CN115211402A - Construction method and application of CV-B3 infected rodent model - Google Patents
Construction method and application of CV-B3 infected rodent model Download PDFInfo
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Abstract
The invention belongs to the technical field of animal model construction, and particularly provides a construction method and application of a CV-B3 infected rodent model, wherein the construction method comprises the steps of infecting the rodent model with CV-B3 in an intraperitoneal injection or nasal cavity instillation mode, and establishing the CV-B3 infected rodent model. The invention establishes a rodent model for Coxsackie group B3 (CV-B3) pathogen research. The modeling method accords with the enterovirus propagation mode and the morbidity rule of clinical hand-foot-and-mouth disease patients, and is simple and feasible, short in modeling period, high in repeatability and stable in model. Has important use value in the aspects of developing the way and mechanism research of Coxsackie group B3 pathogen infected central nerve, drug evaluation, vaccine research and development evaluation and the like.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a CV-B3 infected rodent model.
Background
In recent years, the general epidemic trend of the hand-foot-and-mouth disease in China shows a phenomenon of high incidence. According to the technical scheme, the infection review survey of the hand-foot-and-mouth disease in areas such as Shijiazhuang and Jiangsu in China shows that the positive detection rate of the CVB3 in the hand-foot-and-mouth disease related enteroviruses shows a trend of gradual increase, and the epidemiological survey result prompts that the CVB3 is likely to gradually become the main pathogens of the hand-foot-and-mouth disease following EV71, CVA16, CVA10 and CVA 6.
CV-B3 virus belongs to the enterovirus genus of picornaviridae, generally causes respiratory symptoms like influenza, and can cause severe inflammation of the heart, pancreas, and central nervous system diseases in newborns, infants, and immunodeficient adults. There are many studies reported on animal models of CV-B3, and the animal species can be divided into mouse and non-human primate. At present, a mouse animal model is constructed by intraperitoneal injection, and researches on the research on CV-B3 and various acute complications, such as acute viral myocarditis and encephalitis, are carried out. Non-human primates have high similarity to humans in terms of anatomical structure, physiological metabolism, immune system, etc., but their versatility as model animals is limited due to their high price and special requirements for feeding and environmental facilities.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a construction method and application of a CV-B3 infected rodent model.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
provides a construction method of a CV-B3 infected rodent model, which infects CV-B3 to the rodent model in an intraperitoneal injection or nasal drip mode to establish the CV-B3 infected rodent model.
Further, the method comprises the following steps: the rodent is selected from a 4-week-old female syrian hamster.
Further, the method comprises the following steps: the virus titer of the intraperitoneal injection infection is 10 6.75 CCID 50 mL, infectious dose 100. Mu.L.
Further: the viral titer of the nasal drip infection was 10 6.75 CCID 50 mL, infectious dose 100. Mu.L.
Use of a rodent model of CV-B3 infection for screening or identifying drugs capable of preventing, ameliorating or treating CV-B3 infection is provided.
Use of a rodent model of CV-B3 infection for screening or identifying vaccines capable of preventing CV-B3 infection is provided.
The beneficial effects of the invention are as follows:
the invention establishes a rodent model for researching Coxsackie group B type 3 (CV-B3) pathogeny. The modeling method accords with the enterovirus propagation mode and the morbidity rule of clinical hand-foot-and-mouth disease patients, and is simple and feasible, short in modeling period, high in repeatability and stable in model. Has important use value in the aspects of developing the research on the path and mechanism of Coxsackie group B3 pathogen for infecting central nerve, evaluating medicaments, researching and developing vaccines and the like.
Drawings
FIG. 1 is a graph of the change in body temperature of the intraperitoneal injection group, the nasal cavity infusion group and the control group of Syrian hamsters infected with the coxsackie group B type 3 virus in example 2;
FIG. 2 is a diagram of herpes labialis after infection of the Syrian hamster with the coxsackie group B type 3 virus of example 2; wherein A and B: intraperitoneal injection of a group of syrian hamsters infected with viral herpes postzurini; C. d: nasal drip group syrian hamster infected herpes virus posterior horn mouth;
FIG. 3 is a graph showing the change in serum lactate dehydrogenase content in blood after infection of Coxsackie group B3 virus in the intraperitoneal injection group, nasal cavity infusion group and control group of Syrian hamsters in example 2;
FIG. 4 is a graph showing the changes in the creatine kinase levels in blood after infection of Coxsackie group B3 virus in the intraperitoneal injection group, nasal cavity infusion group, and control group of Syrian hamsters in example 2;
FIG. 5 is a graph showing the changes in the creatine kinase isoenzyme content in blood after infection of Coxsackie group B3 virus in the intraperitoneal injection group, nasal cavity infusion group and control group of Syrian hamsters in example 2;
FIG. 6 is a graph showing the detoxification of throat swab samples of the i.p. group, the nasal drip group, and the control group of Syrian hamsters infected with Coxsackie group B type 3 virus of example 2;
FIG. 7 is a graph showing the detoxification of nasal lavage fluid samples from the intraperitoneal injection group, nasal drip group, and control group of syrian hamsters infected with coxsackie group B type 3 virus in example 2;
FIG. 8 is a graph of stool sample detoxification following infection of Coxsackie group B type 3 virus by the intraperitoneal injection group, nasal drip group, and control group of Syrian hamsters in example 2;
FIG. 9 is a graph of the viral load in the nervous system of the intraperitoneal injection group, the nasal cavity infusion group, and the control group of Syrian hamsters infected with the Coxsackie group B type 3 virus in example 2;
FIG. 10 is a graph showing the viral load of the major organs of the intraperitoneal injection group, the nasal cavity infusion group and the control group of Syrian hamsters infected with the Coxsackie group B3 virus in example 2;
FIG. 11 is a graph showing the viral load in the digestive tract after infection of Coxsackie group B type 3 virus by intraperitoneal injection, nasal drip and control syrian hamsters in example 2;
FIG. 12 is a graph of the viral load in the respiratory tract of the intraperitoneal injection group, nasal drip group, and control group of Syrian hamsters infected with the coxsackie group B type 3 virus in example 2;
FIG. 13 is a graph showing the viral load on the nasal mucosa, labial and tracheal tubes after infection of Coxsackie group B type 3 virus by the intraperitoneal injection group, nasal cavity infusion group and control group of Syrian hamsters in example 2;
FIG. 14 is a graph showing the histopathological changes of the intraperitoneal injection group, nasal cavity infusion group and control group of Syrian hamsters after infection with Coxsackie group B3 virus in example 2; wherein A: is the heart; b: is the liver; c: spleen is selected; d: the lung is the lung; e: is the brain;
FIG. 15 is a graph of the histopathological changes following infection of the Coxsackie group B type 3 virus by the intraperitoneal injection group, nasal drip group, and control group of Syrian hamsters in example 2; wherein F: is a smell ball; g: is the duodenum; h: is jejunum; i: is the ileum; j: the colon is shown.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1
A CV-B3 infected rodent model construction method comprises the steps of infecting CV-B3 to the rodent model in an intraperitoneal injection or nasal drip mode, and establishing the CV-B3 infected rodent model.
In this example, 4 weeks old Syrian hamsters were used as experimental animals, female, 15, SPF grade, body weight 65-85g; divided into an intraperitoneal injection group (6), a nasal drip group (6) and a control group (3). Feeding in a barrier environment, feeding the same fodder, freely feeding, and feeding water. The feed and drinking water required every day are sterilized.
CV-B3 virus: stored in the institute of medical biology of Chinese academy of medical sciences. After Vero cell proliferation culture, the harvested virus liquid is purified to determine half of the cell culture infected dose, namely the infectious titer (CCID) (50 percent of cell culture infectious dose) 50 ) Is 1 x 10 6.75 CCID 50 /mL。
The mode of toxic attack and infection: and (3) an intraperitoneal injection group: infecting Syrian hamster by intraperitoneal injection at an infection dose of 10 6.75 CCID50/mL,100 microliter/mouse; nasal drip group: nasal instillation to challenge Syrian hamster with infection dose of 10 6.75 CCID50/mL, 100. Mu.l/pellet. The control group (Blank group) was fed with 100. Mu.l of physiological saline through each nasal cavity, and was kept normally separately from the intraperitoneal injection group and the nasal cavity infusion group.
Example 2
Rodent models of CV-B3 infection constructed in example 1 were evaluated and analyzed:
clinical symptoms including diet, mental state, herpes, defecation, activity state of limbs, hair state, death, etc. were observed three times a day during the test period of days 0-7 and recorded. In the morning of 9:00 rectal temperature and body weight of Syrian hamsters were measured and recorded.
The experimental animal is killed on the 7 th day of infection, a non-anticoagulation blood sample is collected and placed at 4 ℃, the standing is carried out for 30min, after serum is naturally separated out, the centrifugation is carried out for 10min at 4 ℃ and 3500rpm, the serum is collected into a 1.5mL centrifuge tube, and the biochemical index of the sample is detected by using an automatic biochemical analyzer.
And (3) detecting the toxin expelling condition of the pharynx, the nose and the excrement after infection and the tissue virus load by using a Real-time quantitative PCR (Real-time quantitative PCR) technology.
1. Viral load of pharyngeal and nasal feces
Collecting throat swab, nasal lavage liquid and excrement every day after toxin attack, and extracting RNA by a TRIzol method, which comprises the following steps:
1) Throat and nasal lavage liquid, and fecal samples are added into 1mLTRIzol, shaken, mixed evenly and placed on ice.
2) And sucking the shaken sample by 200 mu L, adding the shaken sample into 1mLTRIzol, adding 200 mu L of chloroform solution, shaking vigorously for 15S, and standing for 10min.
3) The sample was placed in a centrifuge at 12000rpm for 10min.
4) And putting the supernatant into a new 1.5mL centrifugal tube, adding an isopropanol solution with the same volume, reversing, uniformly mixing, and standing for 10min.
5) The sample was placed in a centrifuge at 12000rpm for 10min.
6) The solution was discarded and 1mL of 75% enzyme free ethanol solution was added.
7) The sample was placed in a centrifuge at 12000rpm for 5min.
8) Discard the solution and dry the centrifuge tube. The RNA is dissolved by adding non-enzyme water.
9) The viral load in the samples was detected by RT-qPCR reaction using total RNA as template. RT-qPCR reaction was performed on the extracted sample RNA using One Step TB Green PrimeScript TM PLUS RT-PCR Kit from TaKaRa, primer sequence F: GCTCACCTTCGTTTATTACTAG, R is AATGCTCAAGAATGGAATGG. The reaction system is as follows: 2 Xone Step TB Green RT-PCR Buffer 4.5. Mu.L, ex Taq HS Mix1.5. Mu.L, primeScriptPLUS RTase Mix0.5. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, RNA 2. Mu.L, nuclease-free water 6.5. Mu.L. The reaction conditions are as follows: the dissolution curve reaction is carried out for 39 cycles of 42 ℃ for 5min,94 ℃ for 10s,95 ℃ for 5s, and 60 ℃ for 30s, and the dissolution curve reaction is carried out for 65 ℃ for 5s and 95 ℃ for 5 s.
10 Statistics and analysis of PCR results.
2. Blood biochemical index detection
And (3) placing the non-anticoagulation sample at 4 ℃ for half an hour, after serum is naturally separated out, centrifuging at 4 ℃ at 3500rpm for 10min, collecting the serum into a 1.5mL centrifuge tube, and detecting the biochemical index of the sample by using an automatic biochemical analyzer.
3. Tissue viral load detection
Cardiac blood samples were sacrificed after narcotization of syrian hamsters. The sheared tissue was weighed and soaked in 400. Mu.L of LTRIzol, and after sufficiently grinding the tissue into a homogenate using a tissue grinder, 200. Mu.L of the homogenate was added to 700. Mu.L of LTRIzol, and total RNA was extracted by the TRIzol method according to 2.3.2. The viral load in the samples was detected by RT-qPCR reaction using total RNA as template.
4. Histopathological examination
Taking a picture of macroscopic tissue lesions, recording, cutting lesion tissues, fixing by 4% paraformaldehyde, making pathological sections, carrying out HE staining, and analyzing pathological change conditions.
5. Result processing and analysis
Clinical symptoms of Coxsackie group B type 3 pathogens infected by Syrian hamsters are observed, detoxification conditions and histopathological changes are detected, and the virus infection process and the success or failure of modeling are analyzed. Clinical observations showed that elevated body temperature occurred in both nasal drip and intraperitoneal syrian hamsters after infection over time (fig. 1) and white herpes occurred in the lips (fig. 2). RT-qPCR results indicated that there was a detectable load in pharyngeal swabs, nasal lavage and fecal samples from both Syrian hamsters (FIGS. 6-8). The serum biochemical indices myocardial enzyme were significantly altered (fig. 4). Gross dissection can show that the abdominal cavity injection group has the phenomena of hepatomegaly, lung hemorrhage, cerebral thrombosis, stomach wall congestion, submaxillary lymph node hemorrhage and the like; the nasal drip group has the phenomena of peritoneal effusion, pulmonary hemorrhage, liver edge hemorrhage, bleeding spots of cervical lymph nodes and the like. The tissue viral load results showed that viral nucleic acid was detected in the major visceral organs, nervous tissue, respiratory tract, and digestive tract tissues of both groups of animals (fig. 9-13). Based on clinical manifestations and data results, and combined with pathological examination results (fig. 14-15), it was determined that syrian hamster had typical symptoms of hand-foot-and-mouth disease and associated complications such as viral myocarditis, encephalitis, liver injury, lung injury, etc. after CV-B3 infection.
In conclusion, the Coxsackie type B3 pathogen is infected by means of intraperitoneal injection and nasal drip, clinical symptoms and index observation are carried out, and model evaluation and analysis are carried out by combining a PCR detection technology, so that the two methods can be used for constructing the Syrian hamster model for researching the Coxsackie type B3 pathogen. The modeling method accords with the enterovirus propagation mode and the morbidity rule of clinical hand-foot-and-mouth disease patients, and is simple and feasible, short in modeling period, high in repeatability and stable in model. Has important use value in the aspects of developing the way and mechanism research of Coxsackie group B3 pathogen infected central nerve, drug evaluation, vaccine research and development evaluation and the like.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (6)
1. A construction method of a CV-B3 infected rodent model is characterized in that: infecting CV-B3 to the rodent model by means of intraperitoneal injection or nasal cavity instillation, and establishing the CV-B3 infected rodent model.
2. The method of claim 1, wherein the rodent model infected with CV-B3 is selected from the group consisting of: the rodent is selected from a 4 week old female syrian hamster.
3. The method of claim 1, wherein the rodent model infected with CV-B3 is selected from the group consisting of: the virus titer of the intraperitoneal injection infection is 10 6.75 CCID 50 mL, infection dose 100. Mu.L.
4. The method of constructing a rodent model of CV-B3 infection as claimed in claim 1, wherein: the virus titer of the nasal drip infection is 10 6.75 CCID 50 mL, infectious dose 100. Mu.L.
5. Use of a rodent model of CV-B3 infection prepared by a method of construction according to any of claims 1 to 4 in the screening or identification of a drug capable of preventing, ameliorating or treating CV-B3 infection.
6. Use of a rodent model of CV-B3 infection prepared by the method of construction according to any of claims 1 to 3 in screening or identifying vaccines capable of preventing CV-B3 infection.
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Application publication date: 20221021 |