阅读说明：本技术 一种新型小鼠原位胸膜间皮瘤模型及其建立方法 (Novel mouse in-situ pleural mesothelioma model and establishment method thereof ) 是由 韩丹 白崔巍 沈莎莎 王培� 江杰 谢晓洁 康绍磊 赵迅冉 黄益龙 张正华 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种新型小鼠原位胸膜间皮瘤模型及其建立方法,包括以下步骤：步骤一：取BLAB/C裸鼠90只作为实验组,对照组30只,统一放入培养箱中进行正常培养；步骤二：在BLAB/C裸鼠呼吸和心跳等正常的情况下,将小鼠麻醉并以仰卧位固定在动物手术台上进行实验；步骤三：在小鼠的左侧肋骨5/6肋间隙与左侧胸壁腋前线的交点处,使用显微镊提拉该处的皮肤,随后使用22G无菌锐针头对该处皮肤进行破口,随后使用30G无菌钝枕头沿上述破口对壁层胸膜进行破溃,本发明的移植瘤方法可适用胸膜肿瘤的原位移植。可最大限度的还原胸膜肿瘤的生长方式、侵袭能力以及肿瘤转移效果,为研究胸膜肿瘤构建了一个良好的小鼠模型。(The invention discloses a novel mouse in-situ pleural mesothelioma model and an establishment method thereof, and the novel mouse in-situ pleural mesothelioma model comprises the following steps: the method comprises the following steps: taking 90 BLAB/C nude mice as experimental groups and 30 control groups, and uniformly putting the experimental groups and the control groups into an incubator for normal culture; step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment; step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, the skin at the intersection is lifted by using microscopic forceps, then the skin at the intersection is crevassed by using a 22G sterile sharp needle, and then the parietal pleura is crevassed along the crevasse by using a 30G sterile blunt pillow. The growth mode, the invasion capacity and the tumor metastasis effect of the pleural tumor can be reduced to the maximum extent, and a good mouse model is established for researching the pleural tumor.)

1. A novel mouse in-situ pleural mesothelioma model and a building method thereof are characterized in that: the method comprises the following steps:

the method comprises the following steps: taking 90 BLAB/C nude mice as experimental groups and 30 control groups, and uniformly putting the experimental groups and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt needle to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating needle head to insert needle in parallel at the inner side of left chest wall, making the needle head direction point to left sternoclavicular joint, observing the position of needle head on body surface, stopping inserting needle at the position 1mm away from left sternoclavicular joint, and injecting tumor cell suspension 60-80 μ l;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

2. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the first step, the BLAB/C nude mice are selected to have the standard of 16-18g of body weight, normal body functions and half of sex, and 90 mice in the experimental group are divided into three groups which are respectively named as a large-dose group, a medium-dose group and a small-dose group.

3. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the third step, when the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse is crevassed, the parietal pleura at the crossing point is not damaged.

4. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the fourth step, the small dose group is injected with 60 mul of tumor cell suspension, the medium dose group is injected with 70 mul of tumor cell suspension, and the large dose group is injected with 80 mul of tumor cell suspension.

5. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the fourth step, the distance of needle insertion should be less than 6 mm.

6. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the fourth step, the experimental group was injected once a month with 60-80 μ l each time for two months, and the control group was injected with sterilized saline once a month with 60-80 μ l each time for two months according to the same method and location.

7. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the fifth step, the mice in the experimental group and the control group are weighed 1 time per month and the weight data is recorded when observed, and the whole experiment lasts for two years.

8. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the sixth step, 10-20 mice in the experimental group and 3-5 mice in the control group are scanned each time, and the mice in the experimental group are not immediately treated after each scanning is finished until the mice die naturally.

9. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: in the sixth step, chest CT scanning is performed on the dying or dead mice, and then autopsy pathology is checked, so as to observe whether tumors are induced in pleura of the mice.

10. The novel mouse in-situ pleural mesothelioma model and the establishment method thereof according to claim 1, wherein the model comprises the following components: and in the sixth step, the data of the tumor induction rate, the mouse mortality rate and the like of the three groups of experimental groups and the three groups of control groups are recorded in real time and a table is made.

Technical Field

The invention belongs to the technical field of in-situ transplantation of pleural tumors, and particularly relates to a novel mouse in-situ pleural mesothelioma model and an establishing method thereof.

Background

Pleural mesothelioma is a primary tumor of pleura and is a tumor derived from four pleural regions, namely visceral layer, parietal layer, mediastinum or diaphragm. The foreign disease incidence is higher than that of the domestic disease incidence, and the foreign disease incidence is 0.07-0.11% and 0.04% respectively. Mortality accounts for less than 1% of all tumors worldwide. More than 50 years old, the ratio of male to female is 2: 1. The disease is associated with asbestos exposure. There is a lack of effective treatment for malignant forms.

The pleura is serous membranes covering the mediastinum on the inner surfaces of the chest walls of the left and right lungs, and the medical science expresses various functional activities of the zang-fu organs as "qi", and the theory of lung qi is considered to be an important point in traditional Chinese medicine, so that the study is worthy of depth. The pleura is wrapped outside the lung, so that the pleura is beneficial to the lung to complete the function of governing the qi and governing the respiration from the aspect of traditional Chinese medicine, belongs to the category of lung qi, has the function of defending the body, is related to the abundance or insufficiency of vital qi of an organism, possibly has a certain relation with the modern immune function, is not recorded in medicine for the pleura, and belongs to the category of pleural effusion generated due to pleural lesion. The pathological mechanism has been shown to be that asbestos can lead to inflammatory progressive infiltration, or can lead to immune escape effects. In addition, due to the particularity of asbestos substances, cells can influence a series of chromosome arm deletion effects such as spindle yarn and the like in the division process, but only research reports the influence of pleural mesothelial cells on the hyperplasia reaction of thymus and splenocytes in relation to the action of pleura and an immune system, in vitro culture of normal pleural mesothelial cells is an important means for carrying out relevant experimental research and researching the characteristics of the pleural mesothelial cells, has profound significance for researching mouse models of pleural in-situ tumors, and has not been reported in the field of pleural mesothelioma in mouse chest cavity in-situ tumors.

At present, the pleural mesothelioma nodulation mode of animal experiments is mainly that axillary or ventral wall nodulation is common, but the tumor formed by the nodulation mode cannot simulate the damage and invasion effect of the tumor on surrounding tissues (such as visceral pleura, parietal pleura and adjacent lung tissues) in the growth process.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the existing defects, and provide a novel mouse in-situ pleural mesothelioma model and an establishment method thereof, so as to solve the problems that the pleural mesothelioma nodulation mode of the existing animal experiment proposed in the background technology is mainly that axillary or ventral wall nodulation is common, but the tumor formed by the nodulation mode cannot simulate the damage and invasion effect of the tumor on peripheral tissues (such as visceral pleura, parietal pleura and adjacent lung tissues) in the growth process.

In order to achieve the purpose, the invention provides the following technical scheme: a novel mouse in-situ pleural mesothelioma model and an establishment method thereof comprise the following steps:

the method comprises the following steps: taking 90 BLAB/C nude mice as experimental groups and 30 control groups, and uniformly putting the experimental groups and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt pillow to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating needle head to insert needle in parallel at the inner side of left chest wall, making the needle head direction point to left sternoclavicular joint, observing the position of needle head on body surface, stopping inserting needle at the position 1mm away from left sternoclavicular joint, and injecting tumor cell suspension 60-80 μ l;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

Preferably, in the first step, the BLAB/C nude mice are selected to have a standard weight of 16-18g, normal body functions and half male and female functions, and 90 mice in the experimental group are divided into three groups, namely a large dose group, a medium dose group and a small dose group.

Preferably, in the third step, when the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary line of the mouse is creased, the parietal pleura at the crossing point is not damaged.

Preferably, in the fourth step, the small dose group is injected with 60 μ l of tumor cell suspension, the medium dose group is injected with 70 μ l of tumor cell suspension, and the large dose group is injected with 80 μ l of tumor cell suspension.

Preferably, in the fourth step, the distance of needle insertion should be less than 6 mm.

Preferably, in the fourth step, the experimental group is injected once a month, 60-80 μ l each time, and continuously injected for two months, and the control group is injected with the sterilized saline solution once a month, 60-80 μ l each time, and continuously injected for two months according to the same method and position.

Preferably, in the fifth step, when the mice of the experimental group and the control group are observed, the body weight is weighed for 1 time per month, and the body weight data is recorded, and the whole experiment lasts for two years.

Preferably, in the sixth step, 10-20 mice in the experimental group and 3-5 mice in the control group are scanned each time, and after the completion of each scanning, the mice in the experimental group are not immediately treated and are naturally killed.

Preferably, in the sixth step, the moribund or dead mice are subjected to chest CT scanning and then to autopsy pathology examination, and then the mice are observed whether tumors are induced in pleura.

Preferably, in the sixth step, the data of tumor induction rate, mouse death rate and the like of the three experimental groups and the control group are recorded in real time and tabulated.

Compared with the prior art, the invention provides a novel mouse in-situ pleural mesothelioma model and an establishment method thereof, and the novel mouse in-situ pleural mesothelioma model has the following beneficial effects:

1. the tumor transplantation method can be suitable for in-situ transplantation of pleural tumors, can reduce the growth mode, invasion capacity and tumor metastasis effect of the pleural tumors to the maximum extent, provides a reliable animal model for the research of clinical diagnosis and treatment of the pleural mesothelioma, and effectively avoids the problem that the pleural mesothelioma tumor formation mode of the current animal experiment is mainly common in axillary or ventral wall tumor formation, but the tumor formed by the tumor formation mode cannot simulate the damage and invasion effect of the tumor on peripheral tissues (such as visceral pleura, parietal pleura and adjacent lung tissues) in the growth process;

2. according to the invention, through the mouse in-situ pleural mesothelioma model, the problems of high tumor malignancy, fast disease progress, late stage in discovery, no effective treatment method, short disease course (about 6-18 months), high fatality rate and poor prognosis are effectively solved, and because few clinical cases, special diseased people and difficult collection of cases and specimens are still lack of systematic research on pathology, image and molecular biology in the generation and development processes, the clinical prevention, early diagnosis and treatment are still a big problem;

3. the inconsistency of the course and the form of the induced tumor just makes up the defect that a human is difficult to obtain fresh tumor tissue specimens at different tumor stages clinically, is suitable for researching the etiology, mechanism, pathology, image, genetics and the like of the occurrence and development of the tumor, and can be used for establishing a strain and carrying out passage by utilizing the induced tumor;

4. the mouse tumor cell of the invention is a human tumor cell, the disease part and the generating tissue of the induced tumor are the same as those of human, the differentiation degree and the tissue type have greater similarity with human, the mouse tumor cell has better correspondence with clinic, can be used as an experimental tumor model to simulate the change of human body, study the disease mechanism, discuss the prevention and treatment measures, guide the clinic diagnosis and treatment, the material is cheap, the induction method is relatively simple and convenient, the invention is easy to master, compared with other induction methods, the invention has fast disease, high induction rate and typical pathological changes, and is a valuable animal model for studying pleural tumor.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention without limiting the invention in which:

FIG. 1 is a flow chart of steps of a novel mouse in-situ pleural mesothelioma model and a method for establishing the same according to the present invention;

FIGS. 2 and 3 show that after the mouse is anesthetized and fixed on an animal operating table in a supine position, the intersection point of the intercostal space of the left rib 5/6 of the mouse and the axillary anterior line of the left chest wall is selected as a needle insertion position;

FIG. 4 shows the skin being pulled up with micro-forceps, and the skin being subsequently breached with a 22G sterile sharp needle and the parietal pleura being breached along the breach with a 30G sterile blunt pillow;

FIG. 5 shows that the intercostal muscles and parietal pleura are broken by about 0.5-0.6mm, the needle is inserted into the left chest wall in parallel by rotating the needle head, the direction of the needle head is pointed to the left sternoclavicular joint, the position of the needle head is observed on the body surface, the needle insertion is stopped at a position 1mm away from the left sternoclavicular joint, and 60-80ul crystal violet is injected;

fig. 6 shows that the nude mice are sacrificed by cervical dislocation and dissected to further verify that crystal violet smoothly enters the thoracic cavity, which indirectly shows that the mouse in-situ pleural mesothelioma model can be successfully established by the method.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, 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.

Referring to fig. 1-6, the present invention provides a technical solution: a novel mouse in-situ pleural mesothelioma model and an establishment method thereof comprise the following steps:

the method comprises the following steps: taking 90 BLAB/C nude mice as experimental groups and 30 control groups, and uniformly putting the experimental groups and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt pillow to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating needle head to insert needle in parallel at the inner side of left chest wall, making the needle head direction point to left sternoclavicular joint, observing the position of needle head on body surface, stopping inserting needle at the position 1mm away from left sternoclavicular joint, and injecting tumor cell suspension 60-80 μ l;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

In the present invention, preferably, in the first step, the black/C nude mice are selected to have a body weight of 16-18g, normal body functions and half male and female, and 90 mice in the experimental group are divided into three groups, which are named as a large dose group, a medium dose group and a small dose group.

In the present invention, it is preferable that, in step three, the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse is lacerated without damaging parietal pleura.

In the present invention, preferably, in step four, the small dose group is injected with 60. mu.l of tumor cell suspension, the medium dose group is injected with 70. mu.l of tumor cell suspension, and the large dose group is injected with 80. mu.l of tumor cell suspension.

In the present invention, preferably, in step four, the distance of needle insertion should be less than 6 mm.

In the present invention, preferably, in step four, the experimental group is injected once a month with 60-80 μ l each time for two months, and the control group is injected with the sterilized saline once a month with 60-80 μ l each time for two months according to the same method and location.

In the present invention, preferably, in step five, the mice in the experimental group and the control group are observed, and the body weight is weighed 1 time per month, and the body weight data is recorded, and the whole experiment lasts for two years.

In the present invention, preferably, in step six, 10 to 20 mice in the experimental group are scanned each time, 3 to 5 mice in the control group are scanned, and after each scanning of the mice in the experimental group is completed, the mice are not immediately disposed until the mice die naturally.

In the present invention, preferably, in step six, chest CT scanning is performed on dying or dead mice, and then autopsy pathology is examined to see whether tumors have been induced in pleura of the mice.

In the present invention, preferably, in the sixth step, data of tumor induction rate, mouse mortality, etc. of the three experimental groups and the control group are recorded in real time and tabulated.

Example one

A novel mouse in-situ pleural mesothelioma model and an establishment method thereof comprise the following steps:

the method comprises the following steps: taking 30 BLAB/C nude mice as an experimental group and 30 control groups, and uniformly putting the experimental group and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt pillow to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating the needle head to insert needle in parallel at the inner side of the left chest wall, making the needle head direction point to the left sternoclavicular joint, observing the position of the needle head on the body surface, stopping inserting needle at a position 1mm away from the left sternoclavicular joint, and injecting tumor cell suspension by 60 μ l;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

In the first step, the BLAB/C nude mouse is selected to have the standard of weight of 16-18g, and each function of the mouse body is normal and half of the mouse body is male and female.

In step three, when the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse is crevasse, the parietal pleura at the crossing point is not damaged.

In the fourth step, the distance of needle insertion should be less than 6 mm.

In step four, the experimental group was injected once a month with 60. mu.l each time for two months, and the control group was injected with sterilized saline once a month with 60-80. mu.l each time for two months according to the same method and location.

In step five, the mice in the experimental group and the control group were weighed 1 time per month and the weight data was recorded for two years for the whole experiment when observed.

And in the sixth step, 10 mice in the experimental group are scanned each time, 3-5 mice in the control group are scanned, and the mice in the experimental group are not immediately processed after the mice in the experimental group are scanned each time and die naturally.

And step six, performing chest CT scanning on the moribund or dead mice, and then performing autopsy pathology examination to observe whether tumors are induced in pleura of the mice.

And step six, recording the data of the tumor induction rate, the mouse mortality rate and the like of the experimental group and the control group in real time and making a table.

Example two

A novel mouse in-situ pleural mesothelioma model and an establishment method thereof comprise the following steps:

the method comprises the following steps: taking 30 BLAB/C nude mice as an experimental group and 30 control groups, and uniformly putting the experimental group and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt pillow to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating the needle head to insert needle in parallel at the inner side of the left chest wall, making the needle head direction point to the left sternoclavicular joint, observing the position of the needle head on the body surface, stopping inserting needle at a position 1mm away from the left sternoclavicular joint, and injecting 70 μ l of tumor cell suspension;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

In the first step, the BLAB/C nude mouse is selected to have the standard of weight of 16-18g, and each function of the mouse body is normal and half of the mouse body is male and female.

In step three, when the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse is crevasse, the parietal pleura at the crossing point is not damaged.

In the fourth step, the distance of needle insertion should be less than 6 mm.

In step four, the experimental group was injected once a month at 70. mu.l each time for two months, and the control group was injected with sterilized saline once a month at 60-80. mu.l each time for two months according to the same method and location.

In step five, the mice in the experimental group and the control group were weighed 1 time per month and the weight data was recorded for two years for the whole experiment when observed.

And in the sixth step, 10 mice in the experimental group are scanned each time, 3-5 mice in the control group are scanned, and the mice in the experimental group are not immediately processed after the mice in the experimental group are scanned each time and die naturally.

And step six, performing chest CT scanning on the moribund or dead mice, and then performing autopsy pathology examination to observe whether tumors are induced in pleura of the mice.

And step six, recording the data of the tumor induction rate, the mouse mortality rate and the like of the experimental group and the control group in real time and making a table.

EXAMPLE III

A novel mouse in-situ pleural mesothelioma model and an establishment method thereof comprise the following steps:

the method comprises the following steps: taking 30 BLAB/C nude mice as an experimental group and 30 control groups, and uniformly putting the experimental group and the control groups into an incubator for normal culture;

step two: under the normal conditions of BLAB/C nude mice breathing, heartbeat and the like, anesthetizing the mice and fixing the mice on an animal operating table in a supine position for carrying out an experiment;

step three: at the intersection of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse, using a micro-forceps to pull the skin, then using a 22G sterile sharp needle to lacerate the skin, and then using a 30G sterile blunt pillow to lacerate the parietal pleura along the laceration;

step four: inserting needle into the nude mouse chest along the reverse direction of lifting skin, breaking intercostal muscles and parietal pleura by about 0.5-0.6mm, rotating the needle head to insert needle in parallel at the inner side of the left chest wall, making the needle head direction point to the left sternoclavicular joint, observing the position of the needle head on the body surface, stopping inserting needle at a position 1mm away from the left sternoclavicular joint, and injecting 80 μ l of tumor cell suspension;

step five: after the injection is finished, withdrawing the needle head, and observing the vital signs of the mouse;

step six: weighing the mice of the experimental group and the control group once every 1 month and recording data, wherein the mice of the experimental group are subjected to chest CT scanning on the mice in batches every 1-3 months from the third month after induction, and the change condition of the tumor in the mice is observed in real time.

In the first step, the BLAB/C nude mouse is selected to have the standard of weight of 16-18g, and each function of the mouse body is normal and half of the mouse body is male and female.

In step three, when the crossing point of the left rib 5/6 intercostal space and the left chest wall axillary anterior line of the mouse is crevasse, the parietal pleura at the crossing point is not damaged.

In the fourth step, the distance of needle insertion should be less than 6 mm.

In step four, the experimental group was injected once a month, 80. mu.l each time, for two months, and the control group was injected with sterilized saline once a month, 60-80. mu.l each time, for two months, according to the same method and location.

In step five, the mice in the experimental group and the control group were weighed 1 time per month and the weight data was recorded for two years for the whole experiment when observed.

And in the sixth step, 10 mice in the experimental group are scanned each time, 3-5 mice in the control group are scanned, and the mice in the experimental group are not immediately processed after the mice in the experimental group are scanned each time and die naturally.

And step six, performing chest CT scanning on the moribund or dead mice, and then performing autopsy pathology examination to observe whether tumors are induced in pleura of the mice.

And step six, recording the data of the tumor induction rate, the mouse mortality rate and the like of the experimental group and the control group in real time and making a table.

In summary, the body weights of the mice in the experimental group and the control group are basically the same within two years, when the injection amount of the tumor cell suspension to the mice in the experimental group is 90 μ l, the survival rate of the mice in the experimental group is the highest, and is about 83% at the highest, and 80% of the mice in 83% can successfully induce the tumor in vivo, so that medical observation and research can be carried out, and the tumor transplantation method of the invention is proved to be applicable to the in-situ transplantation of pleural tumor. The growth mode, the invasive ability and the tumor metastasis effect of pleural tumors can be reduced to the maximum extent, a good platform is established for researching the traditional Chinese medicine visceral manifestation theory and the function and effect of pleura, and the problem that the tumor formation mode of the pleura mesothelioma in the current animal experiment is mainly that axillary or ventral wall tumors are common, but the tumor formed by the tumor formation mode cannot simulate the injury and the invasive effect of the tumor on peripheral tissues (such as visceral pleura, parietal pleura and adjacent lung tissues) in the growth process is solved.

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.