Mouse BHDS pulmonary focal degenerative pulmonary bullous model construction method and application

Abstract

The invention discloses a mouse BHDS pulmonary focal degenerative pulmonary bullous model construction method and application, and the method comprises the following steps: adaptively feeding a healthy mouse for one week, then carrying out HCl injury treatment, and giving two microRNA fitting substances to the mouse twice in a nasal drop mode to obtain the BHDS pulmonary focal degenerative pulmonary bullous model. Expression changes of characteristic molecules in BHDS clinical sample lung tissues exist in the lung tissues of the model constructed by the invention, and the model is most suitable for BHDS-like lung tissue pathology so far. The invention provides a proper tool model for the effectiveness, safety and drug screening of BHDS pulmonary focal degenerative pulmonary bullous (BHD syndrome pulmonary cystic disease) treatment drugs, and ensures the repeatability and high efficiency of the model. Compared with a model constructed by gene knockout, the construction method provided by the invention is more stable in model formation, simpler and more convenient in operation method and experimental technology, and lower in cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a mouse BHDS lung focal regressive bulla model construction method and application. BACKGROUND

[0002] BHD syndrome (Birt-Hogg-Dube Syndrome, BHDS) is an autosomal dominant genetic disease, and a currently known pathogenic gene is FLCN. Folliculin The single copy mutation of the gene can cause lung bulla and / or spontaneous pneumothorax, facial and neck skin follicle fibroma, and kidney cancer. From the clinical practice, BHDS shows racial difference in phenotype: in Europe, most BHDS patients have early skin follicle fibroma; and in Asia, especially in China and Japan, the early phenotype of the patients is multiple focal lung bulla / pneumothorax. Compared with common lung bulla, the lung bulla of BHDS patients is more common, is often found in lung parenchyma, can be found throughout the lung, and the patients have a 7 times higher risk of kidney cancer than normal people, and their first-degree relatives are also high-risk groups.

[0003] At present, the pathological formation mechanism of BHDS lung bulla is still unclear. It has been found in research that the homozygous knockout of FLCN is embryonic lethal, and a model cannot be constructed. When the FLCN of type II alveolar epithelial cells is conditionally knocked out, the increase in epithelial cell apoptosis mediated by the mTOR signaling pathway is observed in the lung tissue of the mouse, and the space in the alveolus is large; the deletion of Flcn in the mesenchymal cells of the mouse leads to the decrease in cell proliferation in the lung tissue of the mouse after birth, the down-regulation of the expression of extracellular collagen Col3a1 and elastin, and the cystic-like change in morphology. The above-mentioned existing researches all observe the extensive tissue "regressive" characteristics in the lung tissue of the animal, do not form the "focal" typical lesion, and are not consistent with the pathological characteristics found in the histopathological study of the BHDS patients, such as the lining of the inner surface of the lung bulla with complete epithelial cells, the thin bulla wall, and the absence of typical "inflammatory reaction" around the bulla. Therefore, until now, an explicit BHDS focal regressive bulla experimental animal model has not been established. SUMMARY

[0004] In view of the defects and deficiencies in the prior art, the purpose of the present application is to provide a mouse BHDS lung focal regressive bulla model construction method.

[0005] Another purpose of the present application is to provide the application of the mouse BHDS lung focal regressive bulla model constructed by the construction method.

[0006] The technical scheme of the present application is as follows: The application discloses a method for constructing a mouse BHDS pulmonary focal regressive bulla model, and the method comprises the following steps: after healthy mice are adaptively fed for one week, the mice are treated by HCl, and two microRNA analogues are given to the mice in a nose drop manner to obtain the mouse BHDS pulmonary focal regressive bulla model.

[0007] Further, the two microRNA analogues comprise mmu-let-7d-5p agomir and mmu-miR-322-5p agomir.

[0008] Further, the two microRNA analogues are given to the mice in the nose drop manner, and the dose of each microRNA analogue is the minimum dose that can induce a bulla phenotype, and the mice are fed for one week; then the two microRNA analogues are given to the mice in the nose drop manner, and the dose of each microRNA analogue is the minimum dose that can induce a bulla phenotype, and the mice are fed for one week.

[0009] Further, the dose of each microRNA analogue is 1.25 nmol per mouse.

[0010] Further, the HCl treatment is 0.1M HCl nose drop treatment for one day, and the dose is 1.5 ml / kg.

[0011] Further, the healthy mice are 6-8-week-old SPF C57BL / 6 mice.

[0012] The mouse BHDS pulmonary focal regressive bulla model obtained by the method according to any one of the above has the following beneficial effects: (1) the occurrence and development process of BHD syndrome pulmonary focal regressive bulla are researched; (2) the process of BHD syndrome pulmonary focal regressive bulla inducing pneumothorax is researched; (3) the pathological mechanism of BHD syndrome pulmonary focal regressive bulla is researched.

[0013] The mouse BHDS pulmonary focal regressive bulla model obtained by the method according to any one of the above is applied to the preparation of a targeted drug for treating BHD syndrome pulmonary focal regressive bulla.

[0014] The application has the following beneficial effects: (1) Existing methods for constructing mouse models of pulmonary bullae have resulted in lungs exhibiting widespread tissue "degeneration" without forming the typical "focal" lesion. However, the mouse BHDS focal degenerative bullae model constructed in this invention produces significant focal lesions on the basis of widespread tissue "degeneration." Moreover, molecular detection has revealed that the lung tissue of the mouse BHDS focal degenerative bullae model constructed in this invention shows changes in the expression of characteristic molecules found in the lung tissue of clinical BHDS samples, making it the most consistent model with BHDS-like lung tissue pathology to date.

[0015] (2) This invention provides a suitable tool model for the efficacy, safety and drug screening of drugs for the treatment of focal degenerative bullae in the lungs of BHD syndrome (also known as "cystic lesions of the lungs in BHD syndrome, which are essentially the same). It ensures the reproducibility and efficiency of the model and has broad application prospects.

[0016] (3) In addition, compared with gene knockout models, the method of constructing the mouse BHDS focal degenerative bullous model of the present invention is more stable, the operation method and experimental technique are simpler, and the construction cost is lower. Attached Figure Description

[0017] Figure 1 The following is a schematic diagram of the operation process for constructing the mouse BHDS focal degenerative bullous model: The operation process for constructing a BHDS-like bullous mouse model using low-concentration hydrochloric acid combined with FLCN-miRNAs is as follows: After purchasing 7-week-old male mice, they were maintained for one week. After 24 hours of nasal treatment with low-concentration HCl, they were treated with two types of agomiRs. Seven days later, they were treated with two types of agomiRs again. After one week of feeding, the mice were sacrificed and samples were collected. Figure 2 This is a pathological feature image (HE staining) of the BHD-like pulmonary bullous mouse model of Example 1. Figure 3 Molecular pathological examination of the BHDS-like bullous mouse model of Example 1: (a) Western blot (WB) detection of α-SMA expression and statistical analysis in lung tissue; (b) Western blot (WB) detection of cleaved caspase 3 (Cl.Cas3) expression and statistical analysis in lung tissue; Figure 4 IHC detection of α-SMA expression in mouse lung tissue for Example 1; Figure 5 Clinical trial data for Example 2: (A) Western blot detection of lung tissue protein levels (Cl.Cas3, α-SMA); (B) HE staining of BHDS, PSP and NC, immunohistochemical detection of α-SMA expression, and TUNEL assay. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments. Example

[0019] I. Reference Figure 1 The method for constructing the mouse BHDS focal degenerative bullous model of the lungs in this embodiment includes the following: (1) Select 6-week-old SPF-grade C57BL / 6 mice (purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd.) and acclimatize them for one week; (2) Treat with low concentration HCl (0.1M) nasal drops for 1 day, at a dose of 1.5 ml / kg; (3) Two microRNA fitters, namely mmu-let-7d-5p agomir and mmu-miR-322-5p agomir (both microRNAs are regulated by the FLCN gene and were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.), were administered to mice via nasal drops at a dose of 1.25 nmol / mouse for each fitter and fed for 1 week. (4) Repeat step (3) again; (5) A mouse model of focal degenerative bullae in the lungs of patients with BHDS was obtained. Referring to the above Figure 1 Table 1 shows the model group, control group, and injury group:

[0020] II. The model group, control group, and injury group mice in the above embodiments were identified by HE staining to determine the pulmonary bullae phenotype, such as... Figure 2 As shown, the pathological features of a mouse model of BHDS-like focal degenerative bullae are observed, and HE shows BHDS-like bullae in the lung tissue.

[0021] III. The characteristic pathological molecular expression of mice in the model group, control group, and injury group of the above embodiments was identified, such as... Figure 3 and Figure 4 As shown.

[0022] Figure 3Molecular pathological examination of a BHDS-like pulmonary bullous mouse model was performed. (a) Western blotting (WB) analysis of α-SMA expression in lung tissue showed that α-SMA expression in the lung tissue of BHD-like focal degenerative bullous mouse model mice was significantly lower than that in the blank control group and the HCl-induced damage group. (b) Western blotting analysis of cleaved caspase 3 (Cl.Cas3) expression in lung tissue showed that Cl.Cas3 expression in the lung tissue of BHD-like focal degenerative bullous mouse model mice was significantly higher than that in the blank control group and the HCl-induced damage group.

[0023] Figure 4 IHC analysis of α-SMA expression in mouse lung tissue showed that α-SMA expression in the lung tissue of mice with BHD-like focal degenerative bullae was significantly lower than that in the blank control group and the HCl-damaged group. Example

[0024] Clinical trial: The expression of damage repair-related characteristic molecules in lung tissues of patients with clinical BHD syndrome, lung tissues of patients with non-FLCN-mutant pneumothorax (PSP), and adjacent tissues of lung cancer patients was detected. This was used to demonstrate that the characteristics of the model constructed in Example 1 above are consistent with those of the clinical trial.

[0025] The samples for this clinical trial came from Nanjing Chest Hospital, Taizhou Hospital in Zhejiang Province, and Jiangsu Cancer Hospital, and were the remaining samples after clinical pathological examination of surgical patients.

[0026] like Figure 5 As shown, A. WB detection of lung tissue protein levels revealed that the apoptosis marker cleaved caspase 3 (Cl.Cas3) was increased in the lung tissue of patients with BHD syndrome, while the expression of the damage repair factor α-SMA was significantly decreased in the lung tissue of patients with BHD syndrome.

[0027] B. HE staining of lung tissues from BHD syndrome, PSP, and control (first row) shows the pathological features of focal degenerative bullae in BHDS; immunohistochemical detection of α-SMA expression (second row) shows that key factors for damage repair in the lung tissues of BHDS patients are suppressed, and TUNEL assay (third row) shows increased apoptosis in the lung tissues of BHDS patients.

[0028] In summary, by comparing the relevant characteristics of the clinical trial in this embodiment with the relevant characteristics of the model constructed in Example 1, it was found that the characteristics of the mouse BHDS focal degenerative bullous pulmonary model constructed in this invention are consistent with the clinical trial characteristics. That is, the mouse BHDS focal degenerative bullous pulmonary model constructed in this invention exhibits changes in the expression of characteristic molecules in the lung tissue of BHDS clinical samples, and is the model that best matches the pathology of BHDS-like lung tissue to date.

[0029] The present invention has been disclosed above with reference to preferred embodiments, but these are not intended to limit the present invention. Anyone skilled in the art can make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the scope of the claims in this application.

Claims

1. A method for constructing a mouse model of focal degenerative pulmonary bullae in the lungs of patients with bronchiectasis (BHDS), characterized in that, The method includes: after acclimatizing healthy mice for one week, treating them with HCl, and administering two microRNA fitters to the mice via nasal drops to obtain the BHDS focal degenerative bullous lung model.

2. The method for constructing a mouse BHDS focal degenerative bullous model of the lungs as described in claim 1, characterized in that, The two microRNA fitters include mmu-let-7d-5p agomir and mmu-miR-322-5p agomir.

3. The method for constructing a mouse BHDS focal degenerative bullous model of the lungs as described in claim 2, characterized in that, The mice were given two microRNA fitters via nasal drops, with each microRNA fitter being administered at the minimum dose required to induce a bullous phenotype in the lungs, and were fed for one week. The two microRNA fitters were then administered to mice via nasal drops. The dose of each microRNA fitter was the minimum dose that could induce the bullous phenotype in the lungs. The mice were fed for one week.

4. The method for constructing a mouse BHDS focal degenerative bullous model of the lungs as described in claim 3, characterized in that, Each microRNA fitter was 1.25 nmol / mouse.

5. The method for constructing a mouse BHDS focal degenerative bullous model of the lungs as described in any one of claims 1-4, characterized in that, The HCl damage treatment was administered as follows: 0.1M HCl nasal drops for 1 day, at a dosage of 1.5ml / kg.

6. The method for constructing a mouse BHDS focal degenerative bullous model of the lungs as described in any one of claims 1-4, characterized in that, The healthy mice were 6-8 week old SPF-grade C57BL / 6 mice.

7. The application of the mouse BHDS focal degenerative bullae model obtained by the method according to any one of claims 1-6 in the study of the pathogenesis of focal degenerative bullae in BHD syndrome: (1) To study the occurrence and development of focal degenerative bullae in the lungs of patients with BHD syndrome; (2) To study the process of pneumothorax induced by focal degenerative bullae in the lungs of patients with BHD syndrome; (3) To study the pathological mechanism of focal degenerative bullae in the lungs of BHD syndrome.

8. The use of the mouse BHDS focal degenerative bullous model obtained by the method according to any one of claims 1-6 in the preparation of targeted drugs for treating focal degenerative bullous lung disease in BHD syndrome.

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