CN113080136B - Method for establishing retroperitoneal sarcoma mouse orthotopic xenograft animal model - Google Patents
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- CN113080136B CN113080136B CN202110208759.9A CN202110208759A CN113080136B CN 113080136 B CN113080136 B CN 113080136B CN 202110208759 A CN202110208759 A CN 202110208759A CN 113080136 B CN113080136 B CN 113080136B
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- 201000004846 retroperitoneal sarcoma Diseases 0.000 title claims abstract description 49
- 238000010171 animal model Methods 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 15
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 26
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 23
- 210000000509 perirenal space Anatomy 0.000 claims abstract description 9
- 238000011580 nude mouse model Methods 0.000 claims abstract description 8
- 241000699660 Mus musculus Species 0.000 claims abstract description 7
- 238000002054 transplantation Methods 0.000 claims abstract description 7
- 238000002689 xenotransplantation Methods 0.000 claims description 9
- 210000003734 kidney Anatomy 0.000 claims description 8
- 206010039491 Sarcoma Diseases 0.000 claims description 7
- 210000003195 fascia Anatomy 0.000 claims description 6
- 201000008808 Fibrosarcoma Diseases 0.000 claims description 5
- 239000006285 cell suspension Substances 0.000 claims description 5
- 206010024627 liposarcoma Diseases 0.000 claims description 5
- 206010002091 Anaesthesia Diseases 0.000 claims description 4
- 241000699670 Mus sp. Species 0.000 claims description 4
- 230000037005 anaesthesia Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000011065 in-situ storage Methods 0.000 abstract description 13
- 238000011161 development Methods 0.000 abstract description 11
- 238000011160 research Methods 0.000 abstract description 10
- 230000007547 defect Effects 0.000 abstract description 7
- 238000003745 diagnosis Methods 0.000 abstract description 4
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- 238000003384 imaging method Methods 0.000 abstract description 3
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000004303 peritoneum Anatomy 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- 231100000588 tumorigenic Toxicity 0.000 description 3
- 230000000381 tumorigenic effect Effects 0.000 description 3
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 2
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- 206010061598 Immunodeficiency Diseases 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a retroperitoneal sarcoma mouseThe method for establishing orthotopic xenograft animal model adopts common nude mice, the transplantation position is determined in the perirenal space, the number of transplanted cells is 1 multiplied by 107And (3) establishing a retroperitoneal sarcoma mouse orthotopic xenograft animal model by using the retroperitoneal sarcoma cells, and feeding the mouse for 10-30 days to form an obvious tumor. The invention makes up the defects in the prior art, can be used for researching the occurrence and development of the retroperitoneal sarcoma, screening drugs, quickly diagnosing and exploring an operation method, and provides a retroperitoneal in-situ tumor drug screening model, an imaging research model for the in-situ development of the retroperitoneal sarcoma, and a research model of novel diagnosis molecules and methods for the retroperitoneal sarcoma.
Description
Technical Field
The invention particularly relates to a method for establishing a retroperitoneal sarcoma mouse orthotopic xenograft animal model.
Background
Soft tissue sarcomas are a highly heterogeneous group of rare tissues originating from mesenchymal tissue. Although it only accounts for 1.5% of all human tumors. Retroperitoneum is 15-20% of the primary sites of soft tissue sarcomas, with a poorer prognosis relative to soft tissue sarcomas elsewhere. Among the retroperitoneal sarcomas are mainly differentiated/dedifferentiated liposarcomas, fibrosarcomas, leiomyosarcomas, undifferentiated polymorphic sarcomas and peripheral nerve sheath tumors. Retroperitoneal sarcomas tend to be hidden deep behind the peritoneum, and they often develop into large tumors and invade surrounding organs before symptoms appear, often difficult to find in the early stages. Surgery is difficult to remove intact and has a high recurrence rate, while being insensitive to both radiotherapy and chemotherapy. Early diagnosis and interventional treatment of malignant retroperitoneal tumors are global challenges.
The basic and clinical research in the field of retroperitoneal tumors is very poor relative to other tumor types, especially in the absence of suitable animal models. The existing models comprise a human tumor tissue xenotransplantation model, a human tumor cell xenotransplantation model under the skin and a transgenic mouse. However, the existing three models have certain defects, for example, the problem that specimens need to be strictly ethically examined exists in human tumor tissue source xenograft, the procedure is complex, and the availability is poor; the existing human tumor cell xenograft has the defects that a tumor-bearing mouse is a naked mouse with serious immunodeficiency, the breeding condition is harsh and the price is high, and meanwhile, the transplantation position is not the primary part of the retroperitoneal sarcoma and can not well simulate the generation and development of the retroperitoneal sarcoma. The genetic background of the transgenic mice is simple, and the genetic heterogeneity of the clinical retroperitoneal sarcoma cannot be truly reflected.
It can be seen that the previously reported patient-derived tumor xenograft model, cell-derived xenograft and transgenic animal model play an important role in the retroperitoneal sarcoma study, but they have the disadvantages of difficult specimen acquisition and complex procedure, harsh SCID mouse feeding conditions, simple genetic background, etc. Therefore, an in-situ tumorigenesis model which is simple, convenient and fast and can accurately simulate the retroperitoneal tumorigenesis and development is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for solving the defects of three models in the background technology, aims to provide an animal model for in-situ generation and development of retroperitoneal sarcoma, aims to provide a screening model for retroperitoneal in-situ tumor drugs, aims to provide an imaging research model for in-situ development of retroperitoneal sarcoma, aims to provide a research model for novel diagnosis molecules and methods of retroperitoneal sarcoma, and explores generation and development of retroperitoneal tumor and related mechanisms thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: provides a method for establishing a retroperitoneal sarcoma mouse orthotopic xenograft animal model, which comprises the following steps:
(1) culturing retroperitoneal sarcoma cells 1X 107A plurality of;
(2) anaesthetizing a common nude mouse, adopting a supine position, opening the abdomen, and exposing the kidney;
(3) the transplantation position is determined in the perirenal space, a 1mL syringe needle is tightly attached to the left fat of the kidney, the needle is inserted downwards by oblique puncture and then horizontally inserted for 5mm, and 100uL of cell suspension is injected into the perirenal space; the cell suspension contains 1X 10 of the step (1)7Retroperitoneal sarcoma cells;
(4) closing the abdomen of the mouse after the xenotransplantation, suturing and removing an anesthesia machine, establishing a retroperitoneal sarcoma mouse in-situ xenotransplantation animal model, and feeding the mouse for 10-30 days to form an obvious tumor.
In a preferred embodiment of the present invention, in the step (1), human retroperitoneal sarcoma cells are used as the retroperitoneal sarcoma cells, and other retroperitoneal sarcoma cells can be used instead.
In a preferred embodiment of the present invention, in the step (2), 5-6 weeks of normal nude mice are used.
In a preferred embodiment of the present invention, the step (2) specifically includes the following steps:
the isoflurane respiratory anesthesia method comprises the following steps of (1) anesthetizing a mouse in a supine position, fixing four limbs of the mouse by using a paper adhesive tape, sterilizing the peritoneum, opening the abdomen, deviating from the left side by 4-6 mm along the midline of the abdomen, and cutting from the groin by 5mm to the rib lower edge by 5 mm; the skin spreader spreads the abdominal cavity, the intestinal tract moves to the left side of the abdominal cavity, and the kidney is exposed.
In a preferred embodiment of the present invention, in the step (3), the perirenal space is located between the anterior fascia and the posterior fascia.
In a preferred embodiment of the present invention, the visceral reduction in step (4) comprises bowel reduction.
In a preferred embodiment of the present invention, in the step (4), the mice have a time to develop a significant tumor, and the fibrosarcoma HT1080 is within 15 days; liposarcoma SW872 is available in less than 30 days.
Compared with the background technology, the technical scheme has the following advantages:
1. the animal model established by the invention can accurately simulate the in-situ tumorigenesis model of retroperitoneal tumorigenesis development;
2. the invention solves the defect problems of the existing model:
the invention is planted with cells, is not a clinical patient sample, can be purchased by a company, has easy source and simple culture;
secondly, ordinary nude mice are adopted, and tumor-bearing mice with harsh feeding conditions and high price are not needed; the defect that the transgenic mouse cannot truly reflect the genetic heterogeneity of the clinical retroperitoneal sarcoma is also avoided;
the position of the xenotransplantation is perirenal clearance which is retroperitoneal clearance, and the xenotransplantation can well simulate the generating and developing microenvironment of retroperitoneal tumors and has high repeatability;
3. the animal model can be used for researching the generation and development of the retroperitoneal sarcoma, drug screening, rapid diagnosis and operation method exploration, further provides a retroperitoneal in-situ tumor drug screening model, an imaging research model of the retroperitoneal sarcoma in-situ development, a research model of novel diagnosis molecules and methods of the retroperitoneal sarcoma and the like, has wide application range, can enable people to know the retroperitoneal tumor more deeply, and can break through on early diagnosis, treatment and relapse prevention.
Drawings
FIG. 1 is a schematic diagram of the site of inoculation of a retroperitoneal sarcoma cell mouse, a-before inoculation, b-after inoculation;
FIG. 2 is a photograph of retroperitoneal sarcoma mouse xenograft chemiluminescence;
FIG. 3 is a nuclear magnetic resonance image of a retroperitoneal sarcoma in situ xenograft solid tumor;
FIG. 4 is a tumor anatomy of a retroperitoneal sarcoma in situ xenograft model;
FIG. 5 is a pathological diagram of retroperitoneal sarcoma in situ xenograft tumor.
Detailed Description
Example 1
The animal model construction method for human retroperitoneal sarcoma cell xenograft of the embodiment comprises the following specific steps:
(1) culturing human retroperitoneal sarcoma cells 1X 107A plurality of;
(2) general nude mice of 5-6 weeks are anesthetized by isoflurane breathing, the four limbs of the mice are fixed by paper adhesive tapes in a supine position, the peritoneum is disinfected and the abdomen is opened, the left side of the central line of the abdomen is 5mm, and the limbs are cut from the upper 5mm of the groin to the lower 5mm of the rib. The skin spreader spreads the abdominal cavity, the intestinal tract moves to the left side of the abdominal cavity, and the kidney is exposed.
(3) The transplantation position is determined by placing perirenal space between anterior fascia and posterior fascia of kidney, placing kidney, adrenal gland, renal blood vessel, renal pelvis, ureter and fat sac in the space, tightly attaching left side fat of kidney with 1ml syringe needle, obliquely inserting needle downwards, and adding waterThe needle is horizontally inserted by 5 mm; will contain 1X 107100ul of cell suspension of sarcoma cells was injected into the perirenal space.
(4) The intestinal tract is reset and the abdomen is closed and sutured. The anesthesia machine is removed.
In the above steps, the animal model is a normal nude mouse xenogeneic orthotopic transplantation tumorigenic model. As shown in FIG. 2, the time for the mouse to form a distinct tumor can be observed, and the fibrosarcoma HT1080 is within 15 days; liposarcoma SW872 is available in less than 30 days. As shown in fig. 3, by establishing a normal control group to be compared with the animal model of this embodiment, and by establishing the retroperitoneal sarcoma orthotopic xenograft model tumor anatomy map of fig. 4 and the pathological section of fig. 5, it can be seen that the transplantation tumorigenic states of fibrosarcoma HT1080 and liposarcoma SW872 in the model are very similar to the clinical retroperitoneal sarcoma tumorigenic positions and forms, and the model can not only well simulate the microenvironment for generating and developing retroperitoneal tumors, but also play an auxiliary role in the fundamental research related to retroperitoneal sarcomas, including molecular mechanism, drug screening, cellular immunotherapy, diagnostic molecule screening, image material development, clinical surgical excision, etc., and can effectively promote the preclinical research of retroperitoneal sarcomas, so that more research results can be served to retroperitoneal sarcoma patients as soon as possible.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A retroperitoneal sarcoma mouse orthotopic xenograft animal model, comprising: the establishing method comprises the following steps:
(1) culturing retroperitoneal sarcoma cells 1X 107A plurality of;
(2) anaesthetizing common nude mice, and adopting supine position xenotransplantation;
(3) the transplantation position is determined in the perirenal space, a 1mL syringe needle is tightly attached to the left fat of the kidney, the needle is inserted downwards by oblique puncture and then horizontally inserted for 5mm, and 100uL of cell suspension is injected into the perirenal space; the cell suspension contains 1X 10 of the step (1)7Retroperitoneal sarcoma cells;
(4) and (3) removing the anesthesia machine from the mouse after the xenotransplantation, establishing a retroperitoneal sarcoma mouse orthotopic xenotransplantation animal model, and feeding the mouse for 10-30 days to form an obvious tumor.
2. The retroperitoneal sarcoma mouse orthotopic xenograft animal model of claim 1, wherein: in the step (1), human retroperitoneal sarcoma cells are adopted as the retroperitoneal sarcoma cells.
3. The retroperitoneal sarcoma mouse orthotopic xenograft animal model of claim 1, wherein: in the step (2), 5-6 weeks of ordinary nude mice are used.
4. The retroperitoneal sarcoma mouse orthotopic xenograft animal model of claim 1, wherein: in the step (3), the perirenal space is located between the anterior fascia and the posterior fascia.
5. The retroperitoneal sarcoma mouse orthotopic xenograft animal model of claim 1, wherein: in the step (4), the time for the mice to form obvious tumors is within 15 days of fibrosarcoma HT 1080; liposarcoma SW872 is available in less than 30 days.
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| WO2005058028A2 (en) * | 2003-12-16 | 2005-06-30 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
| CN101185764A (en) * | 2007-10-17 | 2008-05-28 | 南京大学 | Orthotopic transplantation rat liver cancer model and preparing method and application thereof |
| CN110476891A (en) * | 2019-09-09 | 2019-11-22 | 中南大学湘雅二医院 | A kind of construction method of synovial sarcoma xenograft mouse model and its application |
| CN110495424A (en) * | 2019-09-09 | 2019-11-26 | 中南大学湘雅二医院 | A kind of construction method of synovial sarcoma xenograft mouse model |
| CN110547250A (en) * | 2019-09-23 | 2019-12-10 | 中南大学湘雅二医院 | Construction method and application of synovial sarcoma xenograft mouse model with healthy immunity |
| CN110637783A (en) * | 2019-09-23 | 2020-01-03 | 中南大学湘雅二医院 | Construction method of synovial sarcoma xenograft mouse model with healthy immunity |
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| GB201120989D0 (en) * | 2011-12-06 | 2012-01-18 | Mars Inc | Genetic test |
| US10709120B2 (en) * | 2017-01-23 | 2020-07-14 | University Of South Carolina | Peromyscus animal model for cancer |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005058028A2 (en) * | 2003-12-16 | 2005-06-30 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
| CN101185764A (en) * | 2007-10-17 | 2008-05-28 | 南京大学 | Orthotopic transplantation rat liver cancer model and preparing method and application thereof |
| CN110476891A (en) * | 2019-09-09 | 2019-11-22 | 中南大学湘雅二医院 | A kind of construction method of synovial sarcoma xenograft mouse model and its application |
| CN110495424A (en) * | 2019-09-09 | 2019-11-26 | 中南大学湘雅二医院 | A kind of construction method of synovial sarcoma xenograft mouse model |
| CN110547250A (en) * | 2019-09-23 | 2019-12-10 | 中南大学湘雅二医院 | Construction method and application of synovial sarcoma xenograft mouse model with healthy immunity |
| CN110637783A (en) * | 2019-09-23 | 2020-01-03 | 中南大学湘雅二医院 | Construction method of synovial sarcoma xenograft mouse model with healthy immunity |
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