Mouse model for treating in-situ pulmonary nodules by using bacterial lysate as well as construction method and application of mouse model

Abstract

The invention discloses a mouse model for treating in-situ pulmonary nodules by using bacterial lysates as well as a construction method and application of the mouse model. The method comprises the following steps: constructing an in-situ pulmonary nodule KT mouse model, instilling adenovirus, randomly dividing the model into an in-situ pulmonary nodule control group and a treatment group, performing bacterial lysate administration on mice in the treatment group at the maximum treatment dosage of 35mg / kg / d, and administering the same dosage of PBS solution to the control group. According to the invention, the possibility of killing cancer cells and preventing precancerous lesion progression by promoting autoimmunity of mice through oral immunostimulation drugs is provided for the first time, namely, the curative effect and mechanism of new drugs are researched through a mouse model, so that the clinical indications of the drugs are widened, and the blank field of clinical pulmonary nodule drug treatment is supplemented.

Description

Technical Field

[0001] The present invention relates to biomedical technology, and in particular to a bacterial lysate-treated in situ pulmonary nodule mouse model, a construction method and an application thereof. Background Art

[0002] Bacterial lysate (OM-85) is an oral immunostimulatory drug that has been marketed. It contains extracts from eight common pathogenic bacterial species (Haemophilus influenzae, Pneumococcus, Klebsiella pneumoniae, Klebsiella odorifera, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus viridans, and Neisseria catarrhalis). It is mainly used clinically to prevent respiratory tract infections and is a promising chemical prevention strategy. Its principle is to stimulate the activity of peripheral blood monocytes and alveolar macrophages, accelerate T lymphocyte maturation, correct the imbalanced ratio of T suppressor / T helper cells, increase T lymphocyte reactivity, and increase NK cell activity. However, the therapeutic effect on in situ pulmonary nodules has not been reported yet.

[0003] Bacterial lysate (OM-85) is commonly used to prevent and treat respiratory infections. Its main mechanism of action is to activate the innate immune system and enhance the self-defense ability of immune cells. However, the application of OM-85 in the prevention and treatment of in situ pulmonary nodules is a completely new idea, involving the regulation of the "self-surveillance" function of the immune system. The treatment of respiratory infections focuses on enhancing the ability of the immune system to resist foreign pathogens, while the management of in situ pulmonary nodules is more complicated, involving the identification and removal of abnormal cells. This transformation requires a breakthrough in existing technologies and theoretical frameworks, including in-depth research on the immunomodulatory mechanism of drugs and specific designs targeting the pathological characteristics of pulmonary nodules.

[0004] The active ingredients in bacterial lysates are extracted from eight common respiratory pathogens. They activate the host defense mechanism through the mucosal immune system, have specific and non-specific immunostimulatory effects, stimulate and activate cellular immunity and humoral immune responses in the body, activate mucosal lymphoid tissue, stimulate complement and cytokine production, and promote the secretion of secretory immunoglobulin by the tracheal mucosa. Clinically, it can be used as an oral immunostimulant to prevent repeated respiratory infections and acute attacks of chronic bronchitis. In human experiments, bacterial lysates can stimulate the activity of peripheral blood monocytes and alveolar macrophages, accelerate the maturation of T lymphocytes, increase the level of sIgA in salivary and bronchoalveolar lavage, and correct the imbalanced ratio of T suppressor / T helper cells, and increase the reactivity of T lymphocytes and the activity of antiviral interferons. Oral Fanfushu has achieved good results in the treatment of acute attacks of chronic bronchitis. It can enhance the patient's immunity, reduce the number of acute attacks of chronic bronchitis, shorten the course of the disease, alleviate symptoms, reduce the number of people suffering from bronchopneumonia or pneumonia and the number of hospitalizations, and achieve the purpose of preventing and treating acute attacks of chronic bronchitis. It significantly reduces the incidence of acute exacerbations in patients with a history of chronic bronchitis and mild COPD, and is well tolerated.

[0005] Ground glass opacity (GGO) is becoming an important clinical dilemma in oncology as its diagnosis is increasing in clinical practice. In general, persistent GGO on CT may indicate neoplastic disease, and when solid components are present, the likelihood of malignancy is higher. There are currently no rules for the diagnosis and intervention of GGO in clinical practice, and it is difficult to simplify because there is always variability between observers and measurement reproducibility is particularly difficult. According to the current basic clinical consensus, a higher risk of malignancy will automatically trigger more aggressive follow-up, including more diagnostic tests, increased patient anxiety, and additional radiation exposure and costs. The risk of lung tissue loss caused by lung tissue resection and complications of resection of potential malignant nodules are also of concern. However, to date, no well-established clinical trials have been used to provide preliminary evidence for the treatment of precancerous lesions with chemotherapy. Past studies have mostly focused on compounds based on molecular pathways, and rarely involved immunotherapy. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a bacterial lysate for treating an in situ pulmonary nodule mouse model and its construction method and application.

[0007] The purpose of the present invention is to provide the application of bacterial lysates in the treatment of in situ pulmonary nodule mouse models and related mechanism research. The application of the cell lysates in the treatment of in situ pulmonary nodule mouse models is to solve the technical problem that there is no application of bacterial lysates in the treatment of in situ pulmonary nodule mouse models in the prior art. Bacterial lysates are used to treat in situ pulmonary nodule mouse models, promote early intervention, prevent pulmonary nodule malignancy, and conduct drug efficacy and mechanism research at the same time.

[0008] Technical solution: The first aspect of the present invention provides a method for constructing an in situ pulmonary nodule mouse model for treating an in situ pulmonary nodule with the bacterial lysate. An in situ pulmonary nodule KT mouse model is constructed, adenovirus is instilled, and the mice are randomly divided into an in situ pulmonary nodule control group and a treatment group. The mice in the treatment group are administered bacterial lysate at a maximum therapeutic dose of 35 mg / kg / d, and the control group is given the same dose of PBS solution.

[0009] Furthermore, the administration method is oral, once a day, for 10 consecutive days, followed by a 10-day break, and repeated for 3-5 courses.

[0010] Furthermore, a method for constructing an in situ pulmonary nodule KT mouse model is as follows: (1) hybridizing loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice; (2) airway instillation of an adenovirus (AdCre) carrying a cutting enzyme activity that recognizes a specific gene sequence is performed on the F0 hybrid mice obtained in step (1) or their offspring.

[0011] Furthermore, the adenovirus (AdCre) is used at a concentration of 2.5×10 7 pfu.

[0012] Furthermore, the mice were divided into groups 2 weeks after the adenovirus instillation.

[0013] Further, the mice were 8-10 weeks old.

[0014] Another aspect of the present invention provides a method for treating an in situ pulmonary nodule mouse model using the bacterial lysate obtained by the method.

[0015] The third aspect of the present invention provides the use of the model in treating mice with in situ pulmonary nodules with bacterial lysates.

[0016] Pulmonary nodules are defined as round, focal, solid or subsolid abnormal lung shadows with a diameter of less than 3 cm found in the lungs through routine chest CT scan or low-dose chest CT screening. Currently, except for early surgical treatment, there is no drug treatment for lung nodules. Regular CT follow-up will inevitably bring radiation exposure. At the same time, the invasive puncture biopsy of lung nodules used in clinical practice will also bring some unnecessary postoperative complications.

[0017] There are significant differences in the mechanisms of action for the treatment of chronic bronchitis and in situ pulmonary nodules. Current studies have confirmed that OM-85 can activate the immune system to treat chronic bronchitis, which mainly enhances the immune defense function, reduces airway inflammation, and prevents acute exacerbations. The key to the treatment of in situ pulmonary nodules is to regulate the "immune surveillance" function of the immune system, activate T cells and natural killer cells to identify and eliminate abnormally proliferating cells, and regulate the tumor microenvironment to inhibit nodule development. The functions and mechanisms involved in OM-85 in these two diseases are different. This method focuses more on describing the ability of OM-85 to enhance the immune surveillance and clearance of abnormal cells by the immune system in in situ pulmonary nodules.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention treats the in situ pulmonary nodule mouse model with bacterial lysates, compares the Micro-CT and pathological changes within 15 weeks between the control group and the treatment group, and clarifies that oral bacterial lysates have the effect of inhibiting invasion and malignant transformation of in situ pulmonary nodules. Oral bacterial lysates can improve the survival time of mice with in situ pulmonary nodules and slow down the invasion trend. These high-risk nodules are likely to be precursors of adenocarcinoma, so the biological activity of bacterial lysates on such nodules may become an intermediate endpoint of future chemoprevention trials, and have an impact on the diagnosis and treatment of early lung cancer and future practice guidelines.

[0020] This invention proposes for the first time the possibility that oral immunostimulatory drugs can promote the autoimmunity of mice to kill cancer cells and prevent the progression of precancerous lesions. That is, the efficacy and mechanism of new drugs are studied through mouse models to broaden the clinical indications of the drug and fill the gaps in clinical drug treatment of pulmonary nodules. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flow chart of the experiment for treating orthotopic lung nodules in mice using bacterial lysates;

[0022] Figure 2 CT and pathological results of mice in the control group and treatment group show tumor growth and infiltration. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0024] The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate in this embodiment comprises the following steps:

[0025] (I) Construction of an orthotopic lung nodule KT mouse model (for the specific construction method, see patent CN202310754130.3):

[0026] (1) Cross loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice;

[0027] (2) Performing airway instillation of an adenovirus (AdCre) carrying a cutting enzyme activity that recognizes a specific gene sequence into the F0 hybrid mice or their offspring obtained in step (1).

[0028] In this example, the concentration of adenovirus (AdCre) used was 2.5×10 7 pfu, because low dose (2.5*10 7 The pfu)KT model has the characteristic of slower lesion progression, which provides a good observation time window for observing the process of in situ pulmonary nodules transforming from the benign stage to the malignant stage, especially the pathophysiological changes in the microenvironment of early in situ pulmonary nodules. It can better provide the time for drug efficacy to take effect, and is more in line with the clinical development process of pulmonary nodules.

[0029] Two weeks after the mice were instilled with adenovirus, they were randomly divided into a treatment group and a control group. The treatment group was intervened by oral bacterial lysate, and the control group was given the same dose of PBS buffer. The tumor growth of the mice, histopathology, immunofluorescence, and distribution of immune cells in flow cytometry were observed, and the immune microenvironment was analyzed using single-cell sequencing (scRNA-seq) and spatial transcriptome technology.

[0030] (II) Construction of an orthotopic lung nodule treatment mouse model

[0031] 8-10 week old KT mice were selected and randomly divided into 2 groups, namely the control group and the treatment group. According to the literature, 2 weeks after modeling, the mice in the treatment group were given the maximum therapeutic dose of 35 mg / kg / d, and the control group was given the same dose of PBS solution. The administration method was oral, once a day, for 10 consecutive days, rest for 10 days, and repeated for 3-5 courses ( Figure 1 ).

[0032] The CT follow-up time was 2 weeks, 5 weeks, 10 weeks, and 13 weeks after modeling. The survival curve was obtained by observing the tumor growth of mice, and the immune microenvironment was analyzed using tissue pathology, Micro-CT and other technologies.

[0033] (III) Results

[0034] Micro-CT and pathological HE results showed that the tumor burden in the KT mouse treatment group was reduced compared with the control group ( Figure 2 ).

[0035] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate, characterized in that: A KT mouse model of orthotopic pulmonary nodules was established, and adenovirus was instilled. The mice were randomly divided into an orthotopic pulmonary nodule control group and a treatment group. The mice in the treatment group were administered with bacterial lysate at the maximum therapeutic dose of 35 mg / kg / d, and the mice in the control group were given the same dose of PBS solution.

2. The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate according to claim 1, characterized in that: The administration method is oral, once a day, for 10 consecutive days, rest for 10 days, and repeat 3-5 courses of treatment.

3. The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate according to claim 1, characterized in that: A method for constructing an orthotopic pulmonary nodule KT mouse model: (1) hybridizing loxp-stop-loxp KrasG12D transgenic mice with loxp-Tgfbr2-loxp transgenic mice; (2) airway instilling an adenovirus (AdCre) carrying a cutting enzyme activity that recognizes a specific gene sequence into the F0 hybrid mice obtained in step (1) or their offspring.

4. The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate according to claim 3, characterized in that: The adenovirus (AdCre) was used at a concentration of 2.5×10 7 pfu.

5. The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate according to claim 1, characterized in that: The mice were divided into groups 2 weeks after the adenovirus instillation.

6. The method for constructing a mouse model of in situ pulmonary nodules treated with bacterial lysate according to claim 1, characterized in that: Mice were 8-10 weeks old.

7. Treating an orthotopic pulmonary nodule mouse model with the bacterial lysate obtained by the method according to any one of claims 1 to 6.

8. Use of the model of claim 7 in treating mice with orthotopic pulmonary nodules with bacterial lysates.

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