METHOD FOR CONSTRUCTING LUNG CANCER ANIMAL MODEL

Abstract

A method for constructing a lung cancer animal model. According to the method, a pathogenic substance of a lung cancer is atomized into atomized particles, and an animal inhales the atomized particles in an inhaling mode, so as to construct a required lung cancer animal model.

Claims

1. A method for constructing a lung cancer animal model, comprising an animal subjected to a nebulization after a pathogenic substance of a lung cancer is atomized into atomized particles, so that the animal inhales the atomized particles in an inhaling mode.

2. The construction method according to claim 1, wherein a size of the atomized particle is that: a mass median aerodynamic diameter (MMAD) is 2.9 μm; and a percentage of a particulate smaller than 5 μm is 76%.

3. The construction method according to claim 1, wherein the nebulization is for the animal to continuously inhale for 15 minutes to 20 minutes.

4. The construction method according to claim 1, wherein an atomization amount of the pathogenic substance of the lung cancer ranges from 2 ml to 8 ml.

5. The construction method according to claim 1, wherein after inhaling the atomized particles, the animal is placed in an SPF environment to obtain a required lung cancer animal model.

6. The construction method according to claim 1, wherein the animal is subjected to the nebulization after the pathogenic substance of the lung cancer is atomized into the atomized particles by using an atomization inhalation instrument, and operating parameters of the instrument are as follows: pressure: 0.5 bar/50 kpa to 2.0 bar/200 kpa atomization amount: 2 ml to 8 ml working flow: 3.0 L/min to 6.0 L/min atmospheric pressure: 500 hpa to 1060 hpa atomization rate: 370 mg/min size of particle: mass median aerodynamic diameter (MMAD): 2.9 μm; and percentage of particulate smaller than 5 μm: 76%.

7. The construction method according to claim 1, wherein in the case that a diameter of the atomized particle ranges from 5 μm to 10 μm, the obtained animal model simulates the lung cancer occurring on a main bronchus and a secondary bronchus, in the case that the diameter ranges from 3 μm to 5 μm, the obtained animal model simulates the lung cancer occurring on the secondary bronchus and branched bronchi, and in the case that the diameter is less than 3 μm, the obtained animal model simulates the lung cancer occurring on a terminal bronchiolar epithelium and an alveolar epithelium.

8. The construction method according to claim 7, wherein when the diameter of the atomized particle ranges from 2 μm to 3 μm, the constructed animal model simulates an adenocarcinoma in non-small cell lung cancers.

9. The construction method according to claim 8, wherein the pathogenic substance of the lung cancer is an adenovirus carrying a Cre recombinase capable of activating a Kras oncogene of a lung epithelial cell; in the case that a virus concentration is 5×10.sup.5-5×10.sup.6, the obtained animal model simulates an early stage of the lung cancer; in the case that the virus concentration is 2.5×10.sup.7, the obtained animal model simulates a progressing stage of the lung cancer; and in the case that the virus concentration is 7.8×10.sup.9, the obtained animal model simulates an invasive carcinoma stage of the lung cancer.

10. The construction method according to claim 7, wherein in the case that the pathogenic substance of the lung cancer is an adenovirus carrying a Cre recombinase, and the diameter of the atomized particle ranges from 5 μm to 10 μm, the atomized particles are inhaled by Kras.sup.LSL-G12D;LKB1.sup.fl/fl genetically engineered mice, which are hybrids of Kras.sup.LSL-G12D mice and LKB1.sup.fl/fl mice, to construct mouse models of a squamous carcinoma in non-small cell lung cancers.

11. The construction method according to claim 2, characterized in that, the nebulization is for the animal to continuously inhale for 15 minutes to 20 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic diagram illustrating aerosol inhalation of virus.

[0033] FIG. 2 is a schematic diagram illustrating a generating process of a lung cancer after a genetically engineered mouse inhaled the virus. Kras.sup.LSL-G12D mouse (No. #008179, Jackson Laboratory)

[0034] FIG. 3 illustrates occurrence and development of the lung cancer dynamically observed by small animal CT imaging.

[0035] FIG. 4 illustrates a lung cancer-hypermetabolism area observed by small animal PET/CT imaging.

[0036] FIG. 5 illustrates the occurrence and development of the lung cancer generally observed through a lung tissue.

[0037] FIG. 6 illustrates the occurrence and development of the lung cancer observed by tissue section upon HE staining.

DETAILED DESCRIPTION

[0038] The present invention is further described hereinafter with reference to the drawings and specific embodiments of the description, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and devices used in the present invention are conventional reagents, methods and devices in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

Embodiment 1 Construction of Lung Cancer Animal Model

[0040] 1. Experimental Atomization Instrument

[0041] The atomization instrument is an atomization inhalation instrument Pari-3305-Junior boy SX purchased from Germany, with a mass median diameter (MMD) of 2.9 μm.

[0042] A schematic diagram illustrating aerosol inhalation of virus is shown in FIG. 1.

[0043] When in use, air was compressed to inhale and atomize a solution, a spray was generated by a compressor, and then transmitted to an atomizer through an air hose to spray, and a liquid aerosol was atomized and transmitted to a spray nozzle. An amount of the spray was increased by inhaling an additional air flow generated, so that the inhalation was performed quickly and effectively.

[0044] The atomizer was provided with a PIF control system to help slow absorption, so that atomized particles were uniformly distributed in a bronchiole and an alveolus.

[0045] 2. Experimental Mice

[0046] Kras.sup.LSL-G12D genetically engineered mice aged 8 weeks to 16 weeks (No. #008179, Jackson Laboratory) were used.

[0047] An animal experiment program was approved by the Animal Experiment Ethics Committee of Sun Yat-sen University and Southern Medical University. All the animal experiments conformed to the Guide for the Care and Use of Laboratory Animals issued by National Institutes of Health (NIH Publication, Eighth Edition, 2011).

[0048] 3. Experimental Virus Drug

[0049] Adenovirus carrying Cre recombinase (Shanghai Genechem).

[0050] 4. A method for constructing a lung cancer animal model included the following steps.

[0051] In S1, an adenovirus stock solution with a titer of 8E+10 PFU/mL was diluted with PBS into working concentrations according to experimental requirements, which were: virus titers=5×10.sup.5 PFU/mL, 5×10.sup.6 PFU/mL, 2.5×10.sup.7 PFU/mL, 5×10.sup.7 PFU/mL, 5×10.sup.8 PFU/mL and 7.8×10.sup.9 PFU/mL respectively. An amount of a liquid in an atomization cup of the atomization inhalation instrument ranged from 2 ml to 3 ml.

[0052] In S2, an anesthetized mouse was stably placed on a stage and put on a mask, and the atomization inhalation instrument was started to enable the mouse to inhale atomized particles of the virus carrying the Cre recombinase for 15 minutes to 20 minutes. An atomization air flow was kept stable during the period until the liquid in the atomization cup of the atomization inhalation instrument was completely inhaled.

[0053] Parameters of the atomization inhalation instrument in the step S2 might be set as follows:

[0054] pressure: 0.5 bar/50 kpa to 2.0 bar/200 kpa

[0055] working flow: 3.0 L/min to 6.0 L/min

[0056] atmospheric pressure: 500 hpa to 1060 hpa

[0057] atomization rate: 370 mg/min

[0058] size of atomized particle: mean median diameter (MMAD): 2.9 μm; and percentage of particulate of <5 μm: 76%.

[0059] The atomized particles could be uniformly dispersed on an alveolar epithelium and enter the alveolar epithelium, wherein the Cre recombinase played a role to activate an oncogene, thus generating a lung cancer.

[0060] In S3, the mouse was placed in an SPF environment for observation after waking up, and small animal CT imaging was performed 2, 4 and 5 months after the inhalation to dynamically observe occurrence and development of a tumor. Tissue sampling was performed 4 and 5 months after the inhalation to observe occurrence and development of a lung tumor in situ until the model was successfully constructed.

[0061] 5. Experimental Results

[0062] Since an inhalation delivery system provided an extremely small aerosol liquid drop carrying the virus to the alveolus, the Cre recombinase was expressed in an infected lung cell, and a Kras gene in the Kras.sup.LSL-G12D mouse was activated, which finally caused the tumor in a lung.

[0063] The occurrence and development of the tumor of the mouse inhaling the virus were dynamically observed by small animal CT and PET/CT. The mice were put to death at different time points for the tissue sampling, and the occurrence and development of the lung cancer were pathologically observed. The results were shown in FIG. 2 to FIG. 6.

[0064] FIG. 2 is a schematic diagram illustrating a generating process of a lung cancer after a genetically engineered mouse inhaled the virus. After the Kras.sup.LSL-G12D mouse inhaled the adenovirus carrying the Cre recombinase, the Cre excised two loxP sites, causing failure of a termination codon, thus activating a downstream Kras oncogene. The activated Kras gene could cause the lung cancer of the mouse.

[0065] FIG. 3 illustrates occurrence and development of the lung cancer dynamically observed by mouse CT imaging. In lung scan images of a WT mouse (left, WT) and a Kras.sup.LSL-G12D mouse (right, HET) inhaling and infected with the virus (titers were respectively 5×10.sup.5, 5×10.sup.6, 2.5×10.sup.7, 5×10.sup.7, 5×10.sup.8 and 7.8×10.sup.9), the tumor was a white high intensity area (marked with a circle and an arrow).

[0066] FIG. 4 illustrates a cancer-hypermetabolism area observed by mouse PET/CT imaging for monitoring a metabolic activity of the tumor. The Kras.sup.LSL-G12D mouse was scanned by PET/CT 16-20 weeks after inhaling the virus (7.8×10.sup.9). Compared with a control group, an increase in uptake of glucose F18 was locally shown in a left lung of the mouse. Meanwhile, this area was suggested to be a tumor hypermetabolism area (see the circle mark) combined with CT results.

[0067] FIG. 5 illustrates the occurrence and development of the lung cancer generally observed by sampling a lung tissue, including occurrence of lung tumors of the WT mouse and the Kras.sup.LSL-G12D mouse inhaling and infected with the virus (titers are respectively 5×10.sup.5, 5×10.sup.6, 2.5×10.sup.7, 5×10.sup.7, 5×10.sup.8 and 7.8×10.sup.9).

[0068] FIG. 6 illustrates the occurrence and development of the lung cancer observed by tissue section upon HE staining, including the occurrence of the lung tumors of the WT mouse (left, WT) and the Kras.sup.LSL-G12D mouse (right, HET) inhaling and infected with the virus (titers are respectively 5×10.sup.5, 5×10.sup.6, 2.5×10.sup.7, 5×10.sup.7, 5×10.sup.8 and 7.8×10.sup.9). According to results of histopathological analysis, an early stage of the lung cancer was stimulated at a virus concentration of 5×10.sup.5-5×10.sup.6. A progressing stage of the lung cancer was stimulated at a virus concentration of 2.5×10.sup.7. An invasive carcinoma stage of the lung cancer was stimulated at a virus concentration of 7.8×10.sup.9.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without deviating from the spiritual substance and principle of the present invention shall be equivalent substitute modes, and are all included in the protection scope of the present invention.