CN110402895A - A kind of construction method of osteosarcoma xenograft mouse model - Google Patents
A kind of construction method of osteosarcoma xenograft mouse model Download PDFInfo
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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
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- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2227/105—Murine
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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
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Abstract
The present invention provides a kind of construction method of osteosarcoma xenograft mouse model, the method includes the Patients with Osteosarcoma tumor tissues being stored in liquid nitrogen environment of first recovering, again aseptically by the specimens block xenogenesis orthotopic transplantation of recovery into immunodeficient mouse body, the xenogenesis orthotopic transplantation includes the immunodeficient mouse for selecting the age at 2~6 weeks, mouse gender is consistent with Gender, the specimens block of recovery is immersed in sterile Incubating Solution, it is fixed after mouse anesthesia, it will be in the ossis of specimens block implantation mouse, suture the operative incision on mouse skin, it brings up mouse and makes tumour growth in its ossis.It is in situ to model that situation is truer, and the pharmaceutical admixtures screened be better utilized in patient compared to subcutaneous modeling, and in Modeling Theory in situ for can be easier that metastases occur than subcutaneous modeling.
Description
Technical field
Field is established the present invention relates to tumour transplatation mouse model, and in particular to a kind of osteosarcoma xenograft mouse mould
The construction method of type.
Background technique
Osteosarcoma is the most common primary bone malignant tumour in Hunan Province, and grade malignancy is high, and Yi Fasheng Lung metastases are disabled
Rate is high with the death rate, poor prognosis.75% Patients with Osteosarcoma is teenager, and previously one it is found that take amputation to control more
It treats, nonetheless, 5 annual survival rates are also only 5~20% after patient's amputation.This brings greatly spirit wound for patient and its family
Evil also all brings heavy financial burden for society or even country, brings bigger social concern.Because osteosarcoma tissue is heterogeneous
The presence of property, previously the basic research result based on osteosarcoma cell line only can be in clinical Patients with Osteosarcoma body less than 20%
It is inside reappeared, largely leads to the lag of clinical osteosarcoma treatment level.
Tumor model is the important carrier of tumor research.The study found that cell constantly adapts to external ring in succeeding generations
Border, Biological characteristics are not inconsistent in body tumour cell, and there are larger consistent for Cell Line Xenografts model and patient tumour itself
Property, and existing animal model for tumour lacks immune system, and the defect of model leads to the failure of anti-tumor drug clinical test
Rate is high.
Humanized's tumor xenograft (patient derived xenograft, PDX) model is a kind of In vivo model,
By being grafted directly on immunodeficient mouse after handling the fresh tumor tissue of patient, the ambient growth provided by mouse
With gradually pass on, can be using mouse as carrier massive amplification specimens.PDX model has the following advantages: firstly, it is equivalent to
The corresponding patient of one model, can react the otherness of tumour between Different Individual well, more can compared to conventional model
React the actual conditions of clinical patient；Secondly, PDX model can be passed on, and the tumor tissues passed on can with it is initial
Tumor tissues keep high consistency in gene copy number, gene mutation and expression pattern etc., are equivalent to for patient's tumour
Sample is expanded, this just provides good condition for drug screening and patient's illness Mechanism Study；Again, PDX is cultivated
Tumor mass, can freeze can still construct new PDX model after recovery, be equivalent to and construct the swollen of different patients
Tumor living body library.
Existing technology is mainly by carrying out chopping processing for fresh osteosarcoma tissue or carrying out cell homogenization processing
Afterwards, it is subcutaneously modeling, is being considered the characteristics of destroying tumor tissues microenvironment itself after homogenized, good cannot keep swollen
The consistency of tumor and surrounding interstitial and patient.
Patent application CN201410083459 provides method for building up and its application in a kind of live tumor tissue library, pass through by
Tumor tissues after human carcinoma's patients surgery are transplanted under the kidney envelope of SCID mice, are survived in Mice Body and are passed on guarantor
Deposit, obtain live tumor tissue library, live tumor tissue library be on the basis of single tumor tissues heterograft, will be numerous
Body tumor tissue is saved in animal body in the form of living tissue library, is spare.The live tumor tissue library includes lateral
Kinds of tumors also includes the various hypotypes or various developing stage of certain longitudinal tumour.The live tumor tissue library has
Multiple use, it is special in the tumour of anticancer drug including the application in terms of the personalized therapy program after determining cancer patient's operation
The application of opposite sex screening aspect, and the application in terms of the animal model of building human carcinoma's research.In the disclosure in this patent,
The human tumor can be the human bodies solid malignants such as lung cancer, liver cancer, gastric cancer, osteosarcoma.Wherein embodiment 1 and comparison are real
It applies example 1 and Fig. 1 b is that the situation that glioma cells in tissue block is transplanted under the subcutaneous and kidney envelope of mouse compares schematic diagram, it can
See that glioma cells in tissue block is transplanted to the tumour grown under kidney envelope and is significantly greater than the tumour for being transplanted to the subcutaneous growth of mouse.
The invention solves the problems, such as tumour growth efficiency in xenograft mouse model, but this field also need it is more kinds of
The osteosarcoma xenograft mouse model and its construction method of class.
Summary of the invention
The present invention provides a kind of construction method of osteosarcoma xenograft mouse model, and the method includes first acquiring bone and flesh
Tumor specimens block, then it is aseptically that fresh specimens block xenogenesis orthotopic transplantation is small into immune deficiency
In mouse body, the xenogenesis orthotopic transplantation includes the immunodeficient mouse for selecting the age at 2~6 weeks, mouse gender and Gender
It is consistent, collected fresh specimens block is immersed in sterile Incubating Solution, it is fixed after mouse anesthesia, it will suffer from
Person tumor tissue is implanted into the ossis of mouse, sutures the operative incision on mouse skin, brings up mouse and makes its ossis
Middle tumour growth.
In a kind of specific embodiment, the Patients with Osteosarcoma tumor tissue of acquisition is stored in sterile saline
In, it is placed on ice, is first Patients with Osteosarcoma tumor tissue through definitive pathological diagnosis, then xenogenesis orthotopic transplantation is small into immune deficiency
In mouse body.
In a kind of specific embodiment, the immunodeficient mouse is NOD/SCID mouse.
In a kind of specific embodiment, the unilateral length of the Patients with Osteosarcoma tumor tissue is less than or equal to 3mm,
Preferably smaller than it is equal to 1mm.
Specifically, the unilateral length of tissue block is less than 3mm, i.e. tissue block is less than or equal to 3mm × 3mm × 3mm tumour
Tissue, after tissue in situ is implanted into immunodeficient mouse, the length on every side it is excessive will nutrition supply it is insufficient, in tumor tissues
The heart may necrosis.The tissue shear of the orthotopic transplantation obtains the smaller the better, but cannot centainly be homogenized to it, in order to avoid destroy tissue and periphery
The connection of environment.
In a kind of specific embodiment, the weight of mouse is 8~20g, preferably 12~15g, institute when orthotopic transplantation
Stating includes antibiotic and fetal calf serum in sterile Incubating Solution, includes antibiotic and DMEM culture medium in the preferably described sterile Incubating Solution
Or RPMI-1640 culture medium, the more preferable antibiotic include penicillin and streptomysin.
In a kind of specific embodiment, by the specific steps packet in the ossis of specimens block implantation mouse
It includes: first making skin incision at the femur or shin bone of mouse, then drilled at mouse tibia with dental burr, the shin bone quilt of tapping
Brill rubs into bits, and specimens block is simultaneously implanted into ossis after exposing marrow by aperture, then sutures the operation on mouse skin
Notch.
In a kind of specific embodiment, mouse is brought up after its skin of shin swelling, the method also includes using
Whether small animal living body imager observes mouse tibia sarcoma size, preferably also turned using small animal living body imager observation tumour
Move on to the internal organ of mouse.
In a kind of specific embodiment, the method also includes bringing up mouse to make in its ossis after tumour growth
Mouse is dissected, removing knurl obtains P0 for tumour knurl, takes P0 for tumour knurl xenogenesis orthotopic transplantation into immunodeficient mouse body
P1 is inside obtained for tumour knurl, optionally takes P1 for tumour knurl xenogenesis orthotopic transplantation into obtaining P2 generation in immunodeficient mouse body
Tumour knurl, and so on passage, preferably the method also includes part P0 cools down for tumour knurl program and is stored in liquid
In nitrogen, and optionally includes and part P1 and P2 cools down for tumour knurl program and is stored in liquid nitrogen.
The present invention also provides a kind of construction methods of osteosarcoma xenograft mouse model, and the method includes in sterile item
Under part by the P0 in fresh mouse model for osteosarcoma tumor tissue, P1 for osteosarcoma tumor tissue or P2 for osteosarcoma tumor
Organize the xenogenesis orthotopic transplantation into immunodeficient mouse body, the xenogenesis orthotopic transplantation includes that the age is selected to be immunized at 2~6 weeks
Deficient mice, mouse gender are consistent with Gender, and the osteosarcoma tumor tissue block is immersed in sterile Incubating Solution,
It is fixed after mouse anesthesia, the osteosarcoma tumor tissue block is implanted into Mouse Bone pulp cavity, the operation sutured on mouse skin is cut
Mouthful, it brings up mouse and makes tumour growth in its ossis.
The present invention also provides a kind of mouse models that method as described above obtains in screening prevention or treatment bone and flesh tumor medicine
In application.
The present invention also provides a kind of construction methods of osteosarcoma xenograft mouse model, and the method includes the guarantors that first recovers
There are the Patients with Osteosarcoma tumor tissues in liquid nitrogen environment, then aseptically by the specimens block xenogenesis of recovery
Into in immunodeficient mouse body, the xenogenesis orthotopic transplantation includes selecting the age small in 2~6 weeks immune deficiencies for orthotopic transplantation
Mouse, mouse gender are consistent with Gender, and the specimens block of recovery is immersed in sterile Incubating Solution, mouse fiber crops
It is fixed after liquor-saturated, by the ossis of specimens block implantation mouse, the operative incision on mouse skin is sutured, mouse is brought up
So that tumour growth in its ossis.
In a kind of specific embodiment, the method also includes to collected Patients with Osteosarcoma tumor tissue into
Line program cooling and the process of Liquid nitrogen storage and the process for the recovery that is rapidly heated.
In a kind of specific embodiment, the Patients with Osteosarcoma tumor tissue is stored in containing DMSO, FBS and NEAA
DMEM in, three sections of procedure above are cooled to liquid nitrogen temperature, and save in liquid nitrogen temperature, the recovery that is rapidly heated refer to by
Cryopreserved tissue, which is put into 36~38 DEG C of water bath with thermostatic control, recovers.
The present invention also provides a kind of construction methods of osteosarcoma xenograft mouse model, and the method includes in sterile item
Under part by the P0 in the mouse model for freezing in liquid nitrogen environment and recovering for osteosarcoma tumor tissue, P1 for osteosarcoma tumor group
Knit or P2 for osteosarcoma tumor tissue xenogenesis orthotopic transplantation into immunodeficient mouse body, the xenogenesis orthotopic transplantation includes selecting
Immunodeficient mouse of the age at 2~6 weeks, mouse gender are consistent with Gender, by the osteosarcoma tumor tissue block
It is immersed in sterile Incubating Solution, it is fixed after mouse anesthesia, the osteosarcoma tumor tissue block is implanted into Mouse Bone pulp cavity, suture
Operative incision on mouse skin brings up mouse and makes tumour growth in its ossis.
The present invention at least has the following beneficial effects:
Application for a patent for invention CN201410083459 is mainly after transplanting from the point of view of tumour growth efficiency, in fact
It is modeled under either subcutaneous modeling or kidney envelope, all simulation tumour environment cannot occur very well.Osteosarcoma derives from bone
Pulp cavity, the present invention have been successfully established tissue block orthotopic transplantation mode, have preferably remained the stable relation of tumour Yu periphery interstitial,
Specifically ossis models in situ, remains the consistency of Model Tumor growing environment and patient tumors.It is former compared to subcutaneous modeling
Position modeling situation is truer, and the pharmaceutical admixtures screened can be better utilized in patient, and in Modeling Theory in situ for
Than subcutaneously modeling can be easier that metastases occur.Generally speaking, the foundation of the osteosarcoma model in situ is expected to improve with application
The validity of clinical clinical treatment of osteosarcoma and the prognosis of patient prevent patient from disabling even dead and bringing greatly medical treatment society negative
Load reduces medical expense and government finance expenditure.
Detailed description of the invention
The A and B of Fig. 1 is the osteosarcoma photo of patient.
The C and D of Fig. 2 is subcutaneous modeling schematic diagram, wherein using balb/c nude mice.
The E and F of Fig. 3 is shin bone ossis modeling schematic diagram in situ, wherein using NOD/SCID mouse.
Fig. 4 is that Micro-CT detects mouse original position tumor formation situation.
Fig. 5 is patient tumors pathology (100X, 400X) picture.
Fig. 6 is P0 after orthotopic transplantation for mouse tumor pathology (100X, 400X) picture.
Specific embodiment
It audits and ratifies through Hospital Ethical Committee, patient signs informed consent form, collected tissue before sample collection
By histopathological examination, the Patients with Osteosarcoma tumor tissue through definitive pathological diagnosis is stored in sterile saline, is placed in
On ice.Tissue block is divided into 3 parts, (as detailed below) heterograft is directly handled under part 1 aseptic condition into immune deficiency
In Mice Body；10%DMSO (dimethyl sulfoxide) is added in part 2 merging, and 89%FBS (fetal calf serum) and 1%NEAA (non-must
Need amino acid) DMEM in, DMEM culture medium is a kind of culture medium containing various amino acid and glucose, gradient freezing to -80
DEG C, it is placed in liquid nitrogen and saves；Third portion be put into it is quick-frozen in liquid nitrogen, then be placed in liquid nitrogen in save.Wherein, part 1 is for building
Mould, for second part for recovering and being inoculated with, Part III does genetic test for inspection.
Comparative example and embodiment
Comparative example is mouse hypodermic inoculation, and embodiment is the implantation of mouse original position marrow.
It inoculates: selecting 4 week old NOD/SCID mouse (or balb/c nude mice) 10, NOD/SCID mouse, that is, non-obese
Diabetes/severe combined immunodeficiency mouse, mouse gender and Gender are consistent (in order to avoid by some unknown hormones
Interference), weight about 12-15g, by fresh tumor tissue (or Patients with Osteosarcoma tumor tissues after liquid nitrogen cryopreservation and recovery) leaching
Bubble (the final concentration of 100U/ml of penicillin, final concentration of streptomysin in the sterile Incubating Solution of pure FBS+ penicillin and streptomysin
For 100U/ml), 1*1*1mm fritter is cut into sterile scissors.It is fixed after mouse anesthesia, make skin incision at the nearly oxter in right side,
Tumor tissue is implanted into subcutaneous (P0), suture operation notch.Observation mice tumors grew situation daily.Tumour growth is to length
When more than 5mm.With 2 knurls of vernier caliper measurement and knurl long (a) and wide (b) is recorded weekly, calculates gross tumor volume (V=a*
b2/2).The internal organ of mouse whether are transferred to using small animal living body imager observation tumour simultaneously, are risen to tumor volume V
1500mm3When or 42d after dissect animal, removing knurl weighing, part tumor mass inoculation passage saves in remaining merging liquid nitrogen.
Marrow implantation in situ: 4 week old NOD/SCID mouse 10 are selected, mouse gender is consistent with Gender, body
12-15g is weighed about, fresh tumor tissue (or Patients with Osteosarcoma tumor tissues after liquid nitrogen cryopreservation and recovery) is immersed in pure
In the sterile Incubating Solution of FBS+ penicillin and streptomysin (the final concentration of 100U/ml of penicillin, streptomysin it is final concentration of
100U/ml), 1*1*1mm fritter is cut into sterile scissors, is fixed after mouse anesthesia, makees skin at right side femur (or shin bone)
Skin notch separates shin bone, is drilled with dental burr, and specimens block is implanted into the hand in ossis (P0), on skin suture
Art notch.Gelatine closing can also can be used without processing at circular hole at the mouse tibia after bits using dental burr drilling.Often
Its observation mice tumors grew situation.It is big using small animal living body imager observation shin bone sarcoma every 3 weeks to skin of shin swelling
Small and visceral metastases (internal organ whether tumour is transferred to mouse) situation, rises to 1500mm to tumor volume3When or 42d after solve
Animal is cutd open, removing knurl weighing, the inoculation passage of part tumor mass, remaining is placed in liquid nitrogen and saves.Tissue block or whole is saved in liquid nitrogen
The process of a knurl includes program cooling, specifically 4 DEG C, -20 DEG C, -80 DEG C (dedicated deep freezers) and liquid nitrogen these temperature
Gradient.
The passage of osteosarcoma xenograft mouse tumor tissues: take the P0 to grow fine small for heterograft inoculation tumor formation
Mouse rises to 1500mm to tumor volume3When or when 42d, put to death mouse, completely strip tumour, be inoculated in 10 as described above
NOD/SCID right side of mice oxter, femur (or shin bone) (P1) observe mice tumors grew situation daily, ditto use vernier caliper
Knurl length and width is measured, tumor volume, small animal living body imager observation visceral metastases situation is calculated, dissects animal after 42d, shell
It weighs from knurl, part tumor mass and so on passes on (P2, P3 etc.), saves in remaining merging liquid nitrogen.
Osteosarcoma is primary and the recovery of passage of tumor tissue is inoculated with: taking P0, P1 and P2 for freezing in liquid nitrogen for osteosarcoma tumor
Tissue, rapid rewarming to room temperature are inoculated in 10 in gender NOD/SCID mouse oxter and shin bone respectively, the same institute of method
It states.
Because the osteosarcoma tissue of patient is very precious research material, it is therefore desirable to be carried out to the tumor tissues of tumor-bearing mice
Passage, to generate more experiment tumor-bearing mices.And carrying out cryopreservation resuscitation to tumor tissues is then because experiment is all every time
Have more than is needed so big tumor mass, and the tumor mass for the i.e. cryopreservation resuscitation reserved seed for planting can be used when next time tests.Tumor group
Passage P0, P1 and P2 etc. are knitted, theoretically the tumor tissues will not change, thus the experiment effect in each generation can be considered identical.
This seminar has collected Patients with Osteosarcoma nearly 40, establishes PDX model, tumor formation case by situ and subcutaneous tumor formation
8.In this 8 tumor-bearing mices, have plenty of individually subcutaneous tumor formation success, has plenty of individually tumor formation success in situ, there are also a lotuses
The then all tumor formations in situ and subcutaneous of tumor mouse.And in the successful mouse of tumor formation in situ, has plenty of and select fresh Patients with Osteosarcoma swollen
Tumor tissue block has plenty of the Patients with Osteosarcoma tumor tissue selected through liquid nitrogen cryopreservation and recovery.In the present invention, if P0 generation at
Tumor success, then the passage such as subsequent P1 and P2 and recovery are very simply and readily, almost all to succeed.
FIG. 1 to FIG. 4 is modeling schematic diagram subcutaneous and in situ.Wherein, the A and B of Fig. 1 is the osteosarcoma photo of patient, Fig. 2's
C and D is that subcutaneously modeling schematic diagram, the E and F of Fig. 3 are shin bone ossis modeling schematic diagram in situ, and Fig. 4 detects small for Micro-CT
Mouse original position tumor formation situation.The mouse soft mass of tumor formation in situ is with destruction of bone as seen from Figure 4.Fig. 5 is patient tumors pathology
(100X, 400X) picture.Fig. 6 is P0 after orthotopic transplantation for mouse tumor pathology (100X, 400X) picture.Wherein, P0 is patient
Tumor tissues plant the tumour grown after mouse for the first time.
From photo shown in Fig. 1~6 as it can be seen that the original position in the present invention to mouse in ossis models successfully.In PDX
Modeling in situ is that the success of OPDX model modeling is of far-reaching significance, and modeling gained tumour and patient's primary tumo(u)r homogeney in situ are more preferable,
Due to modeling success can with further investigated osteosarcoma pathogenesis, the achievement based on OPDX research and development can patient's body obtain compared with
Good repetition, while can replace facing using xenograft mouse with limited osteosarcoma sample in massive amplification clinical patients body
Bed patient carries out various combination drug screening, to select most suitable drug, greatly improve validity and reduce drug pair work
With.
Therefore, OPDX model foundation and application can be expected to improve the validity of clinical clinical treatment of osteosarcoma and the prognosis of patient,
It prevents patient from disabling even dead and bringing greatly medical burden on society, reduces medical expense and government finance expenditure, these
Aspect is all of great immediate significance, and has great economic benefit.
The above content is combine specific preferred embodiment to the further description of the invention made, and it cannot be said that originally
The specific implementation of invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, not
Under the premise of being detached from present inventive concept, several simple deductions and replacement can also be made, all shall be regarded as belonging to guarantor of the invention
Protect range.
Claims (10)
1. a kind of construction method of osteosarcoma xenograft mouse model, which is characterized in that the method includes preservations of first recovering
Patients with Osteosarcoma tumor tissue in liquid nitrogen environment, then it is aseptically that the specimens block xenogenesis of recovery is former
Displacement is planted into immunodeficient mouse body, and the xenogenesis orthotopic transplantation includes the immunodeficient mouse for selecting the age at 2~6 weeks,
Mouse gender is consistent with Gender, the specimens block of recovery is immersed in sterile Incubating Solution, mouse anesthesia
After fix, by specimens block implantation mouse ossis in, suture mouse skin on operative incision, bring up mouse make
Obtain tumour growth in its ossis.
2. the method according to claim 1, wherein the method also includes swollen to collected Patients with Osteosarcoma
Tumor tissue block carries out program cooling and the process of Liquid nitrogen storage and the process for the recovery that is rapidly heated.
3. according to the method described in claim 2, it is characterized in that, the Patients with Osteosarcoma tumor tissue is stored in and contains
In the DMEM of DMSO, FBS and NEAA, three sections of procedure above are cooled to liquid nitrogen temperature, and save in liquid nitrogen temperature, described quick
Heating recovery, which refers to put cryopreserved tissue into 36~38 DEG C of water bath with thermostatic control, recovers.
4. the method according to claim 1, wherein the immunodeficient mouse be NOD/SCID mouse, it is described
The unilateral length of Patients with Osteosarcoma tumor tissue is less than or equal to 3mm, is preferably smaller than equal to 1mm.
5. the method according to claim 1, wherein the weight of mouse is 8~20g when orthotopic transplantation, preferably
12~15g, includes antibiotic and fetal calf serum in the sterile Incubating Solution, in the preferably described sterile Incubating Solution comprising antibiotic and
DMEM culture medium or RPMI-1640 culture medium, the more preferable antibiotic include penicillin and streptomysin.
6. the method according to claim 1, wherein by the ossis of specimens block implantation mouse
Specific steps include: first to make skin incision at the femur or shin bone of mouse, then drilled at mouse tibia with dental burr, aperture
The shin bone at place is drilled to rub into bits, and specimens block is simultaneously implanted into ossis after exposing marrow by aperture, then sutures mouse skin
Operative incision on skin.
7. the method according to claim 1, wherein bring up mouse after its skin of shin swelling, the method
Further include that mouse tibia sarcoma size is observed using small animal living body imager, is preferably also observed using small animal living body imager
Whether tumour is transferred to the internal organ of mouse.
8. the method according to claim 1, wherein making in its ossis the method also includes bringing up mouse
Dissect mouse after tumour growth, removing knurl obtains P0 for tumour knurl, takes P0 for tumour knurl xenogenesis orthotopic transplantation into immune
P1 is obtained in deficient mice body for tumour knurl, optionally takes P1 for tumour knurl xenogenesis orthotopic transplantation into immunodeficient mouse body
P2 is inside obtained for tumour knurl, and so on passage.
9. according to the method described in claim 8, it is characterized in that, the method also includes by part P0 for tumour knurl program
Cool down and be stored in liquid nitrogen, and optionally includes and part P1 and P2 cools down for tumour knurl program and is stored in liquid nitrogen
In.
10. a kind of construction method of osteosarcoma xenograft mouse model, which is characterized in that the method includes in aseptic condition
The lower P0 by the mouse model for freezing in liquid nitrogen environment and recovering is for osteosarcoma tumor tissue, P1 for osteosarcoma tumor tissue
Or P2 for osteosarcoma tumor tissue xenogenesis orthotopic transplantation into immunodeficient mouse body, the xenogenesis orthotopic transplantation includes selecting year
Immunodeficient mouse of the age at 2~6 weeks, mouse gender are consistent with Gender, and the osteosarcoma tumor tissue block is soaked
Bubble is fixed after mouse anesthesia in sterile Incubating Solution, by the ossis of osteosarcoma tumor tissue block implantation mouse, sutures
Operative incision on mouse skin brings up mouse and makes tumour growth in its ossis.
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