CN114698595A - Modeling method of tumor in-situ tumor animal model and tumor in-situ tumor animal model - Google Patents
Modeling method of tumor in-situ tumor animal model and tumor in-situ tumor animal model Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a modeling method of a tumor in-situ tumor animal model and the tumor in-situ tumor animal model, which comprises the following steps: anaesthetizing the animal systemically; shaving and disinfecting the target organ area on the animal; placing an ultrasonic probe in a target organ region to obtain parameters of a target organ and peripheral tissues; marking interested body surface puncture needle insertion points and target organ puncture points based on the parameters; connecting a body surface puncture needle inserting point with the target organ puncture point to generate a puncture path; using a micro-injection needle to puncture according to a puncture path to reach the position of a target organ; injecting the tumor cells in the injector into the target organ position through a micro-injection needle; and observing the injection process of the tumor cells in real time by ultrasonic waves, and rotationally withdrawing the needle after all the tumor cells are injected to obtain the tumor in-situ tumor animal model. The invention has the advantages of small wound, high tumor formation rate, quick recovery and stable operation, avoids large wounds such as abdominopexy, pelvic opening, thoracotomy and the like, has no influence on peripheral visceral organs, and has very large application potential.
Description
Technical Field
The application relates to the field of medicines, in particular to a modeling method of a tumor in-situ tumor animal model and the tumor in-situ tumor animal model.
Background
Effective treatment of tumors is a major medical problem. According to the latest published "world cancer report" of the world health organization, the global cancer burden is increasing. The treatment of tumors is thus a major focus of research and study in the medical field.
An important ring recognized in the field of tumor research is the ability to provide model systems for studying cancer and therapeutic treatments. To assess the efficacy of cancer therapy in preclinical and clinical studies, animal models of tumors in which human tumor cell lines or patient-derived tumors are transplanted into immunodeficient mice are commonly used. The tumor animal model provides a good alternative scheme for clinical tests, and is a crucial step before clinical tests are carried out on new medicines, new equipment, new materials, new technologies and the like.
Establishing a tumor animal model is an important means for researching tumor pathogenesis and developing related researches of tumor treatment. The animal model should be as close as possible to the natural biological process of human tumor, and the process and state of in-situ tumor formation are observed, so as to provide experimental animal model for further research on tumor occurrence and development mechanism, tumor resistance, tumor invasion and metastasis, tumor microenvironment, anti-tumor immunity research, and the like. The established animal model has good substitution effect on simulating human body tumors, is beneficial to developing basic research, and most importantly, answers a series of clinical questions based on basic research and study.
The tumor modeling methods commonly used in laboratories at present include spontaneous tumor animal models, induced tumor animal models, transplanted tumor animal models, and transgenic (genetically modified) tumor animal models. Among them, the transplantation tumor animal model is the most widely used modeling method in the current basic research. The transplanted tumor animal model refers to a tumor-bearing animal model obtained by transplanting tumor tissues and cell strains into an experimental animal body. The model has high molding success rate and is often applied to animals such as nude mice, rats, rabbits and the like. The graft type model can be classified into an orthotopic graft tumor and an ectopic graft tumor according to the site of the graft tumor.
The in-situ transplanted tumor model is established by injecting tumor cells into target organs of experimental animals, and the in-situ transplanted tumor can better simulate the growth process of human tumors, can be used for observing in-situ and metastasis of the tumors and can be used for researching tumor formation after gene knockout/overexpression, so that the model is very widely applied to basic research. The ectopic transplantation tumor is mostly formed by injecting a certain amount of tumor cells into the back of an experimental animal subcutaneously, the subcutaneous transplantation tumor is simple and easy to master, the change of the tumor volume is easy to measure, and local intervention is easy to perform. However, xenografts have difficulty exhibiting tumor in situ and metastasis, and lack tumor heterogeneity.
Traditional orthotopic transplantation tumor modeling usually adopts an anatomical method, such as laparotomy of liver cancer, pancreatic cancer and kidney cancer models, laparotomy of rectal cancer and colon cancer, cervical section of thyroid cancer, and thoracic section of lung cancer models. However, the method of establishing a tumor model by dissecting an animal and performing orthotopic transplantation of tumor has many defects of many complications (such as bleeding and infection), large surgical trauma, slow postoperative recovery, long operation time, complex operation and the like.
Disclosure of Invention
In order to overcome the defects of the traditional in-situ transplantation tumor modeling method, the invention provides a tumor in-situ tumor animal model modeling method and a tumor in-situ tumor animal model. The invention provides a novel method with small wound, repeatability, simple operation, fewer complications and high stability. On one hand, the invention can improve the defects of the traditional operation modeling method, and on the other hand, the invention provides a stable, repeatable and high-tumor-forming-efficiency in-situ transplantation tumor modeling method for animal experimental research before clinic, and promotes the development of tumor research model animals.
According to a first aspect of the embodiments of the present application, there is provided a method for modeling a tumor in situ tumor animal model, comprising the steps of:
(1) anaesthetizing the animal and fixing the animal by using an animal fixer;
(2) shaving and disinfecting the target organ area on the animal, paving a hole towel, and fully exposing the operation area;
(3) placing an ultrasonic probe in the target organ region, and acquiring parameters of the target organ and peripheral tissues under the real-time guidance of ultrasonic;
(4) marking the interested body surface puncture needle insertion points and target organ puncture points based on the parameters;
(5) connecting the body surface puncture needle insertion point and the target organ puncture point to generate a puncture path, wherein the puncture path does not intersect with a large blood vessel, and the safety path does not intersect with a bone;
(7) puncturing by using a micro-injection needle according to the puncturing path to reach the position of the target organ;
(8) injecting the tumor cells in the injector into the target organ position through a micro-injection needle;
(9) and observing the injection process of the tumor cells in real time by ultrasonic waves, and rotationally withdrawing the needle after all the tumor cells are injected to obtain the tumor in-situ tumor animal model.
Furthermore, the general anesthesia adopts 1-1.5% of pentobarbital for abdominal cavity anesthesia, and the preferred dosage is 40-45 g/kg.
Further, the parameters mainly include parameters of tumor, blood vessels, bones and body surface.
Further, the tumor cells had a cell count of 1 x 104-1*106。
Further, the ultrasound includes two-dimensional ultrasound and color doppler ultrasound.
Further, the tumor cell is selected from brain tumor, neck tumor, breast tumor, abdominal tumor, pelvic tumor, skin tumor, extremity and bone joint tumor.
Further, the preparation of the tumor cell comprises the following steps:
1) culturing the tumor cells in an incubator at 37 ℃;
2) then, pancreatin is used for digesting the tumor cells, and a culture medium with serum is added to stop digestion;
3) blowing the tumor cells by using a pipette gun, and collecting the digested tumor cells into a centrifuge tube;
4) putting the tumor cells in the centrifugal tube into a centrifugal machine for centrifugation;
5) taking out the centrifuge tube after the centrifugation is finished, discarding the supernatant, and adding a serum-free culture medium for resuspension;
6) and placing the centrifugal tube on ice, adding matrix glue into the centrifugal tube, re-suspending and uniformly mixing to obtain the final tumor cells.
Further, connecting the body surface puncture needle insertion point and the target organ puncture point to generate a puncture path, comprising:
connecting the body surface puncture needle inserting point and the target organ puncture point to generate a plurality of reference paths;
then, translating N safe paths meeting given conditions from at least one reference path, and deleting abandoning paths, wherein the safe paths intersected with the skeleton or with the blood vessel are abandoning paths;
and outputting the N safety paths into M puncture paths, wherein N is more than or equal to M.
Further, the animal is selected from one of rodents, rabbits, dogs, cats, non-human primates, chickens, pigs and fish.
According to a second aspect of the embodiments of the present application, there is provided an animal model of tumor orthotopic tumors prepared by the modeling method described above.
The tumor modeling method for in-situ transplantation under ultrasonic guidance has the great advantages of small wound, stable modeling method, simple operation, repeatability and the like, and is suitable for large-scale popularization and application of tumor models for in-situ transplantation.
The invention also provides an animal model corresponding to the molding method. Preferably, it may be experimental animals such as rodents (including mice, rats, guinea pigs, and gerbils), rabbits, dogs, cats, non-human primates (marmosets, squirrel, cynomolgus monkeys, and baboons), chickens, miniature pigs, and fish.
The animals currently used to construct orthotopic transplantation tumor models are mainly mice, rabbits and monkeys. Factors to be considered for experimental animals are presumably: easy feeding, high reproduction rate, low price, easy acquisition, convenient operation, high hereditary purity, metabolism type and physiological pathology close to human beings as much as possible, and the like. Among them, the advantages of mice in these respects are obvious without exception, and are the most commonly used experimental animals for tumor modeling. The animal in-situ transplantation tumor modeling method prepared by the method can better simulate the state of human tumor, lays a good foundation for clinical service, and realizes the advantages of minimal invasion, simple operation and stable model.
The invention also provides that the target organ corresponding to the modeling method is preferably skin, oral cavity, thyroid, mammary gland, testis, superficial lymph node, nose, pharynx, lung, esophagus, stomach and intestine, kidney, bladder, uterus, ovary, bone, brain, liver, pancreas, spleen, prostate, and the like.
The technical scheme provided by the embodiment of the application can have the following beneficial effects:
according to the embodiments, the specific parameters of the target organ and the surrounding tissues can be observed in real time by an ultrasonic guidance method, so that the stability of the in-situ transplantation tumor modeling is improved. The in-situ transplantation tumor animal model constructed by the method is not limited by the animal species, can realize in-situ transplantation tumor modeling on various animals such as mice, rats, rabbits, monkeys and the like, is not influenced by the organ positions, and can realize in-situ modeling on various organs and various experimental animals within the ultrasonic visualization range. The growth state of the in-situ tumor is better simulated, and a new thought is provided for researching an in-situ tumor model.
The modeling method of the orthotopic transplantation tumor model can reduce complications brought by the traditional anatomical surgery modeling method on the basis of ultrasonic guidance, has no operation of large wound on animals, and has high success rate of tumor formation. The model can be used for observing the process and the state of tumor in-situ tumorigenesis, can further research the aspects of tumor occurrence and development mechanism, tumor drug resistance, tumor invasion and metastasis, tumor microenvironment, anti-tumor immunity research and the like, and has good application prospect.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
Fig. 1 is a flow chart illustrating a method of modeling a tumor in situ tumor animal model, according to an exemplary embodiment.
Fig. 2 is a schematic flow diagram illustrating the preparation of tumor cells according to an exemplary embodiment.
Fig. 3 is a schematic diagram illustrating selection of a secure path according to an example embodiment.
Fig. 4 is a diagram illustrating mouse liver cancer orthotopic tumor modeling under ultrasound guidance according to an exemplary embodiment.
FIG. 5 is a diagram illustrating a modeled qualification map in accordance with an exemplary embodiment.
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The invention discloses an in-situ transplantation tumor modeling method under ultrasonic guidance, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the methods and applications described herein may be made to implement and utilize the techniques of this invention without departing from the spirit and scope of the invention.
The invention is further illustrated by the following examples:
example 1 molding procedure (fig. 1):
a method for modeling a tumor in situ tumor animal model, comprising the following steps, as shown in fig. 1:
1. c57 mice were anesthetized with 1-1.5% pentobarbital (45g/kg) intraperitoneally, and the animals were fixed using a mouse fixing plate;
2. shaving and disinfecting the abdominal region, paving a hole towel, and fully exposing the operation region; wherein, the abdominal region refers to the point from the upper part of the xiphoid process of the mouse to the connecting line of the anterior superior iliac spine, the left part to the left axillary midline and the right part to the right axillary midline;
3. the method comprises the steps of placing an ultrasonic probe in a liver region, and acquiring parameters of liver tissues and peripheral tissues under real-time ultrasonic guidance, wherein the parameters comprise parameters of blood vessels, parameters of bones and parameters of body surfaces. The ultrasound here includes two-dimensional ultrasound and color doppler ultrasound.
4. Marking body surface puncture needle insertion points and liver puncture points based on the parameters;
5. and connecting the body surface puncture needle inlet point and the liver puncture point to generate a puncture path. The puncture path here does not intersect the great vessels and does not intersect the bone.
6. Sucking the prepared tumor cells by using a syringe with the volume of 1-2 ml; the tumor cells preferably have a cell count of 1.0 x 104-1.0*106The volume is 5.0-20.0 μ l.
7. Puncturing through a safe puncturing path to reach a liver target position; the target position of the liver is 2.0mm-6.0mm under the liver capsule.
8. Injecting the tumor cells in the injector into a target position of the liver;
9. and (3) observing the cell injection process in real time by using ultrasonic waves, and rotating and withdrawing the needle after all the cells are injected to complete the construction of the tumor in-situ tumor animal model.
10. The growth of the target tumor was observed with ultrasound every 3 days and recorded.
Example 2 preparation of tumor cells (fig. 2):
the preparation of tumor cells is a key step of modeling in situ transplanted tumors under the guidance of ultrasound, and comprises the following specific steps:
1. culturing tumor cells in an incubator at 37 ℃, and observing the growth speed and the cell density of the cells; the tumor cells include brain tumor, neck tumor, breast tumor, abdominal tumor, pelvic tumor, skin tumor, limb and bone joint tumor, etc.
2. Then, pancreatin is used for digesting the tumor cells, and a culture medium with serum is added to stop digestion; the amount of pancreatin depends on the size of the cell culture dish, for example, a 10.0cm diameter cell culture dish is digested by adding 3.0-4.0ml pancreatin. The digestion of tumor cells was stopped by adding 2 times the volume of pancreatin in serum-containing cell culture medium.
3. Blowing cells by using a pipette gun, and collecting the digested tumor cells into a centrifuge tube; the digestion condition of tumor cells is 2-4 min.
4. Placing the cells in the centrifugal tube into a centrifugal machine for centrifugation; the centrifugal speed of the tumor cells is 1000-2000r, and the centrifugal time is 4-6 min.
5. Taking out the centrifuge tube after the centrifugation is finished, discarding the supernatant, and adding a serum-free culture medium for resuspension; the volume of serum-free medium added here depends on the number of cells, e.g.1.0 x 104-1.0*106The amount of tumor cells can be 50-250. mu.l.
6. Placing the centrifugal tube on ice, adding matrix glue into the centrifugal tube, and re-suspending and uniformly mixing; the amount of Matrige gel added was equal to the volume of serum-free medium.
Example 3 selection of secure path (fig. 3):
(1) and acquiring peripheral parameters of the target visceral organ under the guidance of the ultrasound. The target organ peripheral parameters comprise parameters of blood vessels, parameters of bones and parameters of body surfaces.
(2) Based on the parameters, marking the interested body surface puncture needle insertion points and the target organ puncture points.
(3) And connecting the body surface puncture needle inserting point and the target organ puncture point to generate a plurality of reference paths. The reference path does not intersect the great vessels and the reference path does not intersect the bone.
(4) Then, the N safe paths meeting the given conditions are translated out by at least one reference path, and the abandon path is deleted. The puncture path which does not intersect with the great vessel and does not intersect with the bone is output as a safe path. The puncture path intersecting the bone or intersecting the blood vessel is output as a discard path. The given conditions include that the puncture range is within the puncture region of interest and the safe path region.
(5) And outputting the N safety paths into M puncture paths. The N safety path outputs are M puncture paths, wherein N is larger than or equal to M.
Example 4 modeling of mouse liver cancer in situ tumor under ultrasound guidance (FIG. 4)
1. General anesthesia was performed on the C57 mice, and the C57 mice were fixed using a mouse fixer; the whole anesthesia is performed by abdominal cavity anesthesia with 1-1.5% pentobarbital, preferably at a dosage of 40-45 g/kg.
2. Shaving and disinfecting the abdominal region, paving a hole towel, and fully exposing the operation region; the abdominal region goes up to the xiphoid process, down to the iliac spine junction, left to the left axillary midline, right to the right axillary midline.
3. Placing an ultrasonic probe in a liver region, and acquiring parameters under the real-time guidance of ultrasonic;
4. marking interested body surface skin puncture needle insertion points and liver puncture points based on parameters acquired under ultrasonic guidance; the obtained parameters mainly comprise parameters of blood vessels, parameters of bile ducts, parameters of bones and parameters of body surfaces.
5. Connecting the body surface skin puncture needle insertion point and the liver puncture point to generate a safe puncture path; the safe path does not intersect with the great blood vessel in the liver, the bile duct, the gall bladder and the skeleton.
6. Sucking prepared Hepa1-6 liver cancer cells by using a syringe; the preparation of the Hepa1-6 liver cancer cells comprises the steps of culturing, digesting, centrifuging, resuspending and uniformly mixing with matrix gel.
7. Puncturing through the safe puncturing path, outputting the actual puncturing path to a liver target injection position;
8.injecting the liver cancer cells in the injector; the amount of cells injected with hepatoma cells per mouse was 5 x 104-10*104。
9. Ultrasonically observing the cell injection process in real time, and rotationally withdrawing the needle after all the cells are injected; the rotary needle withdrawing is rotated by 180-360 degrees.
Example 5 identification of modeling (FIG. 5)
Mouse liver cancer model success markers:
1. visual identification: the body surface of the mouse can touch tumor tissues, and liver cancer nodules can be found in the liver tissues after dissection.
2. Ultrasonic identification:
(1) two-dimensional ultrasound: the characteristics of the sound image: a, expanding growth to generate sound halo; b polymorphism: various intensities and different forms coexist; c, polytropy: the form and internal echo are constantly changed; d, growing rapidly; and e, cancer tissue infiltration and cancer embolism appear in the portal vein or hepatic vein at the late stage.
(2) Color Doppler: the liver cancer nodule and its surroundings can obtain various blood flow information due to the abundant blood supply. The color Doppler ultrasonic detection has high tissue blood flow sensitivity and can accurately reflect the blood supply condition of liver cancer. Blood flow around the cancer nodule may appear as a full circle or an arc around it, which may be continuous portal or pulsatile arterial flow as measured by spectral doppler.
(3) Ultrasonic radiography: a ultrasonic contrast representation of typical hepatocellular carcinoma: high enhancement at arterial stage (small nodules less than 3cm are usually uniformly enhanced, large nodules may be non-uniformly enhanced due to liquification necrosis), clear at late portal or delayed stage, and slightly low enhancement. The ultrasonic contrast expression of atypical liver cancer with highly differentiated liver cells: the arterial phase is highly enhanced, the portal and delayed phases are equally enhanced or slightly enhanced, as in the present case.
3. And (3) pathological identification: HE staining is carried out on the liver cancer tissue, and the boundary of the liver cancer tissue and the normal liver tissue is found.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.
Claims (10)
1. A method for modeling a tumor in situ tumor animal model, which is characterized by comprising the following steps:
(1) anaesthetizing the animal and fixing the animal by using an animal fixer;
(2) shaving and disinfecting the target organ area on the animal, paving a hole towel, and fully exposing the operation area;
(3) placing an ultrasonic probe in the target organ region, and acquiring parameters of the target organ and peripheral tissues under the real-time guidance of ultrasound;
(4) marking the interested body surface puncture needle insertion points and target organ puncture points based on the parameters;
(5) connecting the body surface puncture needle insertion point and the target organ puncture point to generate a puncture path, wherein the puncture path does not intersect with a large blood vessel, and the safety path does not intersect with a bone;
(7) puncturing by using a micro-injection needle according to the puncturing path to reach the position of the target organ;
(8) injecting the tumor cells in the injector into the target organ position through a micro-injection needle;
(9) and observing the injection process of the tumor cells in real time by ultrasonic waves, and rotationally withdrawing the needle after all the tumor cells are injected to obtain the tumor in-situ tumor animal model.
2. The modeling method according to claim 1, characterized in that general anesthesia is performed with intraperitoneal anesthesia using 1-1.5% pentobarbital, preferably at a dose of 40-45 g/kg.
3. The modeling method of claim 1, wherein the parameters include primarily parameters of a tumor, blood vessels, bones, and body surfaces.
4. The molding method according to claim 1, wherein the tumor cells have a cell count of 1 x 104-1*106。
5. The molding method according to claim 1, wherein the ultrasound includes two-dimensional ultrasound and color doppler ultrasound.
6. The molding method according to claim 1, wherein the tumor cell is selected from the group consisting of brain tumor, neck tumor, chest tumor, abdominal tumor, pelvic tumor, skin tumor, extremity and bone joint tumor.
7. The modeling method of claim 1, wherein the preparation of the tumor cells comprises the steps of:
1) culturing the tumor cells in an incubator at 37 ℃;
2) then, pancreatin is used for digesting the tumor cells, and a culture medium with serum is added to stop digestion;
3) blowing the tumor cells by using a pipette gun, and collecting the digested tumor cells into a centrifuge tube;
4) putting the tumor cells in the centrifugal tube into a centrifugal machine for centrifugation;
5) taking out the centrifuge tube after the centrifugation is finished, discarding the supernatant, and adding a serum-free culture medium for resuspension;
6) and placing the centrifugal tube on ice, adding matrix glue into the centrifugal tube, re-suspending and uniformly mixing to obtain the final tumor cells.
8. The modeling method according to claim 1, wherein connecting the body surface puncture needle insertion point and the target organ puncture point to generate a puncture path comprises:
connecting the body surface puncture needle insertion point and the target organ puncture point to generate a plurality of reference paths;
then, translating N safe paths meeting given conditions from at least one reference path, and deleting abandoning paths, wherein the safe paths intersected with the skeleton or with the blood vessel are abandoning paths;
and outputting the N safety paths into M puncture paths, wherein N is more than or equal to M.
9. The molding method according to claim 1, wherein the animal is one selected from the group consisting of rodents, rabbits, dogs, cats, non-human primates, chickens, pigs and fishes.
10. An animal model of tumour orthotopic tumours prepared by a modelling method according to any of claims 1 to 9.
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