CN111713453A

CN111713453A - Method for establishing bone metastasis animal model of lung cancer

Info

Publication number: CN111713453A
Application number: CN202010559972.XA
Authority: CN
Inventors: 闫明霞; 柴文君; 潘洪玉; 孙磊; 刘乾; 管宇; 高海河; 刘晓丽; 徐烨
Original assignee: Fudan University Shanghai Cancer Center
Current assignee: Fudan University Shanghai Cancer Center
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2020-09-29

Abstract

The invention relates to the field of animal models, in particular to a method for establishing a lung cancer bone metastasis animal model by ultrasonic-guided intracardiac injection, which optimizes the method for carrying out intracardiac injection operation by traditional blind beating, positions a left ventricle by ultrasonic guidance, and carries out lung cancer cell injection after a needle enters the left ventricle; the method effectively improves the success rate of establishing the lung cancer bone metastasis mouse model, avoids the blindness of intracardiac injection and prevents the acute bleeding of animals. The invention establishes a reliable lung cancer bone metastasis animal model and provides a new experimental tool for clinical diagnosis and treatment and basic research of lung cancer bone metastasis.

Description

Method for establishing bone metastasis animal model of lung cancer

Technical Field

The invention relates to the technical field of animal models, in particular to a method for establishing a lung cancer bone metastasis animal model by ultrasonic-guided intracardiac injection.

Background

Lung cancer is one of the leading causes of death in patients worldwide. Although early diagnosis and clinical treatment of lung cancer have advanced significantly, it is not ignored that most lung cancer patients are diagnosed in the middle and late stages, the 5-year survival rate is less than 15%, and more than 90% of lung cancer patients die of metastasis. Bone metastasis is one of the main causes of death of patients with lung cancer, and local pain, bone and joint movement disorder, pathological fracture, hypercalcemia, nerve compression and other symptoms are often caused due to the damage of bones and periosteum, so that the life quality of the patients is obviously reduced. At present, the occurrence and development mechanism of lung cancer bone metastasis is not clear, and the lung cancer bone metastasis is one of the key problems to be solved urgently in the field of lung cancer research. The preparation of reliable animal models is an important basis for the research of tumor mechanisms, but the animal models related to the bone metastasis of lung cancer are rarely reported at home and abroad at present.

Disclosure of Invention

The invention aims to provide a reliable method for establishing a bone metastasis animal model of lung cancer.

In order to achieve the above object, the present invention provides a method for establishing a bone metastasis animal model of lung cancer, comprising the steps of:

A) taking GFP/Luc labeled human lung cancer cells A549 or NCI-H1299 in logarithmic growth phase, and adjusting the cell density to 1.5 × 10 by using RPMI1640 culture solution without serum⁷Per mL, 0.2mL of cell-containing fraction 3 × 10 was aspirated by a 1mL insulin syringe⁶Preparing a cell suspension for later use;

B) injecting 10mL/kg of 1% sodium pentobarbital solution into abdominal cavity to anaesthetize NOD/SCID mouse or BALB/c-nu/nu nude mouse, fixing mouse in supine position, and wiping mouse anterior chest wall with alcohol cotton for disinfection;

C) a needle point 6mm enters the left ventricle of the mouse under the ultrasonic guidance at a position between a second rib 3mm away from the left side of the sternum and forming an included angle of 45 degrees with the thorax; after the needle point is confirmed to be positioned in the left ventricle, the needle is drawn back, and bright red blood continuously flows into the needle tube, which indicates that the needle is inserted correctly; then slowly inoculating the cells into the left ventricle, slowly pulling out the insulin syringe, pressing with a cotton swab to stop bleeding, and continuously feeding after operation to establish the bone metastasis animal model of the lung cancer.

Further, in the step A, the GFP/Luc labeled human lung cancer cells A549 or NCI-H1299 in logarithmic growth phase are prepared by the following steps: a549 cells or NCI-H1299 cells are paved on a 6-hole culture plate according to the cell density of 60-70%, Green Fluorescent Protein (GFP)/Luc double-label viruses are added for infection on day 2, the solution is changed after 6-8H, and conventional culture is carried out; after the cells are expanded, the GFP expression condition is observed under a fluorescence microscope, and if the GFP expression in the marked cells is weak or the cell marking rate cannot reach more than 90 percent, the cells need to be sorted by flow cytometry.

Further, in the step C, the needle point 6mm enters the left ventricle of the mouse under the ultrasonic guidance; after the needle point is confirmed to be in the left ventricle, the needle is drawn back, and bright red blood continuously flows into the needle tube, which indicates that the needle is inserted correctly. In the step, the left ventricle can be accurately positioned by ultrasound, the inaccurate needle insertion caused by touch beating is avoided, and the modeling success rate of the animal model is effectively improved.

The invention has the advantages that:

the invention establishes a reliable lung cancer bone metastasis animal model by injecting lung cancer cells in the lower heart under the guidance of ultrasound, and provides a new experimental tool for clinical diagnosis and basic research of lung cancer bone metastasis.

Drawings

FIG. 1 human lung carcinoma A549 and NCI-H1299 cells stably and highly expressing GFP and luciferase.

FIG. 2 is an ultrasonic guided live imaging observation of small animals injected with a human lung cancer cell bone metastasis model; a: an ultrasound guidance schematic; b: the bone metastasis condition of the injected animals in the heart is observed by live imaging of the small animals; c: and (3) comparing the total number of the fluorescence photons and the luminous area of chemiluminescence of different mice.

FIG. 3X-ray and Micro-CT observations of a bone metastasis model of mice injected with NOD/SCID human lung carcinoma A549 cells intracardiacally via ultrasound guidance (shown as bone defect destruction at the arrow).

FIG. 4. intracardiac injection of bone metastases from NOD/SCID mice with human lung carcinoma A549 cells guided by ultrasound; a: a shoulder joint bone metastasis; b: a right knee joint bone metastasis; c: left knee joint bone metastasis.

FIG. 5 histopathological observation of bone metastases from intracardiac injected human lung carcinoma A549 cells NOD/SCID mice guided by ultrasound (HE staining, X100, X400).

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example (b):

1 materials and methods

1.1 materials

1.1.1 human Lung cancer cell lines

Human lung cancer cells A549 and NCI-H1299 are from national key laboratories of oncogenes and related genes and are routinely preserved by the laboratories. The cells were cultured in RPMI1640 and DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ in 5% CO₂The incubator of (2) for cultivation.

1.1.2 Experimental animals

6-8 weeks old female NOD/SCID mice and BALB/c-nu/nu nude mice, 8 mice each, were provided by Shanghai City tumor research institute, and the production license of the experimental animals was SCXK (Shanghai) 2017-. Animal experiments and feeding strictly comply with the requirements of national SPF standard, and the license number of experimental animals is SYXK (Shanghai) 2017-0011.

1.1.3 instruments and reagents

Small animal high resolution ultrasound imaging systems were purchased from Visual sonic, Canada, under the model VeVo-770D. Small animal in vivo fluorescence imaging systems were purchased from Berthold Technologies GmbH & Co. KG, Germany, under model LB983(NC320) with analytical software WinLight 32. Small animal Micro-CT was purchased from Bruker, Belgium under the model Skyscan 1076. Fluorescence microscopes were purchased from Olympus, japan. Luciferase (luciferase, Luc) was purchased from GoldBi Technology, USA. RPMI1640 and DMEM media were purchased from Invitrogen, USA. Fetal bovine serum was purchased from Biowest, France. Petri dishes and plates were purchased from Corning, USA.

1.2 methods

1.2.1 human Lung cancer cell transfection

A549 cells and NCI-H1299 cells are paved on a 6-well culture plate according to the cell density of 60-70 percent, Green Fluorescent Protein (GFP)/Luc double-standard viruses (Wangxingmin, Yan Suberxia, Libridge Hui, and the like) are added on day 2, the application of GFP and Luc double-standard technologies in the establishment of mouse tumor models [ J ] experimental animals and comparative medicine, 2010,30(1):2-7.) is adopted for infection, and the solution is changed after 6-8 hours for conventional culture. After the cells are expanded, the GFP expression condition is observed under a fluorescence microscope, and if the GFP expression in the marked cells is weak or the cell marking rate cannot reach more than 90 percent, the cells need to be sorted by flow cytometry.

1.2.2 establishment of animal model of bone metastasis of human Lung cancer cells by ultrasonic guided intracardiac injection

Collecting GFP/Luc labeled human lung cancer cell A549 in logarithmic growth phase, and adjusting cell density to 1.5 × 10 with serum-free RPMI1640 culture solution⁷Perml, 0.2mL of the cell suspension (cell number 3 × 10; contained: 3.78) was aspirated by a 1mL insulin syringe (29G, BD Bioscience)⁶One) for standby. Injecting 1% sodium pentobarbital (10mL/kg) solution into abdominal cavity to anaesthetize NOD/SCID mouse and BALB/c-nu/nu nude mouse, fixing mouse in supine position, and wiping mouse front chest wall with alcohol cotton for disinfection; a needle point 6mm enters the left ventricle of the mouse under the ultrasonic guidance at a position between a second rib 3mm away from the left side of the sternum and forming an included angle of 45 degrees with the thorax; after the needle point is confirmed to be positioned in the left ventricle, the needle is drawn back, and bright red blood continuously flows into the needle tube, which indicates that the needle is inserted correctly; the cells were then slowly seeded into the left ventricle, the insulin syringe was slowly withdrawn, and the cotton swab pressed to stop bleeding. GFP/Luc labeled human lung cancer cell NCI-H1299 is also used for establishing an intracardiac injection bone metastasis animal model by the same method. After the operation, the mice were kept and closely observed for mental state and physical condition at a fixed time every week.

1.2.3 evaluation of bone metastasis model by Small animal Living body imaging System

Mice were periodically imaged in vivo, X-ray and Micro-CT scanned from week 5 after surgery to determine if bone metastasis occurred in the mice. Live imaging of small animals: mice were first anesthetized by intraperitoneal injection with 1% sodium pentobarbital (10mL/kg) solution, followed by intraperitoneal injection of 0.15 mL/mouse of D-Luciferin substrate (abcam, cat # ab143654, 20mg/mL in PBS); after 15min, the mouse is horizontally placed in a darkroom of an imaging system, and a bright field image is shot; after the bright field image acquisition is finished, switching to a chemical biological luminous imaging mode, wherein the exposure time is 20 s; the bright field image is superimposed with the photon image to determine the bioluminescent signal location. Then shooting by adopting a fluorescence imaging mode, and applying 470nm excitation wavelength and 525nm emission wavelength, wherein the exposure time is 5 s. And after the shooting is finished, the fluorescence image and the bright field image are superposed to determine the position of the fluorescence signal.

1.2.4X-ray radiography evaluation bone metastasis model

The mice are anesthetized by intraperitoneal injection with 1% sodium pentobarbital (10mL/kg) solution, and the mice are taken out of the supine position and subjected to X-ray radiography by adopting X-rays carried by a small animal living body imaging device: voltage 50kV, current 45mA, exposure 2 ms.

1.2.5 Micro-CT scanning evaluation bone metastasis model

Mice were anesthetized with 1% sodium pentobarbital (10mL/kg) solution by intraperitoneal injection, followed by monitoring of bone metastasis in mice using Micro-CT. The Micro-CT parameters were as follows: voltage 50k V, current 20 μ a, exposure time 130ms, angular gain 0.7 °. And performing three-dimensional reconstruction after shooting and scanning.

1.2.6 histopathological examination

Fixing the tissue with paraformaldehyde solution, washing with clear water, soaking in 10% EDTA solution, and decalcifying for two weeks. During this period, the solution was changed every 2 days and shaken on a shaker. After decalcification was completed, the tissue was embedded, sectioned and stained with hematoxylin-eosin (HE) after rinsing with running water for 2 hours.

2 results

2.1 GFP/Luc double-labeled tumor cells

GFP/Luc double-labeled human lung cancer A549 and NCI-H1299 cells are polygonal epithelial-like cells and grow adherently. The observation under a fluorescence microscope shows that the fluorescence signal of the cells in the whole visual field is stronger, the fluorescence distribution uniformity of the cells is good, the transfection rate is close to 100 percent, and the fluorescence signal is not obviously faded along with the in vitro long-term subculture (figure 1). The results show that GFP/Luc double-labeled human lung cancer A549 and NCI-H1299 cells can stably express green fluorescent protein in vitro and can be used for subsequent experiments.

2.2 visualization

2.2.1 Small animal in vivo imaging Observation

BALB/c-nu/nu nude mice and NOD/SCID mice all survived when injected with A549 and NCI-H1299 cells. When the model is modeled for 5 weeks, the symptoms of emaciation, slow movement and the like of some mice are found; the animal model is dynamically monitored in real time by a small animal living body imaging system, and the tumorigenesis and metastasis conditions of cells in the animal body are observed (Yan Mingchun, Liu Jie, Guideshui, etc.. dynamic observation of human lung cancer nude mouse model living body imaging [ J ] tumor, 2008,28(10): 833-. The results of the chemiluminescent imaging showed: after the left ventricle is injected with human lung cancer cells under the guidance of ultrasonic waves, Luc luminescence signals appear at the positions of the central axis bones and the limb bones of all 16 mice, which indicates that the mice have tumor cell metastasis after intracardiac injection. Compared with BALB/c-nu/nu nude mice, the noc/SCID mice have larger Luc light emitting area and higher total fluorescence photon intensity, which indicates that tumor cells are easier to grow and transfer in severe combined immunodeficiency mice. Meanwhile, NCI-H1299-GFP/Luc cells have more Luc distribution points, larger luminous areas and higher total fluorescence photon intensity than A549-GFP/Luc cells, which indicates that the in vivo transfer capacity of different human lung cancer cells after intracardiac injection is different, and the in vivo transfer capacity of the NCI-H1299-GFP/Luc cells is stronger than that of the A549-GFP/Luc cells (figure 2).

2.2.2X-ray and Micro-CT observations

When NOD/SCID mice are injected in the heart at 8 weeks under the guidance of ultrasonic wave by adopting human lung cancer A549 cells, the NOD/SCID mice have the behaviors of emaciation, back of the arch, rickets, obvious activity limitation and the like. And observing the bone metastasis condition of the mouse by X-ray photography, and performing three-dimensional recombination on the whole body skeleton of the animal model by adopting Micro-CT shooting. The X-ray radiograph shows that obvious bone destruction and discontinuous change appear on the tibial plateau and the shoulder joint of the NOD/SCID mouse. According to Micro-CT detection of a small animal, obvious defects and damages appear on bones and periosteum of a tibia platform and a shoulder joint of an NOD/SCID mouse, and normal tissue forms of the bones disappear; the knee joint is completely invaded by tumor tissues and is subjected to melting ice and osteolytic destruction; the long bone bones are damaged like spots and pieces, and the shoulder joints are damaged like worm-eaten (fig. 3). This is in good agreement with the clinical manifestations of bone metastasis of lung cancer (Jianting, Mao Xiao Ming, Lin Jiang, etc.. CT diagnosis of metastatic bone tumor [ J ]. J.

2.3 anatomical observations

After the animals are in the state of dying such as back, slow movement, listlessness and emaciation, CO is adopted₂Animals were sacrificed by asphyxiation and dissected. As a result, it was found that: all 16 mice had metastases at the medial and limb bony joints (fig. 4); the knee joint is wrapped by tumor tissues, is obviously protruded, has irregular shape, is gray white, and has slightly hard surface texture; the shoulder joints, the shoulder blades and the middle shaft bones have tumor nodules which obviously protrude from the surfaces of the bone substances, are round, are grey white in color and are slightly hard in texture. All mice have bone metastasis with different degrees, the metastasis position of the bone metastasis is basically consistent with the position displayed by live imaging and Micro-CT of the small animals, the metastasis mainly occurs in bones of limbs mainly including shoulder joints, shoulder blades and knee joints and middle axial bones mainly including spines, and no metastasis foci of livers, spleens, kidneys and other visceral organs are found.

2.4 histopathological Observation

Bone metastasis foci such as scapula, rib, femur, tibia and the like of the mice are taken, embedded, fixed and HE stained, tumor cells visible under an optical microscope penetrate through cortical bone and invade surrounding soft tissues, and the tumor cells are disorderly arranged, irregular in shape, poor in cell differentiation, deep-stained in nucleus and unobvious in nucleolus (figure 5).

Discussion of 3

The lung cancer metastasis is mainly in the bones, brain, liver, and other parts. The bone is the first target organ of lung cancer metastasis, accounts for about 50% -70% of late-stage lung cancer, is mainly distributed in rib, spine, pelvis, limb bones and other parts, and is mostly osteolytic destruction and rarely osteogenic destruction. The formation of bone metastases of lung cancer is a multifactorial, multistep, complex biological process, and the specific mechanism is not clear at present. When tumor metastasis mechanism and prevention and treatment research are carried out, a tumor metastasis animal model is one of indispensable tools. The tumor animal model is different from the clinical actual condition, the animal model generally has less metastasis, and even if the metastasis occurs, the metastasis rate is relatively low. In lung cancer research, the research reports on lung cancer bone metastasis animal models are overall less. At present, according to the reports of the existing documents, there are 4 methods for establishing the bone metastasis animal model of lung cancer: lung orthotopic transplantation, tail vein injection, intra-tibial injection and left ventricular injection. The main site of metastasis for both lung orthotopic transplantation and tail vein injection is the lung, while bone metastasis is relatively rare (Kuchimaru T, Kataoka N, Nakagawa K, et al. A reusable muscle model of bone metastasis by injecting cancer cells [ J ]. Nat Commun,2018,9(1): 2981.). The tibialis injection method is a direct injection of tumor cells into the tibia, which, despite the high success rate of transplantation, lacks the process of tumor cell metastasis via the blood route (Bengeum, Lichun rain, Huosen, etc.. establishment of mouse model of bone metastasis of lung cancer [ J ]. south university of medical science, 2014,34(5): 664. 668.). The left ventricle injection method is to directly inject tumor cells into the left ventricle of a mouse, but lacks the step that the tumor cells break through a basement membrane in the whole tumor metastasis process. In the preliminary experiments, left ventricle injection was performed without ultrasound guidance, and as a result, it was found that when dissecting mice, about 1/3 mice found tumor bodies near the pericardium, pleura and flag cartilage, and the reason for this analysis may be that the needle insertion position was shifted or the needle insertion was too shallow, which resulted in leakage of tumor cells, thereby affecting the success rate of modeling (Cui YQ, Geng Q, YuT, et al. The method optimizes the traditional method for performing intracardiac injection operation by blind beating, positions the left ventricle through ultrasonic guidance, and performs lung cancer cell injection after the needle enters the left ventricle; the method effectively improves the success rate of establishing the lung cancer bone metastasis mouse model, avoids the blindness of intracardiac injection, and prevents the acute bleeding of animals. The intracardiac injection bone metastasis animal model established by the invention can better simulate the process that tumor cells enter blood circulation and then reach a capillary vascular bed of a bone along with blood flow, pass through vascular endothelial gaps to enter a bone tissue, and finally proliferate in the bone and cause bone reconstruction after a series of interactions with a bone tissue microenvironment; the growth and morphology of the bone metastasis of a tumor patient are very similar, and the metastasis of tumor cells to parenchymal organs can be greatly reduced. Therefore, the method for establishing the intracardiac injection bone metastasis animal model through ultrasonic guidance is a method which is feasible in technical operation and more effective in establishing bone metastasis.

In the process of model making, the invention discovers the following factors influencing the success of model making according to practical experience analysis. (1) The modeling method comprises the following steps: the conventional intracardiac injection adopts blind beating operation, so that the position of an inserted needle is deviated, the deviation of the depth of the inserted needle can cause cell leakage, and solid tumor is formed in the thoracic cavity; after the ultrasonic guidance intervention is applied, the modeling success rate can be greatly improved through more accurate positioning. (2) Cell diameter: the morphology and diameter of different cells are different, and when the cells are injected into the left ventricle, the larger the cell diameter is, the easier the arterial embolism is caused, and the death of the animal is caused. (3) Cell state: the better the cell state and the better the activity, the faster the proliferation speed, which is beneficial to improving the success rate of modeling and the occurrence rate of transfer; (4) biological properties of the cells themselves: also, there are different cell lines in cancer species, and there is a large difference in their metastatic potential, which is mainly related to the biological properties of the tumor cells themselves. The in vivo imaging result (figure 2) of the small animal in the invention shows that compared with A549 cells, NCI-H1299 cells have larger luminous area and higher luminous intensity after intracardiac injection, and the cells with relatively higher transfer capacity are selected to be more favorable for manufacturing a transfer animal model. (5) Animal differences: the invention compares immunodeficient animals of BALB/c-nu/nu nude mice and NOD/SCID mice, and finds that the animals with the same cell concentration and the heavy immunodeficiency degree are more easy to transfer. Therefore, when a transfer animal model is established, severe combined immunodeficiency animals can be appropriately selected to improve the tumor transfer rate.

In a word, the establishment of the bone metastasis animal model by injecting the lung cancer cells in the heart under the guidance of the ultrasound is a feasible and effective modeling method, can successfully obtain a bone metastasis focus, accords with the characteristics of clinical lung cancer bone metastasis, can provide a new experimental tool and platform for clinical and basic research of lung cancer bone metastasis, and has certain practical application significance.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

1. A method for establishing a lung cancer bone metastasis animal model is characterized by comprising the following steps:

2. The method for establishing an animal model of bone metastasis of lung cancer according to claim 1, wherein in step a, GFP/Luc-labeled human lung cancer cells in logarithmic growth phase a549 or NCI-H1299 are prepared by: a549 cells or NCI-H1299 cells are paved on a 6-hole culture plate according to the cell density of 60-70%, GFP/Luc double-standard viruses are added to infect on day 2, the solution is changed after 6-8H, and conventional culture is carried out; after the cells are expanded, the GFP expression condition is observed under a fluorescence microscope, and if the GFP expression in the marked cells is weak or the cell marking rate cannot reach more than 90 percent, the cells need to be sorted by flow cytometry.