CN114931125A - Method for constructing mouse lung cancer pleural effusion model - Google Patents
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- CN114931125A CN114931125A CN202210260539.5A CN202210260539A CN114931125A CN 114931125 A CN114931125 A CN 114931125A CN 202210260539 A CN202210260539 A CN 202210260539A CN 114931125 A CN114931125 A CN 114931125A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0271—Chimeric animals, e.g. comprising exogenous cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for constructing a mouse lung cancer pleural effusion model, which comprises the following steps: (1) culturing LLC cells in vitro, and diluting with physiological saline to 2 × 10 when the cells enter exponential growth phase 5 ～5x10 6 Per mL; (2) taking a 6-8 week-old C57BL/6J wild-type mouse, adapting for 3-5 days, anesthetizing by using an isoflurane anesthesia machine, clamping the skin between 5-6 ribs on the left side of the sternum handle of the mouse by using forceps, making a longitudinal incision at the clamped position by using an ophthalmic scissors, injecting a needle tip with the depth of 3-5 mm, and slowly injecting 10 ml/g LLC cell suspension; (3) after the mice are normally fed and cultured for 6-12 days, the lung cancer pleural effusion model of the mice is prepared. The invention has simple experimental equipment, convenient operation and short modeling period, can successfully construct the pleural effusion model of the mouse, has enough survival time for supporting the subsequent further corresponding drug experiments, and has good repeatability and wide application range.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing a mouse lung cancer pleural effusion model.
Background
Pleural effusion is one of the clinical events of advanced lung cancer, and seriously affects the quality of life and clinical drug treatment of patients. With the continuous deepening of national economic reform, the process of urbanization and industrialization is accelerated continuously, and the incidence and the mortality of lung cancer are increased, so that the lung cancer becomes the malignant tumor with the highest incidence and mortality. The advanced lung cancer causes huge physical and economic burden to patients and family members thereof, and especially the occurrence of pleural effusion often seriously affects the life quality of lung cancer patients and restricts the medication selection of clinicians. Because the human body cannot be directly used for testing, the construction of the animal lung cancer pleural effusion model is an important means for the development of related treatment, related medicaments and related diagnostic markers and diagnostic methods.
At present, it has been reported that models of pleural effusion are mainly teaching aids for in vitro medical teaching, for example: chinese patent application CN202601058U discloses a model of pulmonary ventilation dysfunction. Commonly used animal models of lung cancer include mainly induction and transplantation models, such as: chinese patent application CN104826136A discloses a method for constructing a rat transplantation lung cancer model, and chinese patent application CN108721256B discloses a method for constructing a mouse induced lung cancer model. However, no stable pleural effusion model is involved in the current lung cancer animal model construction, and no direct correlation is generated with pleural effusion events of late-stage clinical lung cancer patients, so that the problems of low quality of life, clinical medication limitation and the like of late-stage patients caused by pleural effusion cannot be simulated and promoted to be solved. Therefore, the construction methods of the lung cancer and pleural effusion related models are difficult to meet the related requirements.
Disclosure of Invention
In order to solve the problems in the prior art, the invention successfully constructs a mouse lung cancer pleural effusion model by a mouse pleural local transplantation mode. Transient anesthesia is directly used to create tiny wounds, and lung cancer cells are transplanted locally in the thoracic cavity.
In order to achieve the purpose, the invention adopts the following technical scheme: the invention relates to a method for constructing a mouse lung cancer pleural effusion model, which comprises the following steps of:
(1) culturing LLC cells in vitro, and diluting with physiological saline to 2 × 10 when the cells enter exponential growth phase 5 ～5x10 6 /mL；
(2) Taking a C57BL/6J wild type mouse with the age of 6-8 weeks, adapting for 3-5 days, after anesthesia by using an isoflurane anesthesia machine, clamping the skin between 5-6 ribs on the left side of a sternum handle of the mouse by using forceps, manufacturing a longitudinal incision with the length of 2-3 mm at the clamped position by using an ophthalmic scissors, wherein the injection depth of a needle point is 3-5 mm, and slowly injecting 10 ml/g LLC cell suspension;
(3) after the mice are normally fed and cultured for 6-12 days, the lung cancer pleural effusion model of the mice is prepared.
Further, in step (1), LLC cells are cultured in vitro, and when the cells enter the exponential growth phase, the cell concentration is diluted to 2x10 with physiological saline 5 ～5x10 6 mL, cell culture conditions included: 10% fetal bovine serum, 90% DMED high sugar medium, 37 ℃, 5% carbon dioxide cell incubator, trypsin, sterile super clean bench, centrifuge, inverted microscope.
Further, in the step (2), a C57BL/6J wild-type mouse with the age of 6-8 weeks is taken, the mouse is adapted for 3-5 days, after anesthesia is carried out by using an isoflurane anesthesia machine, the skin between 5-6 ribs on the left side of a sternum handle of the mouse is clamped by using forceps, a longitudinal incision with the length of 2-3 mm is made at the clamped position by using an ophthalmic scissors, the injection depth of a needle point is 3-5 mm, 10 ml/g LLC cell suspension is slowly injected by inserting a needle, after the injection is finished, the needle head is quickly pulled out, the wound is disinfected by proper iodophor, respiratory anesthesia is quickly stopped, the mouse is placed to be recovered after awakening, a mouse cage is placed to observe vital signs, and heat preservation is taken. The mice include male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse culture condition parameters comprise: the temperature is 19-25 ℃, the relative humidity is 50-70%, and the daily incandescent lamp illuminates for 8-10 hours in the day.
Furthermore, in step (1), all of the LLC in vitro culture cells are in exponential growth phase, and the number of living cells reaches more than 90%.
Further, in the step (2), the weight of the mouse for molding is 16-18g, the molding process is carried out on a sterile operating platform, and the mouse is anesthetized by using an isoflurane respiratory anesthesia machine.
Further, in step (2), the mice include male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse condition parameters comprise: the temperature is 19-25 ℃, the relative humidity is 50-70%, and the daily incandescent lamp illuminates for 8-10 hours in the day.
Further, in step (3), the mice are normally fed and cultured for 7-10 days.
Further, in the step (3), the mice are normally fed and cultured for 10-12 days.
Further, in the step (2), a tiny epithelial tissue wound of 2 mm is made between the 5-6 ribs.
Further, in step (3), the mice were normally fed and cultured for 7 days.
The mouse lung cancer pleural effusion model is applied to detecting the state of pleural effusion, screening and developing related medicaments, determining related treatment schemes, finding related diagnostic markers and developing related diagnostic methods.
Has the advantages that: the invention has the advantages of simple required experimental equipment, convenient operation, short modeling period, good repeatability and wide application range, can successfully construct the pleural effusion model of the mouse, and ensures that the survival time of the mouse is enough to support the subsequent further corresponding drug experiments.
Compared with the prior art, the invention has the following advantages:
(1) the method can effectively and quickly simulate the situation of the pleural effusion of the late-stage lung cancer in clinic, the daily activities and the body weight of the mouse cannot be influenced in the model construction process, the development process of the actual primary lung cancer of the human body is met, and the actual situation of the pleural effusion of the late-stage lung cancer patient can be reflected. The method is beneficial to the formulation of a lung cancer pleural effusion related treatment scheme, the screening and research and development of related treatment medicines, and the discovery of lung cancer pleural effusion diagnosis markers and diagnostic reagent research and development.
(2) The method directly constructs the model of the pleural effusion of the late stage lung cancer of the mouse by the way of in vitro transplantation of LLC cells, can effectively correspond to the time condition of the pleural effusion of a patient with the late stage lung cancer in clinic, has short molding period, can reflect the actual occurrence condition of the pleural effusion of the lung cancer in a human body, and is beneficial to research and development of related treatment strategies of the pleural effusion and related new medicines.
Drawings
FIG. 1 is a schematic representation of LLC cells in exponential growth phase prior to molding in accordance with the invention;
FIG. 2 is a micrograph of a mouse pleural effusion after molding in accordance with the present invention;
FIG. 3 is a graph showing the weight change of the model building group of the present invention and the control group of mice during the model building process;
FIG. 4 is a graph showing the body weight change of the model building group and the control group mice after model building;
FIG. 5 is a plot of pleural effusion volume at day 7 of the molding of the model and control mice of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
The invention discloses a method for constructing a mouse lung cancer pleural effusion model, which comprises the following steps: (1) culturing LLC cells in vitro, counting with hemacytometer and trypan blue when the cells enter exponential growth phase, and diluting the cell concentration to 2 × 10 with physiological saline 5 ～5x10 6 Per mL; all LLC in vitro culture cells of the model are in an exponential growth phase, and the number of living cells reaches more than 90%.
(2) Taking 10C 57BL/6J wild-type mice with 8 weeks old, each half of male and female, adapting for 5 days, after shallow anesthesia by using an isoflurane anesthesia machine, clamping the skin between 5 ribs on the left side of a sternum handle of the mice by using forceps, manufacturing a longitudinal incision with the 2 mm at the clamped position by using an ophthalmic scissors, wherein the injection depth of a needle point is 3 mm, slowly injecting 10 ml/g LLC cell suspension by inserting a needle, quickly pulling out the needle after injection, sterilizing the wound by using proper iodophor, quickly stopping respiratory anesthesia, placing the mice, returning the mice to a warm-keeping squirrel cage to observe vital signs after the mice are revived, and paying attention to warm keeping; the mouse culture condition parameters comprise: the temperature is 19 ℃, the relative humidity is 70%, and the daily incandescent lamp illuminates for 10 hours in the day. The mice include male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse culture condition parameters comprise: the temperature is 20 ℃, the relative humidity is 60%, and the daily incandescent lamp illuminates for 9 hours in the day. The weight of the mouse for molding is 17g, the molding process is carried out on an aseptic operating platform, and the mouse is anesthetized by using an isoflurane respiratory anesthesia machine.
(3) After the mice are normally fed and cultured for 8 days, the lung cancer pleural effusion model of the mice is prepared.
The cell culture of the present invention was carried out by observing the cell morphology under a microscope to judge that the cells were in the exponential growth phase (FIG. 1)
The mouse lung cancer pleural effusion model is applied to detecting the state of pleural effusion, screening and developing related medicaments, determining related treatment schemes, finding related diagnostic markers and developing related diagnostic methods.
Example 2
Example 2 differs from example 1 in that: the invention discloses a method for constructing a mouse lung cancer pleural effusion model, which comprises the following steps: in step (1), LLC cells are cultured in vitro, and when the cells enter exponential growth phase, the cell concentration is diluted to 2x10 with physiological saline 5 ～5x10 6 mL, cell culture conditions included: 10% fetal bovine serum, 90% DMED high sugar medium, 5% carbon dioxide cell incubator at 37 ℃, trypsin, sterile super clean bench, centrifuge, inverted microscope.
In the step (2), a C57BL/6J wild-type mouse with the age of 6 weeks is taken, the mouse is adapted for 3 days, after anesthesia is carried out by using an isoflurane anesthesia machine, the skin between 5.5 ribs on the left side of a sternum handle of the mouse is clamped by forceps, a longitudinal incision with the depth of 3 mm is made at the clamped position by using an ophthalmic scissors, the injection depth of a needle point is 5 mm, 10 ml/g LLC cell suspension is injected slowly through a needle, after the needle head is pulled out quickly after the injection is finished, the wound is disinfected by proper iodophor, the respiratory anesthesia is stopped quickly, the mouse is placed to be placed back to a mouse cage to observe vital signs after the mouse is revived, and the mouse is kept warm by paying attention. The mice include male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse culture condition parameters comprise: the temperature is 25 ℃, the relative humidity is 50%, and the daily incandescent lamp illuminates for 8 hours in the day. The weight of the mouse for molding is 16 g, the molding process is carried out on an aseptic operating platform, and the mouse is anesthetized by using an isoflurane respiratory anesthesia machine.
In the step (3), after the mice are normally fed and cultured for 6 days, the mice lung cancer pleural effusion model is prepared.
Example 3
Example 3 differs from example 1 in that: the invention relates to a method for constructing a mouse lung cancer pleural effusion model, which comprises the following steps of: in step (1), LLC cells are cultured in vitro, and when the cells enter exponential growth phase, the cell concentration is diluted to 2x10 with physiological saline 5 ～5x10 6 mL, cell culture conditions included: 10% fetal bovine serum, 90% DMED high sugar medium, 37 ℃, 5% carbon dioxide cell incubator, trypsin, sterile super clean bench, centrifuge, inverted microscope.
In the step (2), C57BL/6J wild-type mice with the age of 7 weeks are taken, are adapted for 4 days, are anesthetized by an isoflurane anesthesia machine, then are used for clamping the skin between 6 ribs on the left side of a sternum handle of the mice, are used for making a longitudinal incision with the depth of injection of a needle point of 3 mm at the clamped position by ophthalmic scissors, and are 5 mm, and the mice comprise male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse culture condition parameters comprise: the temperature is 22 ℃, the relative humidity is 60%, and the daily incandescent lamp illuminates for 9 hours in the day. The weight of the mouse for molding is 18g, the molding process is carried out on an aseptic operating platform, and the mouse is anesthetized by using an isoflurane respiratory anesthesia machine.
In the step (3), after the mice are normally fed and cultured for 12 days, the mice lung cancer pleural effusion model is prepared.
Test example 1
Materials and equipment:
1.1 Experimental animals
Wild type C57 mice (C57 BL/6J wild type mice): SPF rating, 6-8 weeks old, 16-20 g mass, purchased from Calvens laboratory animals Inc., Changzhou.
1.2 test cells
LLC cells are donated by the university of Nanjing, Life sciences college.
1.3 Primary reagents and configurations
DMEM high-sugar culture solution, fetal calf serum, trypsin, trypan blue detection kit and isoflurane.
1.4 Main Experimental Equipment
Super clean bench, inverted microscope, carbon dioxide cell incubator, centrifuge, and animal suction-type anesthesia machine
Treatment of cells
Taking out LLC cells from a liquid nitrogen tank or a-80 ℃ refrigerator, quickly thawing for 1 min at 37 ℃, centrifuging for 3 min at 1000 rpm/min, culturing in a 90% DMEN high-sugar culture solution and 10% fetal calf serum at 37 ℃ in a 5% carbon dioxide incubator until the cells enter an exponential growth period and are paved on the bottom of a culture bottle, and performing enzymolysis on the iron wall cells by using trypsin and washing by using PBS.
Cell counting
When the cells are cultured to be fully paved at the bottom of the incubator, the total number of the cells is calculated by utilizing a blood counting plate technology, and the proportion of the living cells is calculated by adopting a trypan blue detection kit.
6-8 days after the model of the lung cancer pleural effusion of the mouse in example 1 was molded, the mouse was sacrificed by carbon dioxide inhalation, the pleural cavity was opened to remove tumor tissue and pleural effusion, and the cell type in the pleural effusion was observed by a microscope (FIG. 2), and it was found that the pleural effusion contained a large amount of red blood cells and agglomerated tumor cells.
After modeling, the weight change of the mice is observed by weighing every other day, and the weight change of the mice during the modeling period is found to be not statistically different from that of a control group (figure 3), but the weight of the mice is remarkably different 14 days after the successful modeling, and the symptoms of the mice in a model group are remarkably reduced (p is less than 0.05, figure 4) and are consistent with the symptoms of patients with late clinical lung cancer pleural effusion. On day seven of molding, pleural effusion was detected in all molded mice (fig. 5).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A method for constructing a mouse lung cancer pleural effusion model is characterized by comprising the following steps: (1) culturing Lewis lung cancer cell (LLC) in vitro, and diluting with normal saline to 2 × 10 when the cell enters exponential growth phase 5 ～5x10 6 /mL；
(2) Taking a C57BL/6J wild type mouse with the age of 6-8 weeks, adapting for 3-5 days, after anesthesia by using an isoflurane anesthesia machine, clamping the skin between 5-6 ribs on the left side of a sternum handle of the mouse by using forceps, manufacturing a longitudinal incision with the length of 2-3 mm at the clamped position by using an ophthalmic scissors, wherein the injection depth of a needle point is 3-5 mm, and slowly injecting 10 ml/g LLC cell suspension;
(3) after the mice are normally fed and cultured for 6-12 days, the lung cancer pleural effusion model of the mice is prepared.
2. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in step (1), LLC cells are cultured in vitro, and when the cells enter exponential growth phase, the cell concentration is diluted to 2x10 by normal saline 5 ～5x10 6 mL, cell culture conditions included: 10% fetal bovine serum, 90% DMED high sugar medium, 37 ℃, 5% carbon dioxide cell incubator, trypsin, sterile super clean bench, centrifuge, inverted microscope.
3. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in the step (2), taking a 6-8 week old C57BL/6J wild-type mouse, adapting to 3-5 days, anesthetizing by using an isoflurane anesthesia machine, clamping the skin between 5-6 ribs on the left side of a sternum handle of the mouse by using forceps, making a 2-3 mm longitudinal incision at the clamped position by using an ophthalmological scissors, injecting a needle point into the tissue with the depth of 3-5 mm, slowly injecting 10 ml/g of LLC cell suspension into the needle, quickly pulling out the needle after injection, disinfecting the wound by using appropriate iodine, quickly stopping respiratory anesthesia, putting the mouse back to a mouse cage after the mouse is awake to observe vital signs, paying attention to warm keeping, wherein the mice comprise male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse culture condition parameters comprise: the temperature is 19-25 ℃, the relative humidity is 50-70%, and the daily incandescent lamp illuminates for 8-10 hours in the day.
4. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in the step (1), all LLC in vitro culture cells are in an exponential growth phase, and the number of living cells reaches more than 90%.
5. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in the step (2), the weight of the mouse for molding is 16-18g, the molding process is carried out on a sterile operating platform, and the mouse is anesthetized by using an isoflurane respiratory anesthesia machine.
6. The method for constructing a mouse lung cancer pleural effusion model according to claim 5, wherein: in step (2), said mice include male and female mice; the anesthesia machine is an animal suction type anesthesia machine; the syringe is a disposable sterile syringe small size; the mouse condition parameters comprise: the temperature is 19-25 ℃, the relative humidity is 50-70%, and the daily incandescent lamp illuminates for 8-10 hours in the day.
7. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in the step (3), the normal feeding and water supply culture time of the mice is 7-10 days.
8. The method for constructing a mouse lung cancer pleural effusion model according to claim 1, wherein: in the step (2), a tiny epithelial tissue wound of 2 mm is made between the 5-6 ribs; in the step (3), the mice are normally fed and cultured for 10-12 days.
9. The method for constructing a mouse lung cancer pleural effusion model according to claim 7, wherein: in step (3), the mice were normally fed and cultured for 7 days.
10. Use of the mouse pleural effusion model of any of claims 1-9 for detecting pleural effusion status, screening and developing related drugs, determining related treatment regimens, and finding related diagnostic markers and developing related diagnostic methods.
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