the back and the position of the back, which is 1cm away from the tail root part.
3. The method for constructing an animal model of rheumatoid coronary heart disease according to claim 1, wherein in the step (1), the concentration of bovine type II collagen in the bovine type II collagen solution is 2mg/ml; the rotating speed of the high-speed refiner is 30000r/min, and the volume ratio of the bovine type II collagen solution to the complete Freund adjuvant is 1; the volume ratio of the bovine type II collagen solution to the incomplete Freund adjuvant is 1.
4. The method for constructing an animal model of rheumatoid coronary heart disease according to claim 2, wherein in step (1), the injection amount of the complete Freund's adjuvant emulsion of bovine type II is 0.1 mL/body in the left toe of the animal, and 0.02 mL/body in 4 points of the back; the injection amount of the bovine type II incomplete Freund's adjuvant emulsion in the left toe of the experimental animal is 0.1 mL/body, and the injection amount of each point in 4 points on the back is 0.02 mL/body.
5. The method for constructing an animal model of rheumatoid coronary heart disease according to claim 1, wherein the specific process of administering high fat diet to the experimental animal of step (1) in step (2) is as follows: and feeding atherosclerosis high-fat feed every day from day 0 to day 21.
6. The method for constructing the animal model of the rheumatoid coronary heart disease according to claim 5, wherein the formula of the atherosclerosis high-fat feed in the step (2) comprises the following components in percentage by mass: casein 22.2222%, L-cystine 0.3333%, corn starch 23.5556%, maltodextrin 107.8889%, sucrose 12.5556%, cellulose BW 200.5556%, soybean oil 2.7778%, cocoa butter 17.2222%, compound mineral substance 1.1111%, calcium hydrogen phosphate 1.4444%, calcium carbonate 0.6111%, potassium citrate 1.8333%, compound vitamin V10001.1111%, choline bitartrate 0.2222%, cholesterol 1.2500%, sodium cholate 0.5%; wherein the casein is 80 mesh.
7. The method for constructing an animal model of rheumatoid coronary heart disease according to claim 1, wherein the experimental animal is a rat.
8. An animal model of rheumatoid coronary heart disease constructed by the method for constructing an animal model of rheumatoid coronary heart disease according to any one of claims 1 to 7.
9. Use of the animal model of rheumatoid coronary heart disease according to claim 8 in the study of rheumatoid arthritis with coronary heart disease.
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| 邹纯才等: "瓜蒌薤白滴丸抗心肌缺血再灌注损伤作用", 《国际药学研究杂志》 * |
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