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CN114250200A - Animal model construction method for non-small cell lung cancer brain metastasis and spinal cord metastasis - Google Patents

Animal model construction method for non-small cell lung cancer brain metastasis and spinal cord metastasis Download PDF

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CN114250200A
CN114250200A CN202111588327.1A CN202111588327A CN114250200A CN 114250200 A CN114250200 A CN 114250200A CN 202111588327 A CN202111588327 A CN 202111588327A CN 114250200 A CN114250200 A CN 114250200A
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brain
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lung cancer
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张耀帅
李姗姗
刘浩
张语涵
徐廉松
郭宇
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BENGBU MEDICAL COLLEGE
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Abstract

The invention provides a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, and relates to the technical field of biomedicine. According to the method, the PC-9Luc cells fixed by the sodium carboxymethyl cellulose solution are injected into the brain of a nude mouse through a brain stereotaxic instrument to construct an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, so that the accuracy is high, and the reliability is good.

Description

Animal model construction method for non-small cell lung cancer brain metastasis and spinal cord metastasis
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis.
Background
Lung cancer (Lung cancer) is a malignant tumor of the Lung, and refers to a disease that is abnormally grown by Lung tissue cells and may invade adjacent tissues and organs, metastasize and spread to other parts of the body. The common symptoms comprise cough, hemoptysis, weight loss, shortness of breath, chest pain and the like. Lung cancer is classified into Non-Small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC) according to the histopathological type thereof. Among them, non-small cell lung cancer accounts for about 85% of all lung cancer patients, and its 5-year relative survival rate is only 23.3%, which has become a major burden of medical treatment in our country.
Because the brain structure and the spinal cord structure have particularity, tumor inoculation is difficult, and the detection mode is limited, the construction of an animal model is necessary.
Disclosure of Invention
The invention aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which can establish a reliable animal model and has higher use value.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which mainly comprises the following steps:
culturing the PC-9Luc cells to a logarithmic growth phase, and preparing a cell suspension after digestion; centrifuging the cell suspension, and mixing with sodium carboxymethylcellulose solution to obtain a preparation; anaesthetizing a nude mouse, injecting a spare part to the skull position of the nude mouse by using a brain stereotaxic instrument, then feeding the nude mouse, and carrying out the detection of the living body imaging fluorescence signal of the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
The animal model construction method for non-small cell lung cancer brain metastasis and spinal cord metastasis provided by the embodiment of the invention at least has the following beneficial effects:
the invention provides a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which is characterized in that a brain stereotaxic apparatus is used for injecting PC-9Luc cells fixed by a sodium hydroxymethyl fiber solution into a nude mouse brain to establish the animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, so that the accuracy is high, and the reliability is good.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a fluorescence signal detection diagram of the skull injection site on the lower right side of the skull provided by an embodiment of the present invention;
FIG. 2 is a fluorescence signal detection diagram of the skull injection site on the upper right side of the skull provided by an embodiment of the present invention;
FIG. 3 shows the result of brain metastasis at the lower right side of the skull injection site of a nude mouse according to an embodiment of the present invention;
FIG. 4 shows the result of the transfer of the vertebrae of the nude mouse with the skull injection position at the lower right side according to the embodiment of the present invention;
FIG. 5 shows the result of brain metastasis of nude mice with the injection site of skull being the upper right side according to the embodiment of the present invention;
FIG. 6 shows the result of the upper right spinal transfer at the skull injection site of the nude mouse according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
The invention provides a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which mainly comprises the following steps: culturing the PC-9Luc cells to a logarithmic growth phase, and preparing a cell suspension after digestion; centrifuging the cell suspension, and adding a culture medium to prepare a suspension; centrifuging the suspension, and mixing with sodium carboxymethylcellulose solution to obtain a preparation; anaesthetizing a nude mouse, injecting a spare part to the skull position of the nude mouse by using a brain stereotaxic instrument, then feeding the nude mouse, and carrying out the detection of the living body imaging fluorescence signal of the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Specifically, the cell line selected by the application is the lung adenocarcinoma PC-9Luc cell which is cultured as an injection cell. When the lung adenocarcinoma PC-9Luc cells are injected into small animals, the transfer positions of the cells can be detected in a fluorescence detection mode, so that the reliability of establishing a non-small cell lung cancer brain transfer and spinal cord transfer animal model through brain stereotaxic instrument injection can be effectively verified.
In the present application, the obtained PC-9Luc cells were subjected to cell culture. The method comprises the following specific steps: culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 36-37.3 ℃ and CO2Culturing under saturated humidity condition. When the cells are cultured to the logarithmic growth phase under the conditions, the cells can be in a higher activity level, and the phenomenon that the cells die in a large amount when adapting to the internal environment of an animal body is avoided, so that the reliability of a model establishment result is influenced.
After the cells are cultured to the logarithmic growth phase, 0.25% of pancreatin is added for digestion, the cells are blown away, and when the cells in the culture medium are resuspended, the digestion is stopped, so that the cells are prevented from being over digested, the activity of injected cells is prevented from being influenced, and the result of model construction is further influenced.
After the cell suspension is prepared, the cell suspension is centrifuged for 3min to 5min at the speed of 800rpm to 1000rpm, and after supernatant is removed, culture medium is added for blowing, and suspension is prepared after full suspension. In the application, the culture medium is sucked by a pipette gun and then blown out, and the beating effect is achieved after repeating for 50-70 times.
And (3) after the suspension is prepared, taking the suspension with a specific volume, counting the number of the suspensions, and mixing the counted suspensions with sodium carboxymethyl cellulose to prepare the spare article.
In the application, 10 mu L-20 mu L of suspension can be taken for counting, so that the workload of workers is reduced, and the cell amount in the spare article can reach the expected effect.
In addition, in the application, the cell quantity of the spare parts is determined in a counting mode, at the moment, the cell quantity of the spare parts can be used as an influence factor, and the brain metastasis and spinal cord metastasis conditions of the non-small cell lung cancer are researched when the cell quantity of the spare parts is changed, so that a more accurate animal model is established, and the practical value is higher.
In this application, the counting mode adopts ox abalone counting board to count, reaches anticipated count effect.
After counting, the centrifugation is carried out again, and the preparation of the spare article is carried out according to the preset cell concentration.
In the application, the suspension after centrifugation can be mixed with the sodium carboxymethyl cellulose solution to obtain a spare article with preset cell concentration. The aqueous solution of sodium carboxymethylcellulose has viscosity, and can maintain the pH value balance of the system in the pH range of 2-10, so that the sodium carboxymethylcellulose has high use value as the aqueous solution for fixing cells.
In the application, in order to ensure that the cells have a good fixing effect, the concentration of the sodium carboxymethyl cellulose solution is 0.8 wt% -1.2 wt%. When the concentration of the hydroxymethyl cellulose solution is higher, the viscosity of the aqueous solution is higher, which affects the activity of cells, and when the concentration of the hydroxymethyl cellulose solution is lower, the fixation effect of the hydroxymethyl cellulose solution on the cells is not expected, and the use effect is not good.
After cell preparation is complete, animal model establishment can be performed as follows:
in the present application, SPF-grade BALB/C nude mice were selected as a model. The nude mice are mainly characterized by hairless, nude and athymic, and due to the athymic feature, when a tumor recipient is transplanted in vivo, rejection reaction is not generated, so the nude mice are suitable for being used as the animal model of the application.
In the application, pentobarbital sodium of 10 mu g/mL is used as an anesthetic, and the anesthetic is completed by carrying out intraperitoneal injection on a nude mouse according to an injection ratio of 70 mu g/kg.
After anesthesia, the brain skin of the nude mice needs to be cut open. Specifically, the skin of a nude mouse is wiped with an alcohol cotton swab, then the nude mouse is cut at the center of the cranium of the nude mouse by about 1cm-2cm, the fascia is wiped with hydrogen peroxide, and then the forefontanel is exposed by wiping with the cotton swab.
The skull position is then determined. In detail, the anterior bregma is used as a reference position, and a hole is vertically drilled at the position 1mm away from the coronal and 1.5mm away from the sagittal sutures on the upper right side or lower right side of the skull, so as to find the injection position. In the drilling process, brain tissues need to be protected, and the brain tissues are prevented from being damaged by the drilling action, so that the brain metastasis of lung cancer cells and the spinal metastasis are influenced, and the stability of model establishment is further influenced.
After confirming the position, the prepared spare parts are injected to the position of the skull of the nude mouse by using a brain stereotaxic instrument, and an animal model is established. In detail, the needle point of the injector connected with the brain stereotaxic apparatus is arranged corresponding to the drill hole at the skull, then the needle inserting depth is set, and the spare parts are injected.
In the application, the needle insertion depth is 3.5mm downward and 0.5mm upward for injecting cells, the injection volume is 2-4 muL, the injection speed is 0.8-1.2 muL/min, and after the injection is finished, the needle is pulled out after the injection state is kept for 1-2 min, so that the whole injection process is finished.
It should be noted that the brain stereotaxic apparatus can determine the position of some nerve structures under the cortex by using the reference point, thereby completing the cell injection behavior under the condition of non-direct-view exposure, further ensuring the accurate cell injection position and reliable model establishment.
When the injection was completed, the wound was swabbed with a cotton swab, and the nude mice were sutured with a punch in the head and a wound in the skin. And (4) continuing to raise the nude mice after the nude mice revive.
After 2-3 weeks of feeding, the fluorescence signal of the cells can be detected by a small animal living body imaging detection method, and the tumor focus position of the nude mice is confirmed. Then collecting the brain tissue of the nude mouse and stripping the spine, dehydrating and fixing, embedding paraffin and slicing, and then carrying out HE staining to observe the tumor focus of the brain and the spine of the nude mouse.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present embodiment aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which includes the following steps:
culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 37 deg.C and CO2Culturing under saturated humidity condition. Then, digesting the PC-9Luc cells in the logarithmic phase with 0.25% of pancreatin to prepare a cell suspension; centrifuging the cell suspension at 900rpm for 4min, removing supernatant, adding culture medium, blowing, and suspending to obtain suspension;
dripping 15 μ L suspension into Haliotis diversicolor counting plate, counting, centrifuging at 900rpm for 4min, adding 1 wt% sodium carboxymethylcellulose solution to obtain cell with concentration of 1 × 1081.7x10 pieces/mL8one/mL and 2.7x108One spare article one, two spare articles and three spare articles per mL.
Anaesthetizing a nude mouse by a sodium pentobarbital solution of 10 mu g/L, cutting the skin of the cranium top of the nude mouse by 1cm-2cm, and removing the fascia by using hydrogen peroxide to expose bregma; and taking the bregma as a reference position, vertically drilling a hole at the position 1mm away from the bregma coronary suture and 2mm away from the sagittal suture on the lower right side of the skull, and confirming the skull injection position;
then, injecting a first spare product, a second spare product and a third spare product to skull injection positions of three groups of nude mice respectively by using a brain stereotaxic instrument, keeping the injection state for 1-2 min after injection, and pulling out the needle to finish the injection process.
After the nude mouse wakes up, feeding the nude mouse for 2-3 weeks, and carrying out the detection of the imaging fluorescence signal of the living body of the mouse on the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Example 2
The present embodiment aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which includes the following steps:
culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 37 deg.C and CO2Culturing under saturated humidity condition. Then, digesting the PC-9Luc cells in the logarithmic phase with 0.25% of pancreatin to prepare a cell suspension; centrifuging the cell suspension at 900rpm for 4min, removing supernatant, adding culture medium, blowing, and suspending to obtain suspension;
dripping 15 μ L suspension into Haliotis diversicolor counting plate, counting, centrifuging at 900rpm for 4min, adding 1 wt% sodium carboxymethylcellulose solution to obtain cell with concentration of 1 × 1081.7x10 pieces/mL8one/mL and 2.7x108One spare article one, two spare articles and three spare articles per mL.
Anaesthetizing a nude mouse by a sodium pentobarbital solution of 10 mu g/L, cutting the skin of the cranium top of the nude mouse by 1cm-2cm, and removing the fascia by using hydrogen peroxide to expose bregma; and taking the bregma as a reference position, vertically drilling a hole at the position 1mm away from the bregma coronary suture and 2mm away from the sagittal suture on the upper right side of the skull, and confirming the skull injection position;
then, injecting a first spare product, a second spare product and a third spare product to skull injection positions of three groups of nude mice respectively by using a brain stereotaxic instrument, keeping the injection state for 1-2 min after injection, and pulling out the needle to finish the injection process.
After the nude mouse wakes up, feeding the nude mouse for 2-3 weeks, and carrying out the detection of the imaging fluorescence signal of the living body of the mouse on the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Example 3
The present embodiment aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which includes the following steps:
culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 36 deg.C and CO2Culturing under saturated humidity condition. Then, digesting the PC-9Luc cells in the logarithmic phase with 0.25% of pancreatin to prepare a cell suspension; centrifuging the cell suspension at 700rpm for 5min, removing supernatant, adding culture medium, blowing, and suspending to obtain suspension;
then dripping 10 μ L suspension into Haliotis diversicolor counting plate, counting, centrifuging at 700rpm for 5min, adding 0.8 wt% sodium carboxymethylcellulose solution to obtain cell concentrations of 1 × 1081.7x10 pieces/mL8one/mL and 2.7x108One spare article one, two spare articles and three spare articles per mL.
Anaesthetizing a nude mouse by a sodium pentobarbital solution of 10 mu g/L, cutting the skin of the cranium top of the nude mouse by 1cm-2cm, and removing the fascia by using hydrogen peroxide to expose bregma; and taking the bregma as a reference position, vertically drilling a hole at the position 1mm away from the bregma coronary suture and 2mm away from the sagittal suture on the lower right side of the skull, and confirming the skull injection position;
then, a brain stereotaxic apparatus is used for respectively injecting a first spare product, a second spare product and a third spare product to skull injection positions of three groups of nude mice, and after injection, the injection state is kept for 1min, and then the needle is pulled out, so that the injection process is completed.
After the nude mouse wakes up, feeding the nude mouse for 2-3 weeks, and carrying out the detection of the imaging fluorescence signal of the living body of the mouse on the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Example 4
The present embodiment aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which includes the following steps:
culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 37.3 ℃ and CO2Culturing under saturated humidity condition. Then, PC-9Luc cells in the logarithmic growth phase were digested with 0.25% pancreatic enzyme to prepare fine cellsA cell suspension; centrifuging the cell suspension at 1000rpm for 3min, removing supernatant, adding culture medium, blowing, and suspending to obtain suspension;
then dropping 20 μ L of the suspension into a Haliotis diversicolor counting plate, counting, centrifuging at 1000rpm for 3min, adding 1.2 wt% sodium carboxymethylcellulose solution, and making into cell with concentration of 1 × 1081.7x10 pieces/mL8one/mL and 2.7x108One spare article one, two spare articles and three spare articles per mL.
Anaesthetizing a nude mouse by a sodium pentobarbital solution of 10 mu g/L, cutting the skin of the cranium top of the nude mouse by 1cm-2cm, and removing the fascia by using hydrogen peroxide to expose bregma; and taking the bregma as a reference position, vertically drilling a hole at the position 1mm away from the bregma coronary suture and 2mm away from the sagittal suture on the upper right side of the skull, and confirming the skull injection position;
then, a brain stereotaxic apparatus is used for respectively injecting a first spare product, a second spare product and a third spare product to skull injection positions of three groups of nude mice, and after injection, the injection state is kept for 2min, and then the needle is pulled out, so that the injection process is completed.
After the nude mouse wakes up, feeding the nude mouse for 2-3 weeks, and carrying out the detection of the imaging fluorescence signal of the living body of the mouse on the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Example 5
The present embodiment aims to provide a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, which includes the following steps:
culturing PC-9Luc cells in RPMI 1640 medium containing fetal bovine serum and hygromycin B, and placing the PC-9Luc cells at 36.5 ℃ and CO2Culturing under saturated humidity condition. Then, digesting the PC-9Luc cells in the logarithmic phase with 0.25% of pancreatin to prepare a cell suspension; centrifuging the cell suspension at 800rpm for 3.5min, discarding supernatant, adding culture medium, blowing, and suspending to obtain suspension;
then dropping 13 μ L of the suspension into Haliotis diversicolor counting plate, counting, centrifuging at 800rpm for 3.5min, addingAdding 0.9 wt% sodium carboxymethylcellulose solution to obtain cell concentrations of 1 × 1081.7x10 pieces/mL8one/mL and 2.7x108One spare article one, two spare articles and three spare articles per mL.
Anaesthetizing a nude mouse by a sodium pentobarbital solution of 10 mu g/L, cutting the skin of the cranium top of the nude mouse by 1cm-2cm, and removing the fascia by using hydrogen peroxide to expose bregma; and taking the bregma as a reference position, vertically drilling a hole at the position 1mm away from the bregma coronary suture and 2mm away from the sagittal suture on the upper right side of the skull, and confirming the skull injection position;
then, the brain stereotaxic apparatus is used for respectively injecting a first spare product, a second spare product and a third spare product to skull injection positions of three groups of nude mice, and after injection, the injection state is kept for 1.5min, and then the needle is pulled out, so that the injection process is completed.
After the nude mouse wakes up, feeding the nude mouse for 2-3 weeks, and carrying out the detection of the imaging fluorescence signal of the living body of the mouse on the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
Examples of effects
And (3) small animal living body imaging detection:
the animal models established in the embodiment 1 and the embodiment 2 are used for detecting the fluorescent signals of the living body imaging of the small animals in the following specific mode:
1. sequentially turning on a key knob of the X-ray lamp box, a host power supply and a computer;
2. opening software, and selecting fluorescence and white light for shooting;
3. placing the anesthetized nude mouse at the center of the tray, and enabling the part to be shot to be close to the bottom of the tray;
4. clicking the button of the upper left corner of the software, namely the Capture In-ViVo button, entering an image Capture interface, and shooting by fluorescence and white light.
It is noted here that the small animal in vivo imaging apparatus used was purchased from Bruker, USA, model FX-PRO.
The test results of the live imaging of the small animals are shown in fig. 1 and fig. 2, wherein fig. 1 is a fluorescence signal detection diagram of the skull injection position on the lower right side of the skull, and fig. 2 is a fluorescence signal detection diagram of the skull injection position on the upper right side of the skull.
As can be seen from FIG. 1, the cell volume after the injection of the first, second and third preparation was 3x1055x10 pieces5Or 8x105Under these conditions, nude mice developed both brain and spinal cord metastases.
As can be seen from FIG. 2, the cell volume after injection of the first preparation and the second preparation was 3X105Sum of 5x105At this time, only brain metastasis occurred in nude mice, and no spinal cord metastasis occurred; after the three injections of the preparation, the cell amount was 8 × 105Under these conditions, nude mice developed both brain and spinal cord metastases.
And (3) HE staining detection:
the nude mice of example 1 and example 2 were subjected to brain examination and spinal cord examination, respectively, in the following manner:
1. material taking: taking a brain tissue and a spinal cone tissue of a nude mouse, fixing for more than 24 hours by using a fixing solution, taking the tissue out of the fixing solution, flattening the tissue of a target part in a fume hood by using an operating knife, and then placing the trimmed tissue in a dehydration box;
2. dehydrating and wax dipping: the dewatering box is put into a dewatering machine to be sequentially dewatered by gradient alcohol: 4 hours of 75% alcohol, 2 hours of 85% alcohol, 2 hours of 90% alcohol, 1 hour of 95% alcohol, 30 minutes of absolute ethanol I, 30 minutes of absolute ethanol II, 5 minutes to 10 minutes of alcohol benzene, 5 minutes to 10 minutes of xylene I, 5 minutes to 10 minutes of xylene II, 1 hour of paraffin I1h melted at 65 ℃, 1 hour of paraffin II melted at 65 ℃ and 1 hour of paraffin III melted at 65 ℃;
3. embedding: firstly, putting melted wax into an embedding frame, then taking out the tissue from a dehydration box before wax solidification, putting the tissue into the embedding frame according to the requirements of an embedding surface, cooling the tissue at the temperature of minus 20 ℃, taking out the wax block from the embedding frame after solidification, and finishing the wax block;
4. slicing: putting the trimmed wax block into a freezing table at the temperature of minus 20 ℃ for cooling, putting the cooled wax block into a paraffin slicer for slicing, wherein the thickness of the slice is 4 mu m, then floating the slice in a spreading machine, flattening the tissue in warm water at the temperature of 40 ℃, then taking out the tissue by a glass slide, and then baking the slice in an oven at the temperature of 60 ℃.
The results are shown in fig. 3-6, fig. 3 shows the result of brain transfer at the injection position of the skull of the nude mouse, fig. 4 shows the result of spine transfer at the injection position of the skull of the nude mouse, fig. 5 shows the result of brain transfer at the injection position of the skull of the nude mouse, and fig. 6 shows the result of spine transfer at the injection position of the skull of the nude mouse.
As can be seen from FIGS. 3 and 4, in the nude mouse model constructed in example 1, tumor lesions were metastatic at both brain (see FIG. 3) and spinal cord (see FIG. 4),
as can be seen from fig. 5 and 6, in the nude mouse model constructed in example 2, only brain metastasis occurred in the tumor lesion (see fig. 5), and no spinal cord metastasis occurred (see fig. 6).
Comparing the detection result of the small animal living body imaging fluorescence signal with the detection result of HE staining, wherein the results are as follows: the detection results of the fluorescent signals of the live small animal imaging are as follows: when the injection position of a nude mouse is at the right lower side of the skull, the tumor focus has brain metastasis and spinal cord metastasis; and when the injection position of the nude mouse is the upper right side of the skull, at 3x105Sum of 5x105Under the condition of individual cell quantity, the nude mice only have brain metastasis, and the brain metastasis is 8x105Under the condition of cell amount of each mouse, the nude mice can simultaneously transfer brain and spinal cord; the HE staining result shows that when the amount of the PC-9Luc cells is different, the result is consistent with the result detected by the small animal living body imaging, so that the reliability of establishing the non-small cell lung cancer brain metastasis and spinal cord metastasis models through the injection of a brain stereotaxic apparatus can be shown, and the reliability is high.
In conclusion, the invention provides a method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, the method comprises the step of injecting PC-9Luc cells fixed by a sodium hydroxymethyl fiber solution into a brain of a nude mouse through a brain stereotaxic instrument to construct the animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis, and the method has high accuracy and good reliability.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (10)

1. A method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis is characterized by comprising the following steps:
culturing the PC-9Luc cells to a logarithmic growth phase, and preparing a cell suspension after digestion;
centrifuging the cell suspension, and mixing with sodium carboxymethylcellulose solution to obtain a preparation;
after anaesthetizing a nude mouse, injecting the stock solution into the target position of the nude mouse, then feeding the nude mouse, and carrying out the detection of the living body imaging fluorescence signal of the nude mouse; then, brain tissues and vertebrae of nude mice were collected, and tumor metastasis of the brain and spinal cord of nude mice was observed by sectioning.
2. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases as claimed in claim 1, wherein the target sites are specifically as follows: taking bregma as a reference point, and vertically drilling a hole at the position 1mm away from the bregma coronal suture and 1.5mm away from the sagittal suture on the upper right side or the lower right side of the skull, thereby obtaining the position of the skull.
3. The method for constructing an animal model of non-small cell lung cancer brain metastasis and spinal cord metastasis according to claim 2, wherein the stock is injected by using a brain stereotaxic apparatus, and the needle insertion depth of the brain stereotaxic apparatus is 2.9mm to 3.1 mm.
4. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases according to any one of claims 1 to 3, wherein the cell suspension is centrifuged at 800rpm to 1000rpm for 3min to 5 min.
5. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases as claimed in claim 1, wherein 10-20 μ L of cell suspension is counted before mixing with said sodium carboxymethyl cellulose, and then centrifuged.
6. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases according to claim 1, wherein the concentration of the sodium carboxymethyl cellulose is 0.8 wt% -1.2 wt%.
7. The method for constructing animal model of non-small cell lung cancer brain and spinal cord metastasis according to claim 6, wherein the cell concentration of the stock is (1-2.7) x108one/mL.
8. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases as claimed in claim 1, wherein the cell amount of PC-9Luc cells is (3-8) x10 during the injection of the stock preparation5And (4) respectively.
9. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases as claimed in claim 8, wherein the volume of the stock is 2 μ L-4 μ L when the stock is injected to the target site once in the process of injecting the stock.
10. The method for constructing an animal model of non-small cell lung cancer brain and spinal cord metastases according to claim 8 or 9, wherein the injection speed of the spare article is 0.8-1.2 μ L/min during the injection of the spare article.
CN202111588327.1A 2021-12-23 2021-12-23 Animal model construction method for non-small cell lung cancer brain metastasis and spinal cord metastasis Pending CN114250200A (en)

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101919747A (en) * 2009-06-17 2010-12-22 上海中医药大学附属龙华医院 Construction method of an improved orthotopic transplantation model of colon cancer in nude mice
CN103197067A (en) * 2006-02-28 2013-07-10 阿里乌斯研究公司 Cytotoxicity mediation of cells evidencing surface expression of CD44
CN103330604A (en) * 2013-07-16 2013-10-02 蚌埠医学院 Method building mice portable breast cancer tumor cell suspension orthotopic model
CN103976803A (en) * 2014-05-26 2014-08-13 贵阳医学院附属医院 Human colon cancer nude mouse skin subcutaneous transplantation tumor model establishing method
CN106367393A (en) * 2016-08-26 2017-02-01 中国人民解放军第四军医大学 Mouse prostate cancer circulating tumor cell line and prostate cancer circulating tumor cell isolating and culturing method
CN107299084A (en) * 2017-06-27 2017-10-27 中国人民解放军第二军医大学第二附属医院 Occur application of the circulating tumor stem cell of Epithelial and stromal in lung cancer propagation, resistance and transfer disease
WO2018234556A1 (en) * 2017-06-23 2018-12-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for preventing or treating cancer resistance to egfr inhibition
CN110357858A (en) * 2018-04-09 2019-10-22 威尚（上海）生物医药有限公司 With the 5 substitution difluoropiperdin compounds across blood-brain barrier ability
CN110537992A (en) * 2019-05-17 2019-12-06 暨南大学 A method for constructing an improved human nasopharyngeal carcinoma orthotopic xenograft model
CN110663637A (en) * 2019-09-16 2020-01-10 承德医学院 Construction method of human choriocarcinoma nude mouse orthotopic transplantation tumor model
CN111713453A (en) * 2020-06-18 2020-09-29 复旦大学附属肿瘤医院 A method for establishing an animal model of lung cancer bone metastasis
CN213098515U (en) * 2020-07-17 2021-05-04 广州医科大学 Novel experimental animal brain stereotaxic instrument
CN112813032A (en) * 2021-02-08 2021-05-18 中国科学院苏州纳米技术与纳米仿生研究所 Stem cell evaluation multichannel imaging system and preparation method and application thereof

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103197067A (en) * 2006-02-28 2013-07-10 阿里乌斯研究公司 Cytotoxicity mediation of cells evidencing surface expression of CD44
CN101919747A (en) * 2009-06-17 2010-12-22 上海中医药大学附属龙华医院 Construction method of an improved orthotopic transplantation model of colon cancer in nude mice
CN103330604A (en) * 2013-07-16 2013-10-02 蚌埠医学院 Method building mice portable breast cancer tumor cell suspension orthotopic model
CN103976803A (en) * 2014-05-26 2014-08-13 贵阳医学院附属医院 Human colon cancer nude mouse skin subcutaneous transplantation tumor model establishing method
CN106367393A (en) * 2016-08-26 2017-02-01 中国人民解放军第四军医大学 Mouse prostate cancer circulating tumor cell line and prostate cancer circulating tumor cell isolating and culturing method
WO2018234556A1 (en) * 2017-06-23 2018-12-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for preventing or treating cancer resistance to egfr inhibition
CN107299084A (en) * 2017-06-27 2017-10-27 中国人民解放军第二军医大学第二附属医院 Occur application of the circulating tumor stem cell of Epithelial and stromal in lung cancer propagation, resistance and transfer disease
CN110357858A (en) * 2018-04-09 2019-10-22 威尚（上海）生物医药有限公司 With the 5 substitution difluoropiperdin compounds across blood-brain barrier ability
CN110537992A (en) * 2019-05-17 2019-12-06 暨南大学 A method for constructing an improved human nasopharyngeal carcinoma orthotopic xenograft model
CN110663637A (en) * 2019-09-16 2020-01-10 承德医学院 Construction method of human choriocarcinoma nude mouse orthotopic transplantation tumor model
CN111713453A (en) * 2020-06-18 2020-09-29 复旦大学附属肿瘤医院 A method for establishing an animal model of lung cancer bone metastasis
CN213098515U (en) * 2020-07-17 2021-05-04 广州医科大学 Novel experimental animal brain stereotaxic instrument
CN112813032A (en) * 2021-02-08 2021-05-18 中国科学院苏州纳米技术与纳米仿生研究所 Stem cell evaluation multichannel imaging system and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANLONG TAN EY AL.: "Tyrosine kinase inhibitors show different anti-brain metastases efficacy in NSCLC: A direct comparative analysis of icotinib, gefitinib, and erlotinib in a nude mouse model", 《ONCOTARGET》, vol. 8, no. 58, pages 2 - 3 *
崔白苹等: "脑转移瘤动物模型的制备方法及研究进展", 《中国药理学通报》, vol. 32, no. 3, pages 304 - 309 *

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