CN115024280A - Method and model for establishing esophageal cancer in-situ planting tumor formation model - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and discloses a method for establishing an esophageal cancer in-situ implantation tumorigenesis model and a model, wherein the method comprises the following steps: transfecting the mouse esophageal squamous carcinoma cell 1271 by using a lentivirus luciferase (luciferase) expression plasmid to obtain a stable cell; carrying out mouse esophagus in-situ tumor formation experiments, and carrying out neck esophagus tumor formation and abdomen esophagus tumor formation; after the in-situ planting of the tumor cells is completed, the growth state of the esophageal cancer is regularly monitored by a living body imaging system. The invention can directly utilize mice with normal immune function to establish an esophageal cancer in-situ planting tumor model, reduces the operation difficulty, simulates the tumor microenvironment for origin and growth of esophageal cancer more vividly, and is beneficial to further developing the research on the growth and treatment of esophageal cancer subsequently.

Description

Establishment method and model of esophageal cancer in-situ implantation tumor formation model

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a method for establishing an esophageal cancer in-situ implantation tumor formation model and a model.

Background

Esophageal cancer is the eighth most common malignancy and the sixth leading cause of cancer death worldwide, with about 50 million newly diagnosed as esophageal cancer each year, estimated to result in 406,000 deaths each year. Esophageal squamous cell carcinoma and esophageal adenocarcinoma are the two major types of esophageal cancer. Esophageal squamous carcinoma is a major subtype, accounting for approximately 90% of worldwide esophageal cancer cases, and remains the most common type of esophageal cancer in asia, africa, south america, and african americans.

The survival rate of the early esophageal cancer after the conventional treatment for 5 years is more than 90 percent. However, survival rates for patients in the late stages are significantly reduced. Since most patients are hospitalized at an advanced stage, the overall 5-year survival rate for esophageal cancer is only around 20%. The etiology of esophageal cancer is still poorly understood; at present, the treatment means is mainly surgical treatment, and immunotherapy has good treatment prospect.

The tumor microenvironment and the interaction with the tumor play an important role in the occurrence and development process of the tumor, and due to the unique tumor microenvironments of different organ parts, establishing in-situ models of tumors of different organs becomes the best choice for tumor research. The principle of establishing an in-situ tumor model of the esophageal cancer aims to be closer to a microenvironment of tumor origin and provide the best fidelity for researching the esophageal cancer.

In esophageal squamous carcinoma, both the induction of esophageal squamous carcinoma by oral carcinogens has been studied, and of course a limited number of tumor models have been established, such as: subcutaneous graft tumor model: suturing a small subcutaneous tumor to a mechanically damaged esophagus by surgical operation [1 ]; inoculating cancer cells by injection into the esophagus via the esophageal cannula without any visual assistance [2 ]; seeding the esophageal wall with tumor cells through an incision in the stomach near the gastroesophageal junction [3 ]; tumor cells were also injected into the esophageal muscularis under visible conditions by dissociating the abdominal esophagus [4 ]. The current model has great limitation, most experiments adopt human esophageal squamous carcinoma cells to be planted in immunodeficient mice, or some experiments have higher operation requirements on operators.

Disclosure of Invention

The embodiment of the invention aims to provide a method and a model for establishing an esophageal cancer in-situ planting and tumorigenic model, which can directly utilize mice with normal immune functions to realize the establishment of the in-situ planting and tumorigenic model, reduce the operation difficulty, simulate the origin and growth tumor microenvironment of esophageal cancer more realistically, and facilitate the further research on the growth and treatment of esophageal cancer in the follow-up process.

The embodiment of the invention is realized as follows:

the method for establishing the esophageal cancer in-situ implantation tumor formation model comprises the following steps:

step 101, acquisition of esophageal squamous carcinoma cells

Adding carcinogenic 0.001% 4 nitroquinoline-1-oxide (4NQO) into mouse drinking water, inducing for 12 weeks to generate esophageal squamous carcinoma, obtaining esophageal squamous carcinoma tissues for primary culture, and naming primary culture cells as 1271 cells; the cell lines were cultured in a high-sugar medium (GIBCO; Cat #11995500BT) containing 1% penicillin-streptomycin (GIBCO; Cat #15140122) and 10% fetal bovine serum (Thermo Fisher Scientific; Cat #10270), and subjected to monolayer culture in an incubator at 37 ℃ under a humidified condition of 5% CO 2;

transfecting 1271 cells by using a lentivirus luciferase (luciferase) expression plasmid to obtain stably transfected cells, and naming the stably transfected cells as 1271-luc;

firstly, searching for the minimum lethal concentration of puromycin in the empty white cells 1271, setting concentration gradients of 0.5ug/ml, 1ug/ml, 1.5ug/ml, 2ug/ml and 5ug/ml, observing for 3 days under a white light microscope, and taking antibiotics with the concentration as the optimal screening concentration when the empty white cells just can completely die to obtain the minimum lethal concentration of 1.5 ug/ml;

the infection process of the lentivirus particle cells is as follows: taking cancer cells in logarithmic growth phase, digesting and counting the cancer cells by pancreatin, measuring the cell density by using a cell counting plate, inoculating 5 multiplied by 10^5 cells into each hole of a six-hole culture plate for plating, and adding a cell culture solution to 2 ml; observing the growth state of the cells on the next day, and adding 15ul of virus solution with the titer of 1 multiplied by 10^8TU/ml and 1ul of Polybrene infection additive with the titer of 4ug/ml into each well when the cells reach 30-50% fusion degree during infection; sucking out culture solution containing virus particles 12 hours after virus infection, adding the whole culture medium into the culture plate again, and continuing culture; observing whether the cells have abnormality on the fourth day; day five observations to assess lentiviral particle transfection efficiency: cleaning the outer wall of the culture plate by using 70% alcohol, observing fluorescence in an inverted fluorescence microscope, photographing and determining the infection efficiency of the lentivirus particles on cells;

adding puromycin into a transfected cell culture dish, wherein the final concentration is 1.5 ug/ml; changing the solution every 3 days, reducing the puromycin concentration to 0.75ug/ml after one week, continuously culturing, and observing and evaluating by using a microscope until stable plants are generated; obtaining 1271-luc cells;

step 102, mouse esophagus in-situ tumor formation experiment

Taking 30C 57BL/6 mice of 6 weeks old, and randomly dividing the mice into two groups, wherein one group is used for cervical esophageal neoplasia, and the other group is used for abdominal esophageal neoplasia; performing surgical field unhairing and disinfection on the neck or the abdomen of a mouse, performing intraperitoneal injection anesthesia by using 10 percent chloral hydrate according to the weight of 0.1ml/10g of the mouse, performing isoflurane gas anesthesia for 2-2.5L/min, injecting 1 x 10^6/20ul 1271-luc cells and Matrigel into each mouse in equal proportion to form mixed suspension, inoculating 40ul of cancer cell suspension into an esophageal wall peripheral muscle layer, injecting tumor cells into the neck or the abdomen in situ esophagus to observe edema as a sign of successful inoculation, and performing 5-0 non-absorbable suture full-layer suture on incision;

wherein, the muscle layer at the outer edge of the esophageal wall is the outer muscle layer of the neck esophagus or the outer muscle layer of the abdominal esophagus at the position close to the diaphragm, and the injection part of the abdominal esophagus is far away from the cardia part of the gastroesophageal junction as far as possible;

step 103, monitoring of tumor growth

After the in-situ planting of the tumor cells is completed, the body weight and the growth state of the mouse are monitored regularly; the growth state detection uses a Xenogenin living body imaging system IVIS-100 (Perkin Elmer, Mass.) to carry out living body imaging, 150mg/kg of luciferase substrate is injected into the abdominal cavity of a mouse to monitor the in-situ tumor growth kinetics of an ESCC cell line marked by luciferase injected into the mouse, after the injection into the body is carried out for 10-15min, namely, when a light signal reaches the strongest stable platform stage, imaging analysis is carried out, the tumor growth state is detected through signal intensity, and the imaging data of the growth transfer condition of esophageal cancer is obtained through PET-MR examination 4 weeks after planting; mice were sacrificed and gross and histological data for esophageal cancer were obtained.

The model obtained by the method is applied.

According to the embodiment of the invention, an esophageal cancer in-situ tumor model is established in a mouse with normal immune function, and the growth environment of mouse esophageal cancer can be simulated more vividly by using primary esophageal squamous carcinoma cells, and 1271 cells are transfected by using lentiviral plasmids with Green Fluorescent Protein (GFP) and luciferase (luciferase), so that stably transfected cells are obtained, and the development of tumor can be observed; meanwhile, the tumor formation of cancer cells at different parts is compared, and a stable esophageal cancer in-situ tumor model is established and can be used for researching the in-vivo treatment of esophageal cancer.

Drawings

FIG. 1 is a process diagram of an experimental method of the present invention;

FIG. 2 is a schematic diagram of Transwell migration;

FIG. 3 is a schematic diagram of a Transwell invasion;

FIG. 4 is a graph of the proliferation potency of cells tested by CCK8 assay;

FIG. 5 is a schematic representation of the tumor size of 1271 cells;

FIG. 6 is a graph of nodule size;

FIG. 7 is a schematic of a lentiviral particle transfection 1271 cell;

FIG. 8 is a graph showing the effect of FIG. 7 after a 1.5ug/ml Puromycin screen;

FIG. 9 is a dual luciferase assay of transfected cells;

FIG. 10 is a comparison of two sets of procedure time relationships;

FIG. 11 is a statistical graph of two sets of surgical times;

FIG. 12 is a graph of CUSUM 1;

FIG. 13 is a graph of CUSUM 2;

FIG. 14 is a graph of bioluminescent imaging of abdominal groups taken over different weeks;

FIG. 15 is a graph of bioluminescent imaging of different week-count neck groups;

FIG. 16 is a schematic diagram of PET-MR results;

FIG. 17 is a diagram of esophageal in situ tumor formation;

FIG. 18 is a table comparing the tumor rates and distant metastasis rates in the abdominal and neck groups.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

The following detailed description of specific implementations of the invention is provided in conjunction with specific embodiments:

cell lines and cell cultures

Esophageal squamous carcinoma cells are obtained, carcinogenic 0.001% 4 nitroquinoline-1-oxide (4NQO) is added into mouse drinking water to induce esophageal squamous carcinoma, wherein one group of cells are 1271 cells (murine esophageal squamous carcinoma cells 1271 obtained by esophageal carcinoma research institute of Guangdong province), namely the cells used in the experiment. The cell lines were cultured in a high-sugar medium (GIBCO; Cat #11995500BT) containing 1% penicillin-streptomycin (GIBCO; Cat #15140122) and 10% fetal bovine serum (Thermo Fisher Scientific; Cat #10270), and monolayer-cultured in an incubator at 37 ℃ under humidified conditions of 5% CO 2.

For details of the cell line 1271, see the content of CN 113416705A.

As shown in FIG. 1, it is a process diagram of the experimental method of the present invention:

CCK-8 experiment:

inoculating 1000 cells in 96-well plate according to the concentration of 1 x 10^4/ml, and 5 multiple wells;

adding 10ul of CCK8 into each hole every day from the first day of inoculation, and detecting by an enzyme-labeling instrument after 1 h;

the experiment was repeated three times;

migration and invasion:

the Transwell cell migration experiment is completed by adopting a small chamber with a 8um microporous polycarbonate membrane;

the Transwell cell Invasion experiment is carried out by Martrigel Invasion Chambers;

subcutaneous neoplasia:

preparing a cell suspension with the concentration of 1 x 10^7/100ul by using a PBS solution;

selecting 10 male mice C57BL/6 with age of 6 weeks;

injecting 100ul of cell suspension into the underarm subcutaneous part of each mouse;

mice were sacrificed 4 weeks later, tumor specimens were excised and immunohistochemical examination was performed;

lentiviral particle cell infection:

the used lentivirus particles are GeneCopoeia lentivirus vectors, and a lentivirus expression system constructed by an HIV framework has the capacity of dual expression of Green Fluorescent Protein (GFP) and luciferase (luciferase);

d1 six-well culture plate is inoculated with 5 multiplied by 10^5 cells per well for plating;

when the D2 cells reach 30-50% of fusion degree, 15ul of virus solution with the titer of 1 x 10^8TU/ml and 1ul of Polybrene infection additive with the titer of 4ug/ml are added into each well;

12H, sucking out culture solution containing virus particles 12 hours after virus infection, and adding the whole culture medium to the culture plate again;

d4-5, observing whether the cells have abnormality or not, and evaluating the transfection efficiency of the lentivirus particles;

specifically, the lentivirus particles used in the experiment are GeneCopoeia lentivirus vectors, and a lentivirus expression system constructed by an HIV framework has a toxic gene which is deleted and replaced by an exogenous target gene, so that after a host cell is infected, other cells cannot be infected continuously, and the host cell cannot be used for generating new lentivirus particles.

Specifically, cancer cells in logarithmic growth phase are taken and digested and counted by pancreatin, the cell density is measured by a cell counting plate, 5 multiplied by 10^5 cells are inoculated in each hole of a six-hole culture plate, and a cell culture solution is added to 2 ml; observing the growth state of the cells on the next day, wherein the cells reach 30-50% fusion degree when infection is carried out, and 15ul of virus solution with the titer of 1 multiplied by 10^8TU/ml and 1ul of Polybrene infection additive with the titer of 4ug/ml are added into each hole; sucking out culture solution containing virus particles 12 hours after virus infection, adding the whole culture medium into the culture plate again, and continuing culture; continuously culturing the cells on the fourth day, and observing whether the cells are abnormal; day five observations to assess lentiviral particle transfection efficiency: the outer wall of the plate was cleaned with 70% alcohol, fluorescence was observed on an inverted fluorescence fiberscope, and photographs were taken and the infection efficiency of lentiviral particles on cells was estimated.

Puromycin drug concentration screening

After transfection of cells with lentiviral particles, cell selection with puromycin was required and to determine the optimal drug selection concentration for the cells used in the experiment, the minimal lethal concentration of the leukocytes was investigated and concentration gradients of 0.5ug/ml, 1ug/ml, 1.5ug/ml, 2ug/ml, 5ug/ml were set. When the white blood cells just die completely, the antibiotic at this concentration was used as the optimal screening concentration to obtain a minimal lethal concentration of 1.5ug/ml, observed under a white light microscope for 2-3 days.

Mouse esophagus in-situ tumor formation experiment

In the experiment, 30C 57BL/6 mice (mice with normal immunity) with the age of 6 weeks are utilized and are randomly divided into two groups, one group is used for neck esophageal neoplasia, and the other group is used for abdomen esophageal neoplasia; performing surgical field unhairing and disinfection on the neck or the abdomen, and performing intraperitoneal injection anesthesia by using 10% chloral hydrate according to the weight of 0.1ml/10g of the mouse; using cells that have been successfully transfected, 20ul of a 1X 10^6 cell suspension was mixed with Matrigel basement membrane matrix in a ratio of 1: 1, and finally, 40ul of the cancer cell mixture was inoculated to the muscular layer on the outer edge of the esophageal wall, and 5-0 non-absorbable suture line was performed for incision full-layer suture with edema observed as an indication of successful inoculation.

Monitoring of tumor growth

After completing the in situ planting of the tumor cells, monitoring the weight and growth state of the mice every day in the first week after the operation; in vivo imaging was performed weekly using Xenogenin in vivo imaging system IVIS-100 (Perkin Elmer, ma, usa) by intraperitoneal injection of 150mg/kg luciferase substrate into mice to monitor the kinetics of in situ tumor growth of luciferase-tagged ESCC cell lines injected into the mice and to observe metastasis. Mice were sacrificed 4 weeks after the study was completed. Imaging of esophageal cancer was obtained on a pre-PET-CT examination of sacrificed mice.

Specifically, a Xenogenin in vivo imaging system IVIS-100 is used for in vivo imaging every week, 150mg/kg of fluorescein solution (prepared by DPBS and with the concentration of 15mg/ml) is injected into the abdominal cavity of a mouse, and imaging analysis is performed after the mouse is injected into the body for 10-15min (namely, when an optical signal reaches the strongest stable plateau period).

Fasting is carried out for 6 hours, 18F-FDG (fluorodeoxyglucose) is injected through the tail vein, the injection activity is not lower than 100uci, the mice are anesthetized by injecting 10% chloral hydrate 0.1ml/30g into the abdominal cavity after 1 hour, and PET-MR examination is carried out in a horizontal position.

Learning curve analysis for cervical and abdominal surgery, and data statistics and statistical methods

30 mice are divided into a neck group and an abdomen group, 16 cases of abdominal esophagus in situ tumors and 14 cases of cervical esophagus in situ tumors are performed in the 30 mice, and the operation time, the operation mode, the bleeding condition in the operation and the postoperative outcome of each mouse are recorded. The learning curve is drawn by using a cumulative sum analysis (CUSUM), the operation time is selected as an evaluation index, the target value is set as an average value of the operation time, the operation sequence in time sequence is used as an abscissa, and the difference value between each operation time and the target value is summed to be used as an ordinate. Taking α as 0.05, i.e., P <0.05 as a difference, is statistically significant.

In particular, the method comprises the following steps of,

accumulationThe summation analysis method (CUSUM) can convert the change of the original data into the cumulative summation of the difference between each value and the average value, thereby well reflecting the mean deviation of each data, obtaining the continuous change trend of the data and more intuitively representing the learning curve; taking the operation sequence in time sequence as an abscissa and taking the CUSUM value of each operation as an ordinate; the learning curve was fitted with the SPSS 25.0 software. When P is less than 0.05, the learning curve is successfully fitted; the goodness of fit is judged by a decision coefficient R2, the closer R2 is to 1, the higher the goodness of fit is, the better the fitting model is; when the learning curve graph begins to decline, the point is the starting point of the case that the operation time is lower than the average value, and the corresponding abscissa is the number of the operation cases required for passing the learning period.

The data were statistically analyzed using SPSS25 statistical software (SPSS, Inc, Chicago, IL), and the different statistical methods used in the different experiments were presented and interpreted in each experimental result. Quantitative data are expressed by mean ± standard deviation or median (interquartile range); checking whether quantitative data are in accordance with normal distribution and homogeneity of variance, if yes, adopting t test and analysis of variance (ANOVA) to carry out 2 or 3 groups of variable comparison, and if not, adopting rank-sum test (Mann-Whitney test) to carry out comparison; qualitative data were compared by χ 2 or Fisher's exact probability method. P-values below 0.05 were considered statistically significant.

Acquisition and processing of esophageal specimens

After 4 weeks, 20 mice were sacrificed, 10 in each of the abdominal and neck groups, to obtain fresh tissue samples, and the remaining 10 mice were kept on stock. The mouse was resected with the esophagus and stomach, incised along the longitudinal central axis, washed with physiological saline, and examined for tumor formation. The obtained esophagus specimen should be put into 10% formaldehyde solution (neutral buffered formalin) fixing solution in time, and then hematoxylin-eosin (HE) staining and immunohistochemical examination such as CK5/6, P40, P63, ki67 and the like are perfected.

Results of the experiment

As shown in fig. 2, which is a schematic diagram of Transwell migration, fig. 3 is a schematic diagram of Transwell invasion, and fig. 4 is a graph of cell proliferation potency of CCK8 test, it was found that 1271 cells were able to grow exponentially and had good migration and invasion potency.

As shown in fig. 5, which is a schematic view of the size of the tumor of 1271 cells, and fig. 6 is a graph of the size of the tumor, and the horizontal axis represents the number of weeks, it was found that 1271 had the ability to form tumors and the subcutaneous tumor formation rate was 90%.

As shown in FIG. 7, the schematic diagram of 1271 cells transfected with lentiviral particles, FIG. 8 shows the effect of 1.5ug/ml Puromycin screening in FIG. 7, and FIG. 9 shows the dual luciferase assay of transfected cells, indicating that GFP-1271-luc cells obtained after stabilization have luciferase-expressing ability.

As shown in fig. 10, which is a comparison graph of two groups of operation time relationships, and fig. 11 is a statistical graph of two groups of operation time, it can be seen that 30 mice successfully completed the operation, no major bleeding occurred during the operation, no perioperative death, the operation time for the abdomen group was (370.56 ± 90.92) s, the operation time for the neck group was (317.07 ± 123.22) s, the operation time showed an overall decrease trend with the increase of the number of operation cases, and the difference between the two groups of operation time comparisons was not statistically significant.

As shown in fig. 12, it is a coordinate diagram of CUSUM1, where CUSUM1 ═ 299.492+30.556X-3.244X2+0.019X3, goodness-of-fit coefficient R2 is 0.912, fig. 13 is a coordinate diagram of CUSUM2, CUSUM2 ═ 149.429+414.865X-54.463X2+1.824X3, and goodness-of-fit coefficient R2 is 0.985. From this, it can be seen that 5 abdominal or cervical group surgeries cross the learning curve, and the minimum number of cases required for the surgeries is well known.

Fig. 14 shows a different-week abdomen group bioluminescence imaging chart, and fig. 15 shows a different-week neck group bioluminescence imaging chart. Therefore, the tumor growth is successfully monitored by using bioluminescence imaging, a relatively obvious fluorescence signal can be found at 1 week after operation, and the fluorescence signal can be observed to be enhanced and diffused along with the growth or metastasis of the tumor, and the fluorescence signal of the abdominal group is more obvious than the diffusion of the cervical group.

As shown in FIG. 16, which is a schematic diagram of the results of PET-MR, it can be seen that the esophageal tumor invades the affected stomach and the peritoneal metastasis occurs, and the multiple metastatic lesions in the lung are found.

As shown in FIG. 17, it is a diagram showing the formation of esophageal carcinoma in situ, and it can be seen that 4 weeks after surgery, all mice were sacrificed by cervical dislocation, and the formation of esophageal carcinoma in situ was observed, and significant invasion and metastasis of peripheral tissues were observed in abdominal esophageal carcinoma.

As shown in fig. 18, the abdominal tumor formation rate is as high as 93.75% (15/16), the neck tumor formation rate is as high as 85.71% (12/14), and there is no significant difference between the two groups of tumor formation rates (p > 0.05); the incidence rate of distant metastasis of abdominal esophageal tumors is as high as 68.75% (10/16), the metastasis parts are mostly generated in lymph nodes (7/16), stomach (8/16), liver (5/16), lung (1/16), peritoneum (4/16) and the like, the incidence rate of metastasis of cervical esophageal tumors is low and is 21.43% (2/14), the metastasis is limited to the cervical lymph nodes, and by comparison, abdominal esophageal cancers are easier to find distant metastasis than cervical esophageal cancers and have statistical significance (p is less than 0.05).

It should be noted that the Matrigel gel has the ability of rapid gel formation at 22-35 ℃, and is polymerized to form a three-dimensional matrix with biological activity, and the cancer cell suspension and the liquid Matrigel are mixed and injected into the esophageal wall of a mouse, so that the leakage and diffusion of cell sap at the injection part can be effectively avoided, and the formation of solid tumors is promoted. The external muscle layer of neck esophagus and abdominal esophagus near diaphragm is the common tumor occurrence part of esophageal squamous carcinoma specimens in clinic, and chest esophagus cannot be successfully completed due to technical limitation. The abdominal esophageal injection site is as far away as possible from the cardia of the gastroesophageal junction because the gastroesophageal junction is the site where most esophageal adenocarcinoma occurs.

It should be noted that this experiment utilizes bioluminescence, and does not require excitation light. It is generally considered that the luminescence intensity is attenuated by 10 times per centimeter of depth, tissues or organs rich in blood, such as liver and lung, are attenuated more, and the captured fluorescence photon signal is weaker. This may affect the imaging technique to obtain a metastatic condition of the lung, and additional imaging examinations are needed to help determine the metastatic condition, such as PET-MR. In the experiment, a C57BL/6 mouse is used, and the black hair of the mouse has a certain influence on the acquisition of the fluorescence signal, so that the depilation treatment of the predicted tumorous part of the mouse can acquire a better fluorescence signal every week.

In conclusion, 1, the completion of the cervical or abdominal esophageal squamous cell carcinoma in-situ model on the mice with normal immune functions is feasible, and the murine esophageal squamous cell carcinoma cells 1271 are proved to have rapid growth, migration and invasion capabilities, so that the subcutaneous tumor formation rate of the C57BL/6 mice is up to 90%; 2. the 1271 cells after stable transfer have the ability of stably expressing luciferase, the growth of esophageal tumors in vivo can be monitored by using bioluminescence imaging, and lung transfer can be found by using PET-MR examination. 3. The weight of the mice in the neck group and the abdomen group is obviously reduced in the fourth week, and the reduction of the neck group is more obvious; the total tumor rate is 90%, the tumor rate between two groups has no obvious difference (p is more than 0.05), and the abdominal group is easier to have distant metastasis compared with the cervical group and has statistical significance (p is less than 0.05). 4. The operation time of the mice in the cervical and abdominal groups shows an overall descending trend along with the increase of the number of operation cases, the average time comparison difference has no statistical significance, and the difficulty degree of the modeling operation of the cervical or abdominal esophagus is similar; for the abdominal and cervical tumor formation surgeries, 5 cases are learning curve crossing, the minimum number of cases required by the cervical or abdominal surgery is mastered, and the operation difficulty is reduced.

The establishment method of the esophageal cancer in-situ implantation tumor formation model provided by the invention has the advantages that the position environment of the esophagus is more vivid, the operation is simpler, the obtained research data is more accurate, and the establishment method is favorable for making breakthrough progress in immune research and treatment research of esophageal cancer. The establishment of the esophageal cancer in-situ planting tumor formation model can bridge basic research and clinical research, supplement the use of an in-vitro model system, and utilize mice with normal immune function, so that compared with the mice with unique immune deficiency in the prior art, the model has the advantages of more superiority, more vivid tumor formation environment and more benefit for exploring unknown fields of tumor formation randomness.

The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. The method for establishing the esophageal cancer in-situ implantation tumor formation model is characterized by comprising the following steps:

step 101, esophageal squamous carcinoma cell harvesting

Adding carcinogenic 0.001% 4 nitroquinoline-1-oxide (4NQO) into mouse drinking water, inducing for 12 weeks to generate esophageal squamous carcinoma, obtaining esophageal squamous carcinoma tissues for primary culture, and naming primary culture cells as 1271 cells; the cell lines were cultured in a high-sugar medium (GIBCO; Cat #11995500BT) containing 1% penicillin-streptomycin (GIBCO; Cat #15140122) and 10% fetal bovine serum (Thermo Fisher Scientific; Cat #10270), and monolayer-cultured in an incubator at 37 ℃ under humidified conditions of 5% CO 2;

transfecting 1271 cells by using a lentivirus luciferase (luciferase) expression plasmid to obtain stably transfected cells, and naming the stably transfected cells as 1271-luc;

firstly, searching for the minimum lethal concentration of puromycin in the empty white cells 1271, setting concentration gradients of 0.5ug/ml, 1ug/ml, 1.5ug/ml, 2ug/ml and 5ug/ml, observing for 3 days under a white light microscope, and taking antibiotics with the concentration as the optimal screening concentration when the empty white cells just can completely die to obtain the minimum lethal concentration of 1.5 ug/ml;

the infection process of the lentivirus particle cells is as follows: taking cancer cells in logarithmic growth phase, digesting and counting the cancer cells by pancreatin, measuring the cell density by using a cell counting plate, inoculating 5 multiplied by 10^5 cells into each hole of a six-hole culture plate for plating, and adding a cell culture solution to 2 ml; observing the growth state of the cells on the next day, and adding 15ul of virus solution with the titer of 1 multiplied by 10^8TU/ml and 1ul of Polybrene infection additive with the titer of 4ug/ml into each well when the cells reach 30-50% fusion degree during infection; sucking out culture solution containing virus particles 12 hours after virus infection, adding the whole culture medium into the culture plate again, and continuing culture; observing whether the cells have abnormality on the fourth day; day five observations to assess lentiviral particle transfection efficiency: cleaning the outer wall of the culture plate by using 70% alcohol, observing fluorescence in an inverted fluorescence microscope, photographing and determining the infection efficiency of the lentivirus particles on cells;

adding puromycin into a transfected cell culture dish, wherein the final concentration is 1.5 ug/ml; changing the solution every 3 days, reducing the puromycin concentration to 0.75ug/ml after one week, continuously culturing, and observing and evaluating with a microscope until stable strains are generated; obtaining the cells 1271-luc;

step 102, mouse esophagus in-situ tumor formation experiment

Taking 30C 57BL/6 mice of 6 weeks old, and randomly dividing the mice into two groups, wherein one group is used for cervical esophageal neoplasia, and the other group is used for abdominal esophageal neoplasia; performing surgical field unhairing and disinfection on the neck or abdomen of a mouse, performing intraperitoneal injection anesthesia by using 10% chloral hydrate according to the weight of 0.1ml/10g of the mouse, performing 2-2.5L/min isoflurane gas anesthesia, injecting mixed suspension of 1 x 10^6/20ul 1271-luc cells and Matrigel in equal proportion into each mouse, inoculating 40ul of cancer cell suspension to the muscle layer at the outer edge of the esophageal wall, injecting tumor cells into the neck or abdomen in situ esophagus to observe edema as a sign of successful inoculation, and performing 5-0 nonabsorbable suture line incision full layer;

wherein, the muscle layer at the outer edge of the esophageal wall is the outer muscle layer of the neck esophagus or the outer muscle layer of the abdominal esophagus at the part close to the diaphragm, and the injection part of the abdominal esophagus is far away from the cardia at the junction of the stomach and the esophagus as far as possible;

step 103, monitoring of tumor growth

After the in-situ planting of the tumor cells is completed, the body weight and the growth state of the mouse are monitored regularly; the growth state detection uses a Xenogenin living body imaging system IVIS-100 (Perkin Elmer, Mass.) to carry out living body imaging, 150mg/kg of luciferase substrate is injected into the abdominal cavity of a mouse to monitor the in-situ tumor growth kinetics of an ESCC cell line marked by luciferase injected into the mouse, after the injection into the body is carried out for 10-15min, namely, when a light signal reaches the strongest stable platform stage, imaging analysis is carried out, the tumor growth state is detected through signal intensity, and the imaging data of the growth transfer condition of esophageal cancer is obtained through PET-MR examination 4 weeks after planting; mice were sacrificed and gross and histological data for esophageal cancer were obtained.

2. A model obtained using the method of claim 1.

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