CN112790159A - Method for establishing esophageal cancer PDX mouse model - Google Patents
Method for establishing esophageal cancer PDX mouse model Download PDFInfo
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Abstract
The invention discloses a method for establishing a PDX mouse model of esophageal cancer, which comprises the steps of screening clinical cases of patients with esophageal cancer, collecting esophageal tumor specimens and blood samples of the patients in an operating room, centrifuging the blood samples of the patients, sucking serum into a plurality of EP tubes by using a pipette gun in an ultra-clean workbench after ultraviolet disinfection, placing 1ml of serum into each EP tube, placing the EP tubes in which the specimens are placed on ice for transplantation, and storing the rest of serum in a refrigerator with the temperature of minus 80 ℃ for passage; the serum pretreatment is to use the serum of a patient with the same source as the tumor specimen to incubate the tumor specimen to be inoculated for 5 minutes at constant temperature of 37 ℃ and then transplant the tumor specimen into a mouse body; the invention has high success rate, stable passage and short period.
Description
Technical Field
The invention relates to the field of medicine, in particular to a method for establishing a PDX mouse model of esophageal cancer.
Background
Esophageal cancer is one of the six most common malignant tumors in the world, and China is the country with the highest incidence of esophageal cancer in the world. Over the years, a plurality of esophageal cancer prevention and treatment workers have conducted keen innovation, and have attracted attentive achievements in the aspects of epidemiology, early diagnosis, comprehensive treatment and prevention and the like of esophageal cancer, but the esophageal cancer prevention and treatment work still holds pace and difficulty, the total degree of prevention and treatment still does not reach a satisfactory degree, and great breakthroughs are not made in the aspects of drug screening and new drug development.
The research of tumor animal models is indispensable for researching and developing new drugs, finding specific targets of the drugs and accurately predicting the curative effect. Common tumor animal models are cell-line-derived xenotransplantation (CDX) and human tumor tissue-derived transplantation tumor models (PDX), and the main difference between the CDX animal models and the PDX animal models is that the "raw material" is tumor cells, and the PDX animal models are tumor specimens collected from patients. The success rate and the passage rate of CDX which is taken as a tumor animal model derived from pure tumor cells are naturally much higher than those of PDX, but the affinity with patients is greatly reduced, so that the curative effect of the medicine cannot be accurately predicted in the research process of tumor medicines; PDX can just make up for the defect, can completely or partially simulate the survival environment of the tumor in the human body in the immunodeficient mouse body, greatly increases the affinity of the tumor animal model and the human body, and can maintain the biological and histopathological characteristics of the tumor in the mouse body, thereby providing more accurate and reliable prediction basis for the drug effect research of the tumor medicament.
In the existing PDX mouse modeling technology, after a tumor specimen collected from a patient is transplanted to an immunodeficient mouse, although the tumor specimen can be successfully attached to the subcutaneous part of the mouse and grow based on the immunodeficient characteristic of the mouse, because the tumor specimen still has certain immunity of the patient, a certain degree of immune rejection reaction still exists between the tumor and the mouse, the transplanted tumor cannot be completely transplanted to be attached to the subcutaneous part of the mouse in a short period to achieve a normal growth state of the tumor, meanwhile, the tumor which is not completely transplanted is easy to necrotize due to insufficient blood supply and other reasons, the success rate of modeling and passage is finally influenced, and the modeling period is prolonged.
The defects of low success rate, instable passage, long period and the like of the esophageal cancer PDX mouse model based on the prior art cause that the PDX animal model can not be applied in a large scale and in a conventional way at present.
Disclosure of Invention
The invention aims to provide a method for establishing an esophageal cancer PDX mouse model, which aims to solve the problems in the background technology and has the advantages of high success rate, stable passage and short period.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for establishing an esophageal cancer PDX mouse model comprises the following specific steps:
firstly, specimen collection: by clinical esophagus cancer patient case screening, collecting an esophagus tumor sample and a patient blood sample in an operating room;
secondly, specimen transportation: after the sample is separated from the body, the sample is properly washed by saline, then is transferred into a 50ml centrifuge tube containing a cell culture medium RPMI-1640/DMEM, and is placed on ice together with a patient blood sample to be conveyed to an animal center;
thirdly, preprocessing a blood sample: centrifuging a patient blood sample, sucking serum into a plurality of EP tubes by using a pipette gun in an ultra-clean workbench after ultraviolet disinfection, placing 1ml of serum into each EP tube, placing the EP tubes in which the samples are placed on ice for transplantation, and freezing and storing the rest of serum in a refrigerator at minus 80 ℃ for passage;
fourthly, tumor specimen pretreatment: removing part of necrotic tissues of tumors in an ultra-clean workbench after ultraviolet disinfection, cutting the tumors into rice grains after being washed by normal saline, dividing the tumors to be inoculated into two groups, putting the two groups into a culture dish of 100mm containing normal saline, covering the culture dish and putting the culture dish on ice;
fifth, mouse inoculation: selecting BALB/c-nu/nu mice with even numbers of 4-6 weeks old, equally dividing into 2 groups, wherein one group is used as an experimental group, and the other group is used as a control group; the experimental group is inoculated with the tumor pretreated by the serum of a patient, the control group is inoculated with the tumor treated by the normal saline, the inoculation processes are carried out in an ultra-clean workbench after ultraviolet disinfection, the inoculation part is positioned at the position close to the armpit at the subcutaneous left side of the mouse, and the sterilization treatment is carried out on a wound at a part before and after the inoculation;
sixthly, enabling the mice to naturally grow, observing and detecting each index of the two groups of mice every week, and comparing the tumorigenesis conditions of the two groups after 8 weeks;
seventhly, after 8 weeks, the tumors of the two groups of mice are stripped and passaged, and the new BALB/c-nu/nu mice with the even number of 4-6 weeks old are divided into two groups, one group is used as a passage experimental group, and the other group is used as a passage control group; inoculating the tumor pretreated by the serum of the patient according to the passage experimental group, and inoculating the tumor treated by the normal saline into the passage control group in a grouping way, wherein the method is the same as the fourth step and the fifth step;
and eighthly, allowing the mice to naturally grow, observing and detecting each index of the two groups of mice every week, and comparing the tumorigenesis conditions of the two groups after 8 weeks.
In the first step, a blood sample of the patient into which the anesthetic is not injected is collected.
In the fifth step, the serum pretreatment is to incubate the tumor specimen to be inoculated with the serum of the patient from the same source as the tumor specimen at constant temperature of 37 ℃ for 5 minutes and then transplant the tumor specimen into a mouse body.
In the fourth step, the preferred size of the rice grains is 0.2 x0.2x0.2cm.
In the fifth step, the used puncture implantation needle is a 25-gauge trocar.
Compared with the prior art, the invention has the beneficial effects that:
the method for establishing the esophageal cancer PDX mouse model is optimized and improved in the aspects of tumor formation rate, tumor formation volume, tumor formation speed, tumor formation quality and tumor formation weight; meanwhile, in the passage process, the PDX model established by the method is optimized and improved to different degrees in the aspects of passage tumor formation rate, tumor formation volume, tumor formation speed, tumor formation quality and tumor formation weight, the success rate is high, passage can be stably carried out, and the period is short.
Drawings
FIG. 1 is a graph of the change in the tumor formation volume of mice.
FIG. 2 is a graph showing the change in body weight of mice.
Fig. 3 is a graph comparing tumor weights.
FIG. 4 is a graph comparing mouse tumor formation rates.
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. 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.
According to fig. 1-4, the present invention provides a technical solution:
a method for establishing an esophageal cancer PDX mouse model comprises the following specific steps:
(1) collecting a specimen: by clinical esophagus cancer patient case screening, collecting an esophagus tumor sample and a patient blood sample in an operating room; in order to avoid the influence of narcotics in the operation, a blood sample of a patient is collected before the operation;
(2) specimen transportation: the method comprises the following steps that after the sample is separated from the body, the sample is properly washed by saline, then the sample is transferred into a 50ml centrifugal tube containing a cell culture medium RPMI-1640/DMEM, the transferred tissue culture medium has the function of enabling the tissue to maintain certain nutrition in vitro and keep the tissue activity, and the tissue is placed on ice together with a patient blood sample and conveyed to an animal center, wherein the patient blood sample is placed in an anticoagulation tube which is used independently, and the blood is also placed on the ice after being separated from the body so as to prevent the protein component in the blood from being degraded, and the blood can be better preserved when being transported on the ice;
(3) pretreatment of blood samples: centrifuging a patient blood sample, sucking serum into a plurality of EP tubes by using a pipette gun in an ultra-clean workbench after ultraviolet disinfection, placing 1ml of serum into each EP tube, placing the EP tubes in which the samples are placed on ice for transplantation, and freezing and storing the rest of serum in a refrigerator at minus 80 ℃ for passage;
(4) tumor specimen pretreatment: removing part of necrotic tissues of tumors in an ultra-clean workbench after ultraviolet disinfection, cutting the tumors into rice grains after being washed by normal saline, dividing the tumors to be inoculated into two groups, putting the two groups into a culture dish of 100mm containing normal saline, covering the culture dish and putting the culture dish on ice;
(5) mouse inoculation: selecting 10 BALB/c-nu/nu mice with the age of 4-6 weeks, wherein the BALB/c-nu/nu is a commonly used mouse type used for transplanting a tumor model, and is a nude mouse which is caused by the fact that the mice lack mature T lymphocytes due to congenital thymic dysplasia caused by mutation of BALB/c-nude on nu genes and then causes the cell immunologic function to be low; the main characteristics are hairless and athymic, thinning of the skin of the mouse along with the increase of the age of the mouse, head and neck skin folds and growth retardation. Dividing into 2 groups, each group comprises 5 animals, one group is used as experimental group, and the other group is used as control group; the experimental group was inoculated with tumors pretreated with patient serum, and the control group was inoculated with tumors treated with physiological saline. The inoculation process is carried out in an ultra-clean workbench after ultraviolet disinfection, the inoculation part is positioned at the position close to the armpit at the subcutaneous left side of the mouse, and the wound of the inoculation part needs to be disinfected before and after inoculation;
(6) allowing the mice to grow naturally, observing and detecting each index of the two groups of mice every week, and comparing the two groups of mice with each other after 8 weeks;
(7) after 8 weeks, two groups of mice are sacrificed and the tumors are stripped for passage, and the 10 new BALB/c-nu/nu mice with the age of 4-6 weeks are divided into two groups, one group is used as a passage experimental group, and the other group is used as a passage control group; inoculating the tumor pretreated by the serum of the patient according to the experimental group, and inoculating the tumor treated by the normal saline into the control group, wherein the method is the same as the steps (4) and (5);
(8) the mice are allowed to grow naturally, each index of the two groups of mice is observed and detected every week, and the tumorigenesis conditions of the two groups after 8 weeks are compared.
In the step (5), the serum pretreatment is to incubate the patient serum with the same source as the tumor specimen at constant temperature of 37 ℃ for 5 minutes, and then transplant the patient serum into the mouse. Because the serum can provide basic nutrient substances, hormones, various growth factors and the like for the tumor, promotes the growth of the tumor in vivo and the generation and development process of tumor blood vessels, and is an indispensable nutrient transmitter and a component in a tumor microenvironment, the tumor and the serum of a patient are incubated for 5 minutes at a constant temperature of 37 ℃, so that the temperature of a human body can be simulated at 37 ℃, cells in a tumor tissue are still in a stable growth state, various proteins on the tumor can be activated under the action of the serum, some nutrient substances can be supplied to the tumor after the tumor is separated in vitro to the greatest extent, and the microenvironment for the tumor to grow in the human body is greatly reduced in vitro; the effect of incubating in a constant temperature incubator for 5min is to ensure that the tumor and serum can be fully combined, simultaneously can also greatly protect the activity of the tumor cells after the separation and the activity of non-tumor cells in a tumor microenvironment, and prevent the reduction of the cell activity caused by too long in vitro time from further influencing the tumor formation rate.
In the step (4), the rice grains are preferably 0.2X0.2X0.2cm in size. Theoretically, the larger the tumor volume used, the higher the tumor formation rate; in fact, the fact that the larger tumor volume used in the PDX transplantation tumor model construction process means that a larger puncture needle is needed, and the larger puncture needle is used to cause the wound on the mouse to be larger. Therefore, in order to improve the success rate, a puncture needle with a proper size is selected, the wound of the puncture position of the mouse is reduced as much as possible while the large-volume tumor is borne as much as possible, the size of the used tumor is determined according to the aperture of the puncture needle, and the wound of the puncture position of the mouse is reduced as much as possible while the large-volume tumor is borne as much as possible by 0.2 x0.2x0.2cm;
in the step (5) of the invention, the used puncture implantation needle is a 25-gauge trocar, which can load the tumor to the maximum extent and reduce the trauma to the tumor implantation part of the mouse puncture as much as possible.
Example (b):
the materials and instruments used in the following examples and experiments were as follows:
the isolated esophageal cancer tissue specimens and blood specimens are derived from 5 groups of SPF-grade BALB/c-nu/nu nude mice, which are 20 in total and weigh 25 +/-0.25 g (provided by Jinan Pengyue laboratory animal Breeding Co., Ltd.), a centrifuge (Eppendorf-5810R), an SW-CJ-2F ultra-clean bench (Suzhou purification equipment Co., Ltd.), an RPMI 1640-DMEM medium (Solarbio Co., Ltd.), an EP tube (Axygen), an anticoagulated blood collection tube, a pipette gun, a sterile gun head, physiological saline, vernier calipers, ophthalmic scissors, a scalpel, a No. 25 trocar, a sterile 100mm culture dish, iodophor, 75% alcohol and a digital camera in the first subsidiary hospital of Henan science and technology university.
(II) specimen Collection
Esophageal cancer patients were enrolled as standard: the new patients with esophageal cancer have no infectious disease, definite pathological diagnosis, proper tumor size, complete clinical information and signed informed consent; establishing a cohort patient database comprising: name, sex, age, contact information, preoperative data (including blood routine, biochemistry, gastroscopy, imaging examination, etc.), postoperative data (blood routine, biochemistry, pathology diagnosis report, TNM staging, etc.), treatment regimen, treatment effect, survival, etc.
Communicating with clinical doctors as much as possible in the process of collecting the specimen to obtain more and larger tumor tissue specimens, and simultaneously avoiding collecting necrotic or non-tumor tissues as much as possible so as to ensure the success rate as much as possible; in addition, in order to avoid the influence of narcotics in the operation, a blood sample of a patient should be collected before the operation.
(III) specimen transplantation
1. Specimen transport
The specimens were isolated, washed appropriately with saline, and then transferred to 50ml centrifuge tubes containing cell culture medium RPMI-1640/DMEM, which completely submerged the tumor tissue and was transported to the animal center on ice together with the patient blood samples.
2. Pretreatment of blood samples
Centrifuging a patient blood specimen for 5min 1000rpm/min, sucking serum into a plurality of EP tubes (1 ml/tube) in an ultra-clean workbench after ultraviolet disinfection by using a pipette gun, placing the tubes on ice for transplantation, and freezing and storing the rest in a refrigerator with the temperature of minus 80 ℃ for passage;
3. tumor specimen pretreatment
Removing part of necrotic tissues of tumors in an ultra-clean workbench after ultraviolet disinfection, cutting the tumors into rice grains of 0.2x0.2x0.2cm after being washed by normal saline, dividing the tumors to be inoculated into two groups, putting the two groups into a culture dish of 100mm containing normal saline, covering the culture dish with the culture dish, and putting the culture dish on ice. The tumor of the control group is directly inoculated under the skin of the mouse, and the tumor of the experimental group is firstly incubated with the serum of the patient at constant temperature of 37 ℃ for 5min and then directly inoculated under the skin of the mouse.
4. Inoculation of mice
10 BALB/c-nu/nu mice of 4-6 weeks old were selected and divided into 2 groups, i.e., experimental and control groups, of 5 mice each. The experimental group was inoculated with tumors pretreated with patient serum, and the control group was inoculated with tumors treated with physiological saline. The inoculation process is carried out in an ultra-clean workbench after ultraviolet disinfection by using a No. 25 trocar, the inoculation part is positioned at the position close to the armpit at the left side of the subcutaneous skin of the mouse, and the wound at the part needs to be disinfected before and after the inoculation. The mice are allowed to grow naturally, each index of the two groups of mice is observed and detected every week, and the tumorigenesis conditions of the two groups are compared after 8 weeks.
5. Mouse passage
After 8 weeks, the two groups of mice are sacrificed and the tumors are stripped for passage, and the new BALB/c-nu/nu mice with the age of 4-6 weeks are divided into two groups, namely a passage experimental group and a passage control group, the tumors pretreated by the serum of the patient are inoculated according to the experimental group, and the tumors treated by the normal saline are inoculated in the control group in the same way as 3 and 4. The mice are allowed to grow naturally, the indexes of the two groups of mice are observed and detected every week, and the tumorigenesis conditions of the two groups after 8 weeks are compared.
(IV) results of the experiment
10 mice in the primary, i.e. step 4 mice inoculation, andwhen the tumor formation of two groups of esophagus cancer PDX mice at 8 weeks of new mice in the passage of the mice in the step 5 is compared, the tumor formation volume (783.1 mm) of the experimental group (the esophagus tumor pretreated by serum is transplanted under the skin of the immunodeficiency mice) is shown in figure 13) Significantly higher tumor formation volume (442.6 mm) than control group (saline pretreated esophageal tumor implanted subcutaneously in mice)3) (ii) a Similarly, in the passage, the tumor formation volume of the experimental group (783.1 mm) was passaged3) Also significantly higher than the tumor formation volume of passage control (783.1 mm)3）；
② as shown in figures 2 and 3, the weight (23.3 g) and the tumor weight (843.3 mg) of the primary experimental group mice are obviously higher than the weight (22.8 g) and the tumor weight (532.2 mg) of the control group mice; during passage, the weight (23.9 g) and the tumor weight (1387.4 mg) of the mice in the passage experimental group are also obviously higher than those (23.2 g) and the tumor weight (987.6 mg) of the mice in the passage control group;
③ as shown in fig. 4, the tumor formation rate of the experimental group (82.1%) is significantly higher than that of the control group (47.9%); in the passage, the passage tumor formation rate (92.4%) of the passage experimental group is also obviously higher than that of the passage control group (72.31%);
comparing the tumor forms of the primary and passage experimental groups and the control group to find that the tumor formed by the mice in the experimental group has smooth surface, less parts of toughness and necrosis and can show a great amount of blood vessel enrichment; while the control mice developed tumors with rough surface, relatively soft texture, more necrotic parts, and relatively low vascular density.
The primary and passage 8 weeks two groups of esophageal cancer PDX mice had the following neoplasia:
the results show that compared with the conventional method that esophageal tumor modeling is carried out by saline water treatment, the PDX model established by subcutaneous transplantation of the esophageal tumor after serum pretreatment in an immunodeficiency mouse is obviously optimized and improved in the aspects of tumor formation rate, tumor formation volume, tumor formation speed and tumor formation quality; meanwhile, in the passage process, the PDX model established by the method is optimized and improved to different degrees in the aspects of passage tumor formation rate, tumor formation volume, tumor formation speed and tumor formation quality.
By implementing the method for establishing the esophageal cancer PDX mouse model, before the tumor is transplanted in a mouse body, the serum of a patient is used for pretreating the tumor, the occurrence, development and metastasis of the tumor have close relation with the environment of tumor cells in a human body, and the method not only comprises the structure, function and metabolism of the tissue where the tumor is located, but also comprises the self-metabolism regulation and control in the tumor cells, which are collectively called as a tumor microenvironment. The growth of tumor cells is influenced by the tumor cells through the self metabolism regulation, and then the change of functions of metabolism, secretion, immunity and the like of the whole body or local tissues is influenced, and the generation, development and metastasis processes of tumors are influenced. The blood is used as an important transport transmitter in the human body and can transport nutrient substances and metabolic waste so as to ensure the normal metabolism of the human body; blood is an important component of the tumor microenvironment, and is involved in the absorption and metabolism of nutrients. The mechanism provides an important idea for the research of the people, and the serum is used as an important component of blood, provides basic nutrient substances, hormones, various growth factors and the like for the tumor in a tumor microenvironment, and plays an important role in the angiogenesis and development process of the tumor. Therefore, a tumor sample and a blood sample of an esophageal cancer patient are clinically collected, serum separated from the blood sample of the patient after centrifugation is used for pretreatment of a tumor tissue to be transplanted, a tumor microenvironment in the esophageal cancer patient is indirectly brought into an immunodeficient mouse, and the serum of the esophageal cancer patient homologous with the esophageal tumor sample is used for pretreatment of the tumor sample to be inoculated for esophageal cancer PDX animal model modeling experiment, so that the tumor microenvironment is greatly reduced and brought into the immunodeficient mouse, and the activity of tumor cells is protected. Greatly improves the success rate of the PDX tumor animal model of the esophageal cancer patient; meanwhile, the residual serum can be reused in the subsequent passage process, and the tumor tissue to be passaged is pretreated, so that the passage stability is further improved. The method for establishing the esophageal cancer PDX mouse model lays a foundation for screening and further research of tumor drugs, and provides improvement of methodology on establishing tumor animal model experiments.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A method for establishing an esophageal cancer PDX mouse model is characterized by comprising the following steps: the method comprises the following specific steps:
firstly, specimen collection: by clinical esophagus cancer patient case screening, collecting an esophagus tumor sample and a patient blood sample in an operating room;
secondly, specimen transportation: after the sample is separated from the body, the sample is properly washed by saline, then is transferred into a 50ml centrifuge tube containing a cell culture medium RPMI-1640/DMEM, and is placed on ice together with a patient blood sample to be conveyed to an animal center;
thirdly, preprocessing a blood sample: centrifuging a patient blood sample, sucking serum into a plurality of EP tubes by using a pipette gun in an ultra-clean workbench after ultraviolet disinfection, placing 1ml of serum into each EP tube, placing the EP tubes in which the samples are placed on ice for transplantation, and freezing and storing the rest of serum in a refrigerator at minus 80 ℃ for passage;
fourthly, tumor specimen pretreatment: removing part of necrotic tissues of tumors in an ultra-clean workbench after ultraviolet disinfection, cutting the tumors into rice grains after being washed by normal saline, dividing the tumors to be inoculated into two groups, putting the two groups into a culture dish of 100mm containing normal saline, covering the culture dish and putting the culture dish on ice;
fifth, mouse inoculation: selecting BALB/c-nu/nu mice with even numbers of 4-6 weeks old, equally dividing into 2 groups, wherein one group is used as an experimental group, and the other group is used as a control group; the experimental group is inoculated with the tumor pretreated by the serum of a patient, the control group is inoculated with the tumor treated by the normal saline, the inoculation processes are carried out in an ultra-clean workbench after ultraviolet disinfection, the inoculation part is positioned at the position close to the armpit at the subcutaneous left side of the mouse, and the sterilization treatment is carried out on a wound at a part before and after the inoculation;
sixthly, enabling the mice to naturally grow, observing and detecting each index of the two groups of mice every week, and comparing the tumorigenesis conditions of the two groups after 8 weeks;
seventhly, after 8 weeks, the tumors of the two groups of mice are stripped and passaged, and the new BALB/c-nu/nu mice with the even number of 4-6 weeks old are divided into two groups, one group is used as a passage experimental group, and the other group is used as a passage control group; inoculating the tumor pretreated by the serum of the patient according to the passage experimental group, and inoculating the tumor treated by the normal saline into the passage control group in a grouping way, wherein the method is the same as the fourth step and the fifth step;
and eighthly, allowing the mice to naturally grow, observing and detecting each index of the two groups of mice every week, and comparing the tumorigenesis conditions of the two groups after 8 weeks.
2. The method of establishing a PDX mouse model for esophageal cancer according to claim 1, wherein: in the first step, a blood sample of the patient into which the anesthetic is not injected is collected.
3. The method of establishing a PDX mouse model for esophageal cancer according to claim 1, wherein: in the fourth step, the preferred size of the rice grains is 0.2 x0.2x0.2cm.
4. The method of establishing a PDX mouse model for esophageal cancer according to claim 1, wherein: in the fifth step, the serum pretreatment is to incubate the tumor specimen to be inoculated with the serum of the patient from the same source as the tumor specimen at constant temperature of 37 ℃ for 5 minutes and then transplant the tumor specimen into a mouse body.
5. The method of establishing a PDX mouse model for esophageal cancer according to claim 1, wherein: in the fifth step, the used puncture implantation needle is a 25-gauge trocar.
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