A method for constructing a mouse model of severe pneumonia infected by human respiratory syncytial virus and application thereof

Abstract

The application discloses a method for constructing a mouse severe pneumonia animal model infected by human respiratory syncytial virus and application, and relates to the technical field of viral pneumonia animal models. The method comprises the following steps: inoculating human respiratory syncytial virus on humanized IGF1R transgenic BALB / c mice, causing intra-pulmonary infection of bronchioles and / or lung tissues of the humanized IGF1R transgenic BALB / c mice, and obtaining the mouse severe pneumonia animal model infected by human respiratory syncytial virus. The method makes the mice more susceptible to human respiratory syncytial virus, and solves the problems that a conventional BALB / c mouse model cannot reproduce most of the human disease courses and generate high-titer virus proliferation, and cannot cause severe clinical symptoms due to semi-containment infection of inbred mice to human respiratory syncytial virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of viral pneumonia animal models, in particular to a method for constructing a mouse severe pneumonia animal model infected by human respiratory syncytial virus and application thereof. BACKGROUND

[0002] At present, the lack of suitable animal models to simulate the course of respiratory syncytial virus (RSV) in humans has greatly limited the development of RSV vaccines and drugs. How to construct a mouse severe pneumonia animal model infected by human respiratory syncytial virus and determine its consistency with clinical symptoms has become an important direction of current research and development.

[0003] The existing technical means for constructing a mouse severe pneumonia animal model infected by human respiratory syncytial virus mainly include: virus mouse lung adaptation, screening of more virulent mutant strains; and selection of immune-deficient animals for modeling.

[0004] However, the above technical means have limitations. The virus mutation in the virus mouse lung adaptation process has uncertainty, which leads to the fact that there is no stable mouse lung-adapted strain of human respiratory syncytial virus with high virulence. The immune-deficient mouse model has a large gap with the human body, and the course after infection with the virus is different, which has no actual reference value for human clinical infection with respiratory syncytial virus. The above technical means do not fundamentally solve the problem of weak virulence of human respiratory syncytial virus in mice.

[0005] The present application aims to develop a new method for constructing a mouse severe pneumonia animal model infected by human respiratory syncytial virus, which fundamentally solves the problem of difficulty in infection of human respiratory syncytial virus in mice, and uses the model for drug evaluation. SUMMARY

[0006] The present application aims to provide a method for constructing a mouse severe pneumonia animal model infected by human respiratory syncytial virus and application thereof, so as to solve the problems existing in the prior art. The construction method makes the mouse more susceptible to human respiratory syncytial virus, and solves the problem that the inbred mouse semi-containment infection with human respiratory syncytial virus causes the conventional BALB / c mouse model to be unable to reproduce most of the course of the human body and produce high-titer virus proliferation, and also unable to cause severe clinical symptoms.

[0007] To achieve the above object, the present application provides the following scheme:

[0008] The application provides a method for constructing a mouse severe pneumonia animal model infected with human respiratory syncytial virus, comprising the following steps: inoculating human respiratory syncytial virus on humanized IGF1R transgenic BALB / c mice to cause infection and lesions in the lung bronchioles and / or lung tissues of the humanized IGF1R transgenic BALB / c mice, and obtaining the mouse severe pneumonia animal model infected with human respiratory syncytial virus.

[0009] Further, the inoculation is performed by nasal drop infection.

[0010] Further, the human respiratory syncytial virus is inoculated twice continuously.

[0011] Further, the interval between the two inoculations is 2 days.

[0012] Further, the virus titer of single inoculation is 100 TCID 50 .

[0013] The application also provides a mouse severe pneumonia animal model infected with human respiratory syncytial virus, which is constructed by the method.

[0014] The application also provides application of the mouse severe pneumonia animal model infected with human respiratory syncytial virus in drug efficacy evaluation.

[0015] The application discloses the following technical effects:

[0016] The application uses humanized IGF1R transgenic mice to construct a mouse severe pneumonia animal model, so that the mice are more susceptible to human respiratory syncytial virus, and solves the problem that the inbred mouse is semi-containedly infected with human respiratory syncytial virus, so that the conventional BALB / c mouse model cannot reproduce most of the human disease courses and produce high-titer virus proliferation, and also cannot cause severe clinical symptoms. IGF1R is a key receptor of respiratory syncytial virus infection, and the use of humanized IGF1R transgenic BALB / c mice is an innovative measure for constructing a respiratory syncytial virus infection severe pneumonia model suitable for drug evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 Lung images of mice in different groups after being infected with respiratory syncytial virus; wherein, the BALB / c infection group is the model control group; the transgenic BALB / c infection group is the experimental group;

[0019] Figure 2 The lung index, spleen index and thymus index of mice in different groups are shown in the histogram; the BALB / c infection group is the model control group; the transgenic BALB / c infection group is the experimental group; compared with the normal group # P<0.05, ## P<0.01; compared with the BALB / c infection group ** P<0.01;

[0020] Figure 3 The peripheral blood lymphocyte level of mice in different groups is shown in the histogram; the BALB / c infection group is the model control group; the transgenic BALB / c infection group is the experimental group; compared with the BALB / c infection group * P<0.05;

[0021] Figure 4 The blood routine test results of mice in different groups are shown in the histogram; A is the monocyte examination result; B is the lymphocyte test result; C is the neutrophil test result; in A-C, the BALB / c infection group is the model control group; the transgenic BALB / c infection group is the experimental group; compared with the normal group # P<0.05, ## P<0.01;

[0022] Figure 5 The lung tissue pathological staining (HE x 400) of mice in different groups is shown in the histogram; the BALB / c control group is the model control group; the transgenic BALB / c infection group is the experimental group;

[0023] Figure 6 The lung image of mice after administration in different groups;

[0024] Figure 7 The lung index (A), spleen index (B) and thymus index (C) of mice after administration in different groups are shown in the histogram; compared with the normal group # P<0.05, ## P<0.01; compared with the model group * P<0.05, ** P<0.01;

[0025] Figure 8 The lung tissue inflammatory factors TNF-α (A), IL-6 (B) and IL-1β (C) levels of mice after administration in different groups are shown in the histogram; compared with the normal group ## P<0.01; compared with the model group * P<0.05, ** P<0.01;

[0026] Figure 9 Statistical chart of serum antibody IgA (A), IgG (B) and IgM (C) levels of mice in different groups after administration; compared with the normal group ## P<0.01; compared with the model group * P<0.05, ** P<0.01;

[0027] Figure 10 Lung tissue pathological staining chart (HE x 200) of mice in different groups after administration. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation of the present application.

[0029] It should be understood that the terms used in the present application merely describe particular embodiments, and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, as well as any other stated value or intermediate value within the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the content of this specification and that of any document incorporated by reference, the content of this specification controls.

[0031] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean including, but not limited to.

[0033] Terminology

[0034] The “IGF1R” of this invention refers to the insulin-like growth factor 1 receptor, which is a protein found on the surface of human cells. It is the cell surface receptor for the hormone insulin-like growth factor 1 (IGF-1) and belongs to the tyrosine kinase receptor family.

[0035] Example 1

[0036] 1. Experimental Materials

[0037] Human respiratory syncytial virus strain 18537 (VR-1580) TM );

[0038] Ordinary BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and humanized IGF1R transgenic BALB / c mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with the strain number of the humanized IGF1R transgenic BALB / c mice being T056078.

[0039] 2. Experimental grouping and model building

[0040] Ordinary BALB / c mice were randomly divided into a normal control group and a model control group, with 10 mice in each group. Humanized IGF1R transgenic BALB / c mice were used as the experimental group, with 6 mice in each group. Except for the normal control group, the other two groups of mice were administered 100 TCID50 doses of chlorhexidine glycinate. 50 Viral fluid (VR-1580) TM Nasal drops were administered for infection, 35 μL per dose, and the same method was repeated on the 3rd day after infection.

[0041] 3. Indicator Testing and Results

[0042] Lung images of mice were observed using CT scans, and lung, spleen, and thymus indices were calculated. Peripheral blood CD3 was detected by flow cytometry. + CD4 + CD8 + Lymphocyte percentage; percentage of neutrophils, lymphocytes, and monocytes in white blood cells as determined by routine blood tests; HE staining to observe pathological changes in mouse lung tissue, bronchioles, and interstitium.

[0043] 3.1 Effects of respiratory syncytial virus on lung imaging in mice

[0044] Four days after the first intranasal infection, the mice were lightly anesthetized with isoflurane, and lung imaging was performed using a small animal in vivo CT imaging system.

[0045] like Figure 1The normal group of mice showed clear lung texture, no abnormal distribution, and no exudation or space-occupying lesions in the lung parenchyma. The BALB / c infected mice showed massive infiltrating shadows. The humanized IGF1R transgenic BALB / c infected mice showed large patchy consolidation shadows and diffuse ground glass shadows in the lungs.

[0046] 3.2 Influence of respiratory syncytial virus on lung index, thymus index and spleen index of mice

[0047] On the 5th day after the first nasal infection of the mice, the eyeballs were removed for blood collection and dissection after weighing. The lungs, spleen, thymus and other organs were removed and weighed to calculate the lung index, spleen index and thymus index.

[0048] Compared with the normal group, the lung index of the transgenic BALB / c infected mouse group was significantly increased (P<0.01), and the spleen index and thymus index were significantly reduced (P<0.05, P<0.01). Compared with the BALB / c infected group, the lung index of the transgenic BALB / c infected mouse group had no significant difference, and the spleen index and thymus index were significantly reduced (P<0.01), and the statistical results are shown in Figure 2 .

[0049] Organ index = organ wet weight (g) / body weight (g) x 100%.

[0050] 3.3 Influence of respiratory syncytial virus on peripheral blood lymphocyte level of mice

[0051] On the 5th day after the first nasal infection of the mice, 150 μL of blood was taken from the eyeball into an anticoagulant tube, and staining and red blood cell lysis were performed according to the instructions of the flow antibody and red blood cell lysis solution. 10 mL of PBS was added to terminate lysis, and centrifuged at 2000 r / min for 5 min at 4°C, and the supernatant was discarded. 100 μL of PBS containing 2% FBS and 4% formaldehyde solution were added to resuspend the cells, which were stored at 4°C in the dark for the detection of CD3 + , CD4 + , CD8 + T cells.

[0052] Compared with the BALB / c infected group, the CD4 + T cells in the transgenic BALB / c infected group were significantly reduced (P<0.05), and the statistical results are shown in Figure 3 .

[0053] 3.4 Influence of respiratory syncytial virus on blood routine of mice

[0054] On the 5th day after the first nasal infection of the mice, 100 μL of blood was taken from the eyeball into an anticoagulant tube for mouse blood routine detection. Compared with the normal group, the percentage of mononuclear cells in the transgenic BALB / c infected group was significantly increased (P<0.01), and the statistical results are shown in Figure 4.

[0055] 3.5 Effects of respiratory syncytial virus on mouse lung tissue pathology

[0056] The upper lobe of the left lung of a mouse was fixed in a 4% paraformaldehyde solution. After fixation, dehydration, paraffin embedding, sectioning, and HE staining, the extent of damage to the alveoli, interstitium, and bronchi in the mouse lung was observed under a microscope. Figure 5 As shown, in the normal control group, no inflammatory exudate, edema, or other lesions were observed in the lung interstitium, and no inflammation was observed around the bronchioles; the tissue structure was normal. In the BALB / c infection group, the vascular intima shifted, the lung interstitium thickened, the tissue showed mild edema, and B cell proliferation. In the transgenic BALB / c infection group, there was extensive inflammation and congestion around the blood vessels in the lung tissue, mainly lymphocytes, with partial sloughing of the vascular intima and a small number of eosinophils. Compared with the normal group, the lesion grade of the bronchioles and lung tissue in the transgenic BALB / c infection group was significantly increased (P<0.01). Compared with the BALB / c infection group, the lesion grade of the bronchioles in the transgenic BALB / c infection group was significantly increased (P<0.05). The statistical results are shown in Tables 1 and 2.

[0057] Table 1. Effects of respiratory syncytial virus on bronchioles in the lungs of mice.

[0058]

[0059] Table 2 Effects of respiratory syncytial virus on mouse lung tissue

[0060]

[0061] Example 2

[0062] 1. Experimental Materials

[0063] Standard BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), and humanized IGF1R transgenic BALB / c mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). Ribavirin granules (Yaoda Pharmaceutical Co., Ltd.), and Lianhua Qingwen granules (Shijiazhuang Yiling Pharmaceutical Co., Ltd.).

[0064] 2. Experimental grouping and drug administration

[0065] Humanized IGF1R transgenic BALB / c mice were randomly divided into four groups: a model group, a ribavirin group (82.5 mg / kg / d), a high-dose Lianhua Qingwen group (3.3 g / kg / d), and a low-dose Lianhua Qingwen group (1.65 g / kg / d), with eight mice in each group. The mice were administered 100 TCID45 doses of ribavirin. 50 Viral fluid (VR-1580) TM) nose infection, 35 μL per mouse, and nose infection again on the 3rd day of infection in the same way, and the second infection was 2 days later, to get the severe pneumonia model mice. The normal BALB / c mice were used as the normal control group, 8 mice per group. The mice in each group were given the drugs and doses on the first day of infection for 4 consecutive days.

[0066] 3. Drug evaluation

[0067] The lung image of the mice was observed by CT, and the lung, spleen, and thymus indices were calculated. The contents of inflammatory factors TNF-a, IL-6, and IL-1β in the lung tissue and the contents of serum antibodies IgA, IgG, and IgM were detected by ELISA. The pathological changes of the lung tissue, bronchioles, and interstitium of the mice were observed by HE staining.

[0068] 3.1 Effect of Lianhuaqingwen granules on the lung imaging of infected mice

[0069] On the 4th day after the first nose infection, the mice were lightly anesthetized with isoflurane, and the lung imaging of the mice was examined by a small animal live CT imaging instrument. As shown in Figure 6 , the lung texture of the mice in the normal group was clear, and the distribution was normal. No exudation or space-occupying lesions were found in the lung parenchyma. In the model group, large patches of consolidation shadows and diffuse ground glass shadows were visible in the lung, the air density of the lung was reduced, and the image was gray, indicating pulmonary fibrosis and pulmonary edema. Compared with the model group, the lung structure was relatively complete in the high and low dose Lianhuaqingwen groups, and the distribution of ground glass shadows was reduced.

[0070] 3.2 Effect of Lianhuaqingwen granules on the lung, spleen, and thymus indices of infected mice

[0071] On the 5th day after the first nose infection, the mice were weighed and dissected, and the lung, spleen, and thymus were removed and weighed to calculate the lung, spleen, and thymus indices. Compared with the normal group, the lung index of the model group was significantly increased (P < 0.01), and the spleen and thymus indices were significantly decreased (P < 0.05). Compared with the model group, the lung index of the low-dose Lianhuaqingwen group was significantly decreased (P < 0.05), and the spleen and thymus indices showed no significant change. The statistical results are shown in Figure 7 .

[0072] 3.3 Effect of Lianhuaqingwen granules on the inflammatory factors in the lung tissue of infected mice

[0073] The lung tissue inflammatory factors TNF-a, IL-6, and IL-1β were detected according to the mouse enzyme-linked immunosorbent assay kit instructions. Compared with the normal group, the expression levels of TNF-a, IFN-γ, and IL-6 in the model group were significantly increased (P < 0.01); compared with the model group, the low-dose Lianhuaqingwen group could significantly reduce the contents of these three inflammatory factors (P < 0.01), and the statistical results are shown in Figure 8 .

[0074] 3.4 Effect of Lianhuaqingwen Granules on Serum Antibodies in Infected Mice

[0075] The serum antibodies IgA, IgG, and IgM were detected according to the mouse enzyme-linked immunosorbent assay kit instructions. Compared with the normal group, the expression levels of IgA, IgG, and IgM in the model group were significantly increased (P < 0.01); compared with the model group, the high and low dose Lianhuaqingwen groups could significantly reduce the contents of the three serum antibodies (P < 0.01), and the statistical results are shown in Figure 9 .

[0076] 3.5 Effect of Lianhuaqingwen Granules on the Pathology of Lung Tissue in Infected Mice

[0077] The left upper lobe of the lung of the mouse was fixed in 4% paraformaldehyde solution. After the tissue was fixed, dehydrated, paraffin-embedded, sectioned, and HE stained, the damage degree of the alveoli, interstitium, and bronchus in the lung of the mouse was observed under a microscope. As shown in Figure 10 , the lung tissue structure of the normal group was clear, the alveolar epithelial cells were not degenerated or necrotic, the interstitium was not congested, edematous, or infiltrated with inflammatory cells, the bronchial epithelial cells were arranged in order, and no edema, degeneration, or other abnormal changes were observed. The lung tissue of the model group was diffuse or massive hemorrhage, necrosis, and edema, the alveolar cavity had a large number of inflammatory exudates, the alveolar wall was slightly to severely thickened, the interstitium was congested and edematous, accompanied by a large number of inflammatory cell infiltration mainly composed of neutrophils, the bronchial epithelial cells were swollen and degenerated, and part of the cells were necrotic and shed, which had a significant difference compared with the normal control group (P < 0.01). The lung tissue damage of the low dose Lianhuaqingwen group was significantly reduced, the lung inflammation lesion range was reduced, the alveolar wall was slightly or slightly thickened, accompanied by mild inflammatory cell infiltration, and the bronchial epithelial cells were slightly swollen, which had a significant difference compared with the model group (P < 0.05, P < 0.01). The statistical results are shown in Tables 3 and 4.

[0078] Table 3 Effect of Lianhuaqingwen Granules on the Bronchus in the Lung of RSV-Infected Mice

[0079]

[0080] Table 4 Effect of Lianhuaqingwen Granules on the Lung Tissue of RSV-Infected Mice

[0081]

[0082] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for constructing a mouse model of severe pneumonia infected by human respiratory syncytial virus, characterized in that, The step of obtaining the animal model of severe pneumonia in mice infected with human respiratory syncytial virus by inoculating human respiratory syncytial virus into humanized IGF1R transgenic BALB / c mice to induce intra-pulmonary bronchiole and / or lung tissue infection in the humanized IGF1R transgenic BALB / c mice; The inoculation is performed by nasal drop infection; The human respiratory syncytial virus is inoculated twice continuously; The interval between the two inoculations is 2 days; The virus titer of the single inoculation was 100 TCID 50 .

2. Application of the animal model of severe pneumonia in mice infected with human respiratory syncytial virus obtained by the construction method according to claim 1 in the evaluation of drug efficacy.

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