CN113041267A - Construction method of animal model simulating characteristics of various HFRS diseases and application thereof - Google Patents
Construction method of animal model simulating characteristics of various HFRS diseases and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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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- 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 relates to a construction method of an animal model, in particular to a construction method and application of an animal model simulating characteristics of various HFRS diseases. The method is to inject HTNV 76-118 virus into the abdominal cavity to infect NLRC3KO mice, and the infection dose is 5 multiplied by 105PFUs/50. mu.L/mouse; the disease is characterized by: the kidney tubules of the mouse model are diffusely dilated, and the capillary vessel of the renal medulla is bled; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, denatured and necrotic; the number of platelets decreases, the number of leukocytes decreases, and the liver and kidney function is damaged. The animal model constructed by the invention can be used for screening HFRS disease drugs and evaluating the curative effect.
Description
Technical Field
The invention relates to a construction method of an animal model, in particular to a construction method of an animal model simulating characteristics of various HFRS diseases and application thereof.
Background
Hantavirus (Hantavirus) belongs to the genus orthohantavirus of hantaviridae, order bunyaviridae, is a enveloped, single-stranded RNA virus whose genome consists of 3 segments, of which the large (L) segment encodes the viral RNA-dependent RNA polymerase (RNA-dependent RNA polymerase), the middle (M) segment encodes the viral envelope glycoproteins (Gn and Gc), and the small (S) segment encodes the viral Nucleoprotein (NP). Humans are infected with hantavirus, mainly by contact with the excretions of their natural host (rodents) or by direct bites, causing mainly two serious diseases, nephrotic syndrome with renal syndrome Hemorrhagic Fever (HFRS) and hantavirus cardiopulmonary syndrome (HPS), respectively. HFRS is a natural epidemic disease characterized mainly by fever, hemorrhage, and acute renal function injury.
HTNV infects a natural host and does not have obvious clinical symptoms, but is in a continuous toxic state, so that a disease animal model infected by the HTNV is lacked at present, and the establishment of a clinical treatment medicament and an HTNV vaccine evaluation system is restricted. The ideal animal model of viral diseases is used for researching the mechanisms of invasion, replication, transmission, pathogenesis, regression and the like of viruses in a host body, and provides an important tool for clinical treatment drugs and vaccine evaluation. Although it was clear in the 50 s that neonatal newborn mice, vaccinated with HTNV, were capable of causing fatal disease, they were primarily responsible for neurological changes in mice, not consistent with HFRS disease characteristics, and thus were limiting. In addition, SCID mice reported in earlier documents can be used as an evaluation system of HTNV infected animals, and are based on that high-level virus specific antigens can be detected in liver, spleen and lung of mice after virus infection, and meanwhile, obvious pathological changes appear in liver, spleen and lung tissues, but no obvious pathological changes appear in heart, brain and kidney tissues. However, this mouse model of SCID does not mimic well the major disease features of HFRS: acute injury to renal function and thrombocytopenia, with certain limitations.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a construction method and application of an animal model simulating characteristics of various HFRS diseases, and pathological changes induced by the animal model can simulate clinical characteristics of typical HFRS diseases and better accord with a natural infection process of HTNV to human beings.
In order to achieve the above object, the present invention adopts the following technical solutions:
in a first aspect of the invention, a method of constructing an animal model that mimics many of the characteristics of HFRS disease is provided by injecting HTNV 76-118 virus into infected mice at a dose of 4.98X 105-5.02×105 PFUs/50μL/30g；
The disease is characterized by: the kidney tubules of the mouse model are diffusely dilated, and the capillary vessel of the renal medulla is bled; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, denatured and necrotic; the number of platelets decreases, the number of leukocytes decreases, and the liver and kidney function is damaged.
Further, the injection is intraperitoneal injection.
Further, the mouse is an NLRC3KO mouse.
In a second aspect, the invention provides the use of a non-diagnostic and non-therapeutic animal model constructed according to the method, in particular for screening or identifying substances for the treatment or alleviation of a plurality of HFRS diseases, wherein said HFRS clinical disease is characterized by: diffuse dilation of renal tubules, renal medullary capillary hemorrhage; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, denatured and necrotic; the number of platelets decreases, the number of leukocytes decreases, and the liver and kidney function is damaged.
It is a third object of the invention to provide a non-diagnostic and non-therapeutic method for screening or identifying potential therapeutic agents for the treatment of a variety of HFRS disorders, comprising the steps of:
s1, applying candidate substances to the animal model constructed by the method;
s2, performing histopathology, hematology and serum biochemical index determination and analysis on the animal model, and comparing the animal model with a control group;
wherein, if the indicator indicative of the clinical condition of HFRS is improved in the animal model administered the candidate substance as compared to a control, the substance is indicative of a potential therapeutic agent for a plurality of clinical conditions of HFRS.
Compared with the prior art, the invention has the following beneficial effects:
1. NLRC3KO mice have potential advantages in animal model studies of diseases related to viral infection. NLRC3 is NOD-like receptor family protein 3 (NLRC 3) containing a caspase recruitment structure domain, is a member of NOD-like receptor (NLRs) family, is an inherent immune regulator in cytoplasm and mainly plays an immunosuppressive effect in vivo, NLRC3 can be directly combined with STING to inhibit STING transport, so that interaction between STING and TBK1 is blocked, and generation of downstream type I IFN is finally inhibited. And the generation of the type I IFN plays an important role in the process of resisting virus infection, so that the NLRC 3-deficient mouse has important significance in the research of virus infection.
2. The model constructed by the method provided by the invention can simulate various HFRS clinical disease characteristics, such as acute injury of renal function, decrease of platelet number and the like, and is mainly characterized in that the mouse kidney tubule of the model is diffusely dilated, and the capillary vessel of renal medulla is hemorrhagic; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, degenerative and necrotic. In addition, the decrease of the number of circulating platelets accompanied by impairment of the liver and kidney functions is suggested by hematology and serum biochemical index measurement. The results show that the model constructed by the method of the invention has obvious pathological changes such as liver and kidney function damage, spleen hematopoietic disorder and the like, and the model can be used for screening HFRS disease drugs and evaluating the curative effect.
Drawings
FIG. 1 shows the body weight, body temperature and visceral viral load of NLRC3KO and C57BL/6WT mice; wherein FIG. 1-A shows the body weight change of a HTNV infected mouse; 1-B shows the body temperature change of mice after HTNV infection; 1-C shows the distribution of viral load in the spleen, kidney and blood of mice at various time points after HTNV infection.
FIG. 2 shows the variation of blood markers of NLRC3KO and C57BL/6WT mice at different time points after virus infection; wherein FIG. 2-A shows the absolute platelet counts in whole blood of two groups of mice; FIG. 2-B shows the absolute counts of leukocytes in whole blood of two groups of mice; FIG. 2-C shows the AST levels in serum of two groups of mice; FIG. 2-D shows the levels of LDH in serum of two groups of mice; FIG. 2-E shows the amount of CK-MB in the sera of two groups of mice; FIG. 2-F shows the UA content in serum of two groups of mice.
FIG. 3 is a change in renal pathology following HTNV infection in NLRC3KO and C57BL/6WT mice; wherein, FIG. 3-A shows the HE staining results of renal medulla of two groups of mice 6 days after virus infection; FIG. 3-B shows statistics of HE staining at different time points after infection of two groups of mice with virus; FIG. 3-C shows the results of renal cortex HE staining after infection of two groups of mice with virus.
FIG. 4 is a pathological change in spleen, lung and liver following HTNV infection in NLRC3KO and C57BL/6WT mice; wherein, FIG. 4-A shows statistics of spleen HE staining at different time points after infection of two groups of mice with virus; FIG. 4-B shows spleen HE staining results after infection of two groups of mice with virus; FIG. 4-C shows statistics of lung HE staining at different time points after infection of two groups of mice with virus; FIG. 4-D shows lung HE staining results after infection of two groups of mice with virus; FIG. 4-E shows statistics of HE staining of liver at different time points after infection of two groups of mice with virus; FIG. 4-F shows the results of HE staining of liver after infection of two groups of mice with virus.
FIG. 5 is the expression levels of different inflammatory cytokines in the serum of HTNV infected NLRC3KO and C57BL/6WT mice.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
1. Experimental Material
(1) HTNV strain 76-118: the HTNV virus strain is 76-118 (international standard strain) which is a gift from Hangzhou long-life researchers at the Chinese disease prevention and control center and then stored and amplified in the laboratory. HTNV infects Vero-E6 cells after being poisoned by the brain of a suckling mouse, and viruses obtained by the amplification of the Vero-E6 cells are used for the research after the titer is determined.
(2) C57BL/6WT and NLRC3KO mice: 6-8 weeks old, purchased from Shanghai, Square model organisms, Inc.
2. Experimental methods
(1) NLRC3KO mouse infection experiment
HTNV 76-118 virus was injected intraperitoneally to infect NLRC3KO mice at a dose of 5X 105PFUs/50. mu.L/mouse were used as experimental group and uninfected mice as control group.
Continuously monitoring the body temperature and the body weight of the mouse every day after the infection of the virus, collecting peripheral blood and organs (including heart, liver, spleen, lung, kidney and brain) of the mouse at 3,6,9 and 12 days after the infection, extracting RNA by adopting a Trizol method or a QIAGEN virus RNA extraction kit, and then absolutely quantifying HTNV genes in each sample by adopting a TaKaRa one-step fluorescent quantitative PCR kit; in addition, each organ tissue of the mouse was paraffin-embedded, sectioned, stained with HE staining solution, and then the pathological changes of each organ tissue were observed under an optical microscope.
As a result: as shown in figure 1, NLRC3KO mice began to lose weight on day 4 post virus inoculation and on day 6 post infection, weight was lost to a minimum level indicating that HTNV infected mice had completely developed at this time; whereas the WT group mice did not show significant weight loss throughout the infection. The virus load level of mouse organs at different time points after infection is detected by adopting a qRT-PCR method to evaluate the replication and transmission capability of the virus in the mouse. As a result, HTNV replicates efficiently in spleen, kidney and blood of mice, and NLRC3KO mouse visceral viral load was significantly higher than WT group mice. The virus copy number in the blood of NLRC3KO mice at 3 days after virus infection can be as high as 943 +/-140 copies/mL, and the virus copy number of WT mice is 803 +/-277 copies/mL; on day 6 post-infection, the viral copy numbers in the spleen and kidney of NLRC3KO mice were 3033. + -.175 and 500. + -.87 copies/mg, respectively, while those of WT mice were 1520. + -.425 copies/mg and 270. + -.40 copies/mg, respectively.
As shown in fig. 2, the whole blood of the mice is taken for routine blood test, and the results show that the number of platelets of NLRC3KO mice sharply decreases at 3,6 and 9 days after virus infection, while the number of platelets of WT mice significantly decreases at 3 days after infection, and then returns to the normal level; NLRC3KO mice had a significant decrease in leukocyte numbers at day 3 post viral infection, after which they gradually returned to normal levels; the white blood cell counts of WT mice were significantly reduced on days 3,6, and 9 after virus infection. Biochemical test results showed that at day 3 post-viral infection, NLRC3KO mouse serum LDH levels increased significantly and subsequently decreased; while WT mice did not show significant changes in serum LDH levels. Serum AST levels were significantly elevated in NLRC3KO as well as WT mice on both day 6 and day 9 of viral infection; on day 9 of viral infection, the serum CK-MB levels of NLRC3KO mice were significantly increased, while the serum CK-MB levels of WT mice were not significantly changed. Uric Acid (UA) levels were significantly elevated in serum of HTNV-infected NLRC3KO mice; WT mice have elevated serum UA levels later in viral infection. The above results indicate that NLRC3KO mice were accompanied by persistent impairment of hepatic and renal function following HTNV infection.
As shown in fig. 3, it was found by HE infection that bleeding occurred in capillaries in renal medullary zone of NLRC3KO mice on day 6 after virus infection. In addition, on days 9 and 12 of viral infection, the renal cortex of NLRC3KO mice showed obvious renal tubular expansion, and the lesion degree was significantly higher than that of WT mice. As shown in fig. 4A and 4B, spleens of both groups of mice showed a large number of extramedullary hematopoietic foci on days 6,9, and 12 after viral infection, suggesting a hematopoietic disorder. As shown in fig. 4C and 4D, the mouse lungs were significantly thickened in alveolar walls while undergoing inflammatory cell infiltration on day 6 post viral infection. As shown in fig. 4E and 4F, livers of both groups of mice developed significant inflammatory cell infiltration and degenerative necrosis of hepatocytes at both day 6 and day 9 post viral infection. The above results suggest that the body is accompanied by multi-organ multi-system injury in two groups of mice after HTNV infection, and pathological changes such as pathological expansion of renal tubule and renal medullary hemorrhage in NLRC3 mice can mimic clinical disease characteristics of typical HFRS. And finally, evaluating the response level of the cytokine in the HTNV infection host body by detecting the expression level of the inflammatory cytokine in the serum of the mouse. As shown in FIG. 5, the results show that the serum CXCL1, CCL5, IL-6, IL-12, TNF-alpha, IFN-gamma expression levels of NLRC3KO mice are significantly increased at day 6 and day 9 after viral infection after HTNV infection; the WT mouse serum cytokine response level did not change significantly.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. A method for constructing animal model simulating multiple HFRS disease features that HTNV 76-118 virus is injected into infected mouse at 4.98X 10 times5-5.02×105PFUs/50μL/30g；
The disease is characterized by: the kidney tubules of the mouse model are diffusely dilated, and the capillary vessel of the renal medulla is bled; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, denatured and necrotic; the number of platelets decreases, the number of leukocytes decreases, and the liver and kidney function is damaged.
2. The method of constructing an animal model that models a plurality of HFRS disease characteristics according to claim 1, wherein the injection is intraperitoneal.
3. The method of claim 1, wherein the mouse is an NLRC3KO mouse.
4. Use of a non-diagnostic and non-therapeutic animal model constructed according to the method of claim 1, in particular for screening or identifying, treating or ameliorating a plurality of HFRS diseases, wherein said HFRS clinical disease is characterized by: diffuse dilation of renal tubules, renal medullary capillary hemorrhage; a large number of extramedullary hematopoietic foci appear in the spleen, and liver cells in the liver are edematous, denatured and necrotic; the number of platelets decreases, the number of leukocytes decreases, and the liver and kidney function is damaged.
5. A non-diagnostic and non-therapeutic method for screening for or identifying, treating or ameliorating a plurality of potential therapeutic agents for HFRS disease comprising the steps of:
s1, applying the candidate substance to the animal model constructed by the method of claim 1;
s2, performing histopathology, hematology and serum biochemical index determination and analysis on the animal model, and comparing the animal model with a control group;
wherein an improvement in an indicator indicative of a clinical disorder of HFRS in the animal model administered the candidate substance, as compared to the control group, is indicative of the substance being a potential therapeutic agent for a plurality of clinical disorders of HFRS.
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| CN102441174A (en) * | 2002-03-22 | 2012-05-09 | 美国传染性疾病军医研究所 | DNA vaccines against hantavirus infections |
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