CN106754719A - A kind of method of inoculation liquid composition and xenograft tumor animal model of being originated using its structure malignant pleural effusion - Google Patents
A kind of method of inoculation liquid composition and xenograft tumor animal model of being originated using its structure malignant pleural effusion Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The application is related to a kind of inoculation liquid composition and the method for xenograft tumor animal model of being originated using its structure malignant pleural effusion.The inoculation liquid that the present invention is provided includes following components：Hank's balanced salt solutions, hyclone, basement membrane matrix glue, and ROCK protein inhibitors.The step of present invention builds the method for malignant pleural effusion source xenograft tumor animal model including the clinical malignant pleural effusion sample of collection, enriched tumor cell, using inoculation liquid suspension cell of the invention and is seeded to animal to form xenograft tumor.Modeling method of the invention is simple and easy to apply, tumor formation rate is high, therefore can be used for the various cancers with the malignant pleural effusion particularly drug screening of lung cancer and Mechanism Study.
Description
Technical field
The invention belongs to medical model field.More particularly it relates to the xenogenesis in malignant pleural effusion source is moved
Plant the structure of knurl animal model.
Background technology
In China, lung cancer has replaced liver cancer still to exist as the first Death Cause for Malignant Tumors, and morbidity and mortality
Continue rapid rising.The patients with lung cancer that the high mortality of lung cancer is primarily due to 60-70% has been advanced lung cancer when being found, and is lost
Having removed surgical engine cannot can effect a radical cure.
Animal model for tumour is the Important Platform for carrying out tumour related mechanism and drug screening research.At present, it is preclinical swollen
The method for carrying out subcutaneous vaccination more than knurl animal model using tumor cell line, it has the advantages that easily to set up and easily repeat.But it is swollen
Oncocyte system, departing from natural tumor microenvironment, makes its Genomic instability and can not summarize patient tumors because setting up in vitro
Heterogeneity.Therefore the animal model for tumour that the method is set up, it is impossible to reflect the complicated clinical symptoms of cancer patient completely, have
Significant limitations.In order to overcome these problems, researchers establish the xenograft tumours animal model in cancer patient source,
The operation tumor tissues that patient will be derived from are implanted into immunodeficient mouse body.The model is very good must to maintain tumour shifting
Multiple features such as the histological characteristic of plant, gene expression profile, SNP site and copy number variation, therefore can be applied to patient's
Personalised drug screening, biomarker identification, the evaluation of preclinical oncology medicine effect and biological mechanism research etc., are medicine
The prediction of clinical test results provides more accurate result.
At present, the method for building up of patients with lung cancer source Replanting model mice mainly uses the surgical tissue of patient to be implanted into
Immunodeficient mouse is subcutaneous.Although clinically many patients with lung cancer often merge the malignant pleural effusion of generation, other are pernicious
Advanced tumor can also occur together malignant pleural effusion, and for example the malignant tumour such as breast cancer, lymthoma is (referring to woods river great waves etc., pleura is lived
Inspection joint hydrothorax exfoliative cytology checks the application in malignant pleural effusion diagnosis,《Internal medicine Severe acute disease magazine》, 2001,7
(1)).However, due in malignant pleural effusion tumour cell not only containing dead or connect in negligible amounts, and tumour cell
Nearly dead tumour cell, the inflammatory factor that in addition hydrothorax is also produced containing immunocyte and other cells, immunocyte
And itself may killing tumor cell.Due to above-mentioned limitation, it is considered that, it is impossible to build heterograft with Malignant Pleural
Knurl animal model.Research direction currently for lung cancer animal models is mainly and how lays particular emphasis on by the tumor group that collects of performing the operation
Orthotopic transplantation is knitted to (Wang Liwei etc., Lewis lung carcinoma Research of Animal Model for Study is entered to build xenograft tumor animal model in animal
Exhibition,《Interventional radiology magazine》, 2015,24 (7)).
In sum, this area builds the side of xenograft tumor animal model in the urgent need to a kind of utilization malignant pleural effusion
Method.
The content of the invention
According to a first aspect of the present invention, there is provided a kind of malignant pleural effusion source xenograft tumor animal model, it is described
Animal model is built by following methods：
The clinical malignant pleural effusion sample of (a) collection；
B () is enriched with the tumour cell in the malignant pleural effusion sample；And
C () will be if the tumor cell inoculation obtained by step (b) enrichment is to experimental animal, letting animals feed to tumour is formed,
So as to obtain the xenograft tumor animal model in malignant pleural effusion source.
In one preferred embodiment, used in the step (b)FX systems carry out cell enrichment.
In one preferred embodiment, the tumour cell that the step (b) is obtained is carried out into H＆E dyeing, CK is immunized glimmering
Light is dyeed, to detect whether be enriched with cell is tumour cell.
In one preferred embodiment, the tumour cell that the step (b) is obtained is processed with aseptic inoculation liquid, described
Aseptic inoculation liquid is included：The Matrigel glue of the HBSS of 45 parts by volume, the hyclone of 5 parts by volume and 50 parts by volume, adds
Thiazovivin, final concentration of 10uM.
In another preferred embodiment, the experimental animal is severe combined immunodeficiency (SCID) mouse.
In another preferred embodiment, the malignant pleural effusion sample is selected from and contains following genotype：
EGFR20 exons T790M mutation, the exon L858R of EGFR 21 mutation, and/or ALK fusion gene.
According to a second aspect of the present invention, there is provided a kind of aseptic inoculation liquid composition, comprising following components：Hank ' s balance salt
Buffer solution and/or phosphate buffer PBS, hyclone, basement membrane matrix glue, and ROCK protein inhibitors.
In one preferred embodiment, the basement membrane matrix glue is selected from Matrigel glue or Cultrex BME glue；It is described
ROCK protein inhibitors be selected from Thiazovivin, Y-27632, Pinacidil (Pinacidil), Fasudil (Fasudil),
Ripasudil, RKI-1447, GSK429286A, or their any combination.
In another preferred embodiment, the Hank ' s balanced salt solutions (being purchased from Invitrogen, 14170) are
35-55 parts by volume, hyclone is 3-10 parts by volume, and basement membrane matrix glue is 40-60 parts by volume, and ROCK protein inhibitors
Final concentration of 5-20uM.
According to a third aspect of the present invention, there is provided a kind of side for building malignant pleural effusion source xenograft tumor animal model
Method, comprises the following steps：
The clinical malignant pleural effusion sample of (a) collection；
B () is enriched with the tumour cell in the malignant pleural effusion sample；
C () uses the tumour cell being enriched with aseptic inoculation liquid composition resuspending step (b) of the invention；And
D () will be suspended in tumor cell inoculation in aseptic inoculation liquid composition to experimental animal as described in step (c), raise
Support animal to tumour to be formed, so as to obtain the xenograft tumor animal model in malignant pleural effusion source.
In one preferred embodiment, used in the step (b)FX systems carry out cell enrichment.
In another preferred embodiment, the experimental animal is severe combined immunodeficiency (SCID) mouse.
In another preferred embodiment, the malignant pleural effusion sample from lung cancer, breast cancer, lymthoma or
Oophoroma.
According to a fourth aspect of the present invention, there is provided the animal model that a kind of the method according to the invention builds is in clinical or scientific research
Application in research.
In one preferred embodiment, the application is selected from drug screening research, pharmacodynamic study, pharmaceutical research, trouble
Person's response Journal of Sex Research, gene specific Journal of Sex Research, biomarker research, and/or biological mechanism research.
In another preferred embodiment, the animal model is applied to selected from following genotype：EGFR20 extras
Aobvious sub- T790M mutation, the exon L858R of EGFR 21 mutation, and/or ALK fusion gene.
The technical scheme that the present invention is provided can be modeled using malignant pleural effusion, because malignant pleural effusion gathers convenient,
Simultaneously small on minimal invasive treatment's influence the features such as, therefore can repeatedly model, improve success rate.The malignant pleural effusion that the present invention is provided
Modeling technique scheme can be directed to lose surgical engine can Patients with Advanced Lung Cancer Malignant Pleural be modeled so that can clinical and
There is very big actual application value in scientific research.
The present invention set up xenograft tumor animal model method farthest remain tumour cell biology live
Property, verily reduces the correlated characteristic of patient tumors cell, and whole model sets up that process is simple and easy to apply, and tumor formation rate is up to
40%, therefore can be used for the various cancers with the malignant pleural effusion particularly drug screening of lung cancer and Mechanism Study.
The present invention is further elaborated on below with reference to the drawings and specific embodiments, but these elaborations are only to be
More fully understand those skilled in the art and implement the present invention, any type of limitation is not done to the present invention.Unless another
It is described, all scientific and technical terms otherwise used herein have the general technology with correlative technology field belonging to the present invention
The implication that personnel are generally understood that.
Brief description of the drawings
The CK immunofluorescence dyeings of the tumour cell of Fig. 1 display enrichments.
Fig. 2 display enrichment tumor cell transplantation enter mouse it is subcutaneous after tumor growth curve.
Fig. 3 shows the hematoxylin eosin stain of the tumour cell being enriched with from malignant pleural effusion clinical sample.
Gram Zhuo of Fig. 4 display ALK fusion gene MODEL Cs TC15012 replaces Buddhist nun (Crizotinib) effect experiment.
Gefitinib (Gefitnib) drug effect reality of Fig. 5 display EGFR 21 exon L858R catastrophic models CTC15008
Test.
Erlotinib (Erlotinib) drug effect of Fig. 6 display EGFR 20 exon T790M catastrophic models CTC15015
Experiment.
The AZD9291 effect experiments of Fig. 7 display EGFR 20 exon T790M catastrophic models CTC15015.
Specific embodiment
In the present context, term " inoculation liquid composition ", " inoculation liquid ", " aseptic inoculation liquid ", " aseptic inoculation liquid
Composition " is used interchangeably.
In the application context term " including " and "comprising" can be used interchangeably, and cover simultaneously " by ...
Constitute " this statement.
In the present invention, if not otherwise specified, all technical characteristics mentioned in this article and preferred feature
Can be mutually combined to form new technical scheme.
In the present invention, if not otherwise specified, all implementation methods mentioned in this article and preferred embodiment
Can be mutually combined to form new technical scheme.
Xenograft tumor animal model refers to that tumor tissues or cell are transplanted into experimental animal such as mouse, rat etc.
The animal model for tumour for being formed in vivo.Transplantability model due to experiment condition is easily controllable, experimental period is shorter and
Can it is of the same race or with strain animal in continuous transplanting the advantages of and be widely used in cancer research.
The closure lacuna that thoracic cavity is made up of partial pleura and visceral pleura, is negative pressure, normal condition in it
There is the minimal amount of liquid of lubrication between lower two-layer pleura, to reduce during respiratory activity between two-layer pleura
Friction.Pleura is had influence on when there is certain pathologic condition, either partial pleura produces hydrothorax or visceral pleura to absorb chest
The speed of water is changed, and chest intracavity liquid can all increased, and forms so-called pleural effusion (referring to Chinese patent application
CN102676456A)。
The present inventor is targetedly enriched with by by the tumour cell in malignant pleural effusion, so as to obtain pernicious chest
Chamber hydrops source xenograft tumor animal model.The simple method by being enriched with, modeling method tumor formation rate is generally very low, general low
In 10%.
The present inventor has been unexpectedly discovered that, using inoculation liquid of the present invention, can significantly improve and be accumulated with malignant pleural
The cancer cell in liquid source builds the tumor formation rate of xenograft tumor animal model.
It is without being bound by any theory, inventors have surprisingly discovered that, using the method for the present invention, especially with
Inoculation liquid of the present invention, preferred compositions are usedFX systems carry out cell enrichment, can very significantly improve
The cancer cell originated with malignant pleural effusion builds the tumor formation rate of xenograft tumor animal model.
First aspect present invention provides a kind of malignant pleural effusion source xenograft tumor animal model, the animal mould
Type is built by following methods：
The clinical malignant pleural effusion sample of (a) collection；
B () is enriched with the tumour cell in the malignant pleural effusion sample；And
C () will be if the tumor cell inoculation obtained by step (b) enrichment is to experimental animal, letting animals feed to tumour is formed,
So as to obtain the xenograft tumor animal model in malignant pleural effusion source.
In one preferred embodiment, used in the step (b)FX systems carry out cell enrichment.
In one preferred embodiment, the tumour cell that the step (b) is obtained is carried out into H＆E dyeing, CK is immunized glimmering
Light is dyeed, to detect whether be enriched with cell is tumour cell.
In one preferred embodiment, the tumour cell that the step (b) is obtained is processed with aseptic inoculation liquid, described
Aseptic inoculation liquid is included：The Matrigel glue of the HBSS of 45 parts by volume, the hyclone of 5 parts by volume and 50 parts by volume, adds
Thiazovivin, final concentration of 10uM.
In another preferred embodiment, the experimental animal is severe combined immunodeficiency (SCID) mouse.
In another preferred embodiment, the malignant pleural effusion sample from lung cancer, breast cancer, lymthoma or
Oophoroma.
Second aspect present invention provides a kind of inoculation liquid composition, and said composition includes following components：Hank ' s balance salt
Buffer solution, hyclone, basement membrane matrix glue, and ROCK protein inhibitors.
In a kind of preferred embodiment, the present invention provides a kind of inoculation liquid composition, and the inoculation liquid composition is by with the following group
It is grouped into：Hank ' s balanced salt solutions, hyclone, basement membrane matrix glue, and ROCK protein inhibitors.
Hank ' s balanced salt solutions (Hank ' s Balanced Salt Solution, HBSS) are for cell culture
Conventional buffer solution, can be bought by commercial sources, be for example purchased from Sigma-Aldrich, ThermoFisher
The companies such as Scientific.Equally, the hyclone for being used in the present invention can also be bought by commercial sources, for example, be purchased from
The companies such as Sigma-Aldrich.
Basement membrane matrix glue (Basement Membrane Matrix), also referred to as basilar memebrane extracellular matrix, basilar memebrane
Extract, EHS matrix etc., are a kind of soluble basement membrane preparations, extract from Engelbreth-Holm-Swarm (EHS)
Murine sarcoma, rich in extracellular matrix (ECM) albumen such as laminin, collagen iv, nestin and various cell factors
(https://www.corning.com/worldwide/en/products/life-sciences/products/
surfaces/matrigel-matrix.html).Due to containing cells in vitro it is adherent, growth and differentiation needed for various chemistry
Material, basement membrane matrix glue has been widely used for 2D and 3D cell injuring models.At present, in the market provides basement membrane matrix glue
Mainly there are Sigma-Aldrich companies (Matrigel, such as article number E1270), Corning companies (trade name in company), Trevigen companies (trade nameBME).These basement membrane matrix glue can be suitably used for this hair
It is bright, for example preferably useOrBME。
ROCK albumen (Rho related protein kinases) is a kind of protein kinase, belongs to the AGC family of serine-threonine kinase
Race.ROCK albumen is mainly by acting on cytoskeleton come the shape of regulating cell and movement.ROCK protein inhibitors refer to target
To a series of compounds of ROCK albumen, the ROCK protein inhibitors such as Thiazovivin being currently known, Y-27632, pyrrole that
Ground your (Pinacidil), Fasudil (Fasudil), Ripasudil, RKI-1447, GSK429286A etc..The present invention can make
With any suitable ROCK protein inhibitors, such as including but not limited to Thiazovivin, Y-27632, Pinacidil
(Pinacidil), Fasudil (Fasudil), Ripasudil, RKI-1447, GSK429286A, or their any combination.
It is without wishing to be bound by any theory, it is believed by the inventor herein that ROCK protein inhibitors apoptosis capable of inhibiting cell, it is thus possible to improve thin
The survival of born of the same parents, increases the success rate of modeling.
In one preferred embodiment, the Hank ' s balanced salt solutions are 35-55 parts by volume, and hyclone is 3-10
Parts by volume, basement membrane matrix glue be 40-60 parts by volume, and ROCK protein inhibitors final concentration of 5-20uM.It is furthermore preferred that
Hank ' s balanced salt solutions are 40-50 parts by volume, and hyclone is 4-8 parts by volume, and basement membrane matrix glue is 45-55 volumes
Part, and ROCK protein inhibitors final concentration of 8-15uM.Most preferably, Hank ' s balanced salt solutions are 45 parts by volume,
Hyclone be 5 parts by volume, basement membrane matrix glue be 50 parts by volume, and ROCK protein inhibitors final concentration of 10uM.
Therefore, in a preferred embodiment, aseptic inoculation liquid of the invention is composed of the following components：Hank ' s balance salt
Buffer solution, hyclone, Matrigel glue, and Thiazovivin.For example, aseptic inoculation liquid of the invention is by following components group
Into：Hank ' s the balanced salt solutions of 35-55 parts by volume；The hyclone of 3-10 parts by volume；The Matrigel of 40-60 parts by volume
Glue；And the Thiazovivin of final concentration of 5-20uM.Preferably, aseptic inoculation liquid of the invention is composed of the following components：
Hank ' s the balanced salt solutions of 40-50 parts by volume；The hyclone of 4-8 parts by volume；The Matrigel glue of 45-55 parts by volume；
And the Thiazovivin of final concentration of 8-15uM.Most preferably, aseptic inoculation liquid of the invention is composed of the following components：45
Hank ' s the balanced salt solutions of parts by volume；The hyclone of 5 parts by volume；The Matrigel glue of 50 parts by volume；And final concentration
It is the Thiazovivin of 10uM.
The present invention also provides a kind of method for building malignant pleural effusion source xenograft tumor animal model, the method bag
Include following steps：
The clinical malignant pleural effusion sample of (a) collection；
B () is enriched with the tumour cell in the malignant pleural effusion sample；
C () uses the tumour cell being enriched with aseptic inoculation liquid composition resuspending step (b) of the invention；And
D () will be suspended in tumor cell inoculation in aseptic inoculation liquid composition to experimental animal as described in step (c), raise
Support animal to tumour to be formed, so as to obtain the xenograft tumor animal model in malignant pleural effusion source.
Preferably, used in step (b)FX systems (Clearbridge BioMedics companies) are carried out carefully
Born of the same parents are enriched with.FX systems use micro-fluidic sorting technology, and it is based on ClearCell CTChip FR1 chip drives,
Cell need not be marked, you can reach the concentration effect of tumour cell, be that first, the whole world is entered using full-automatic cell searching system
The beneficiation technologies of row tumour cell living.Compared with circulating tumor cell enrichment system conventional in the market,
FR systems have three advantages：The degree of accuracy, susceptibility are higher, and without mark；The circulating tumor cell purity being enriched to is higher；
Tumour cell living is enriched with, tumour cell bioactivity higher is maintained.Present invention preferably usesFX systems are entered
Row cell enrichment, but the separation of cancer cell and enrichment also can be used the technologies such as traditional centrifugation or this area in the present invention
Other any suitable methods of separation and concentration cancer cell from hydrothorax for knowing are (for example, with reference to Chinese patent application
CN102676456A)。
In one preferred embodiment, the tumour cell that the step (b) is obtained is carried out into H＆E dyeing, CK is immunized glimmering
Light is dyeed, to detect whether be enriched with cell is tumour cell.
In another preferred embodiment, the experimental animal is mouse or rat, more preferably mouse, most preferably severe
Combined immunodeficiency (SCID) mouse.
In another preferred embodiment, the malignant pleural effusion sample from lung cancer, breast cancer, lymthoma or
Oophoroma.Malignant Pleural is mainly caused by the malignant tumour of thoracic organ, is especially common in advanced lung cancer (such as non-small cell lung
Cancer), but also see the cancers such as breast cancer, lymthoma, oophoroma.
Preferably, the step of also including whether identify be enriched with cell is tumour cell after step (b), for example, lead to
Cross H＆E dyeing, cytokeratin (Cytokeratin, CK) immunofluorescence dyeing and detect whether be enriched with cell is thin tumour
Born of the same parents.In another implementation method, usingBefore FX systems carry out cell enrichment, PBS cell is used, plus
Enter erythrocyte cracked liquid cracking, and use PBS re-suspended cells.
Fourth aspect present invention provides a kind of animal model of the method according to the invention structure in clinical or scientific research
In application.The animal model that the method according to the invention builds can be applied in various clinical or scientific research, including but not
It is limited to drug screening research, pharmacodynamic study, pharmaceutical research, patient's response Journal of Sex Research, gene specific Journal of Sex Research, biological marker
Thing is studied, and/or biological mechanism research.
Further, since coming across patient with advanced cancer malignant pleural effusion, the hydrops of many patients comes across treats resistance to more
After medicine, therefore it is generally more special gene mutation model, such as EGFR20 exons by the model that this class sample is set up
T790M is mutated and ALK fusion gene resistance.Therefore, in a preferred embodiment, the animal that the method according to the invention builds
Model is applied to selected from following genotype：EGFR20 exons T790M is mutated, the exon L858R of EGFR 21 are mutated,
And/or ALK fusion gene.
Present invention can be suitably applied to TCA especially lung cancer (such as non-small cell lung cancer) occur together malignant pleural effusion
Patient, collecting the malignant pleural effusion of such patient carries out the foundation of allograft mouse model.Because being accumulated by malignant pleural
Liquid modeling success rate it is very low and unstable, so malignant pleural effusion tumour cell carried out using microflow control technique first it is high-purity
Degree enrichment, while the tumour cell of the enrichment that suspended with aseptic inoculation liquid of the invention, at utmost remains tumour cell biological
Activity is learned, severe combined immunodeficiency mouse subcutaneous growth is then seeded to, the correlation of patient tumors cell is verily reduced
Feature, whole model sets up that process is simple and easy to apply, and tumor formation rate is up to 40%, and the mechanism that can be used for lung-cancer medicament screening and lung cancer is ground
Study carefully.The patients with lung cancer malignant pleural effusion source xenograft tumor models set up by the present invention have manufacturing process simple to operate
Convenient, tool repeatability, modeling success rate is high, it is easy to the advantages of promoting, it is adaptable to drug screening and experimental study, and collects chest
The closed drainage of thoracic cavity operation or thoracocentesis implemented during liquid are small to patient effect.
The present invention is expanded on further below in conjunction with specific embodiment.In the examples below, unless otherwise instructed, it is used
Experiment material and reagent can be bought in routine biochemistry Reagent Company.Experimental technique used, unless otherwise specified, is often
Rule method.In addition, these embodiments are merely to illustrate the present invention and limit the scope of the present invention never in any form.It is of the invention
Scope is limited only by the accompanying claims.Those skilled in the art can not depart from after present disclosure has been read
Various changes and change are made in the case of spirit and scope of the invention to the present invention, these are changed and change is all considered as this
The equivalents of invention embodiment and fall within the scope of the present invention.
Embodiment
The structure of the xenograft tumor mouse model in the lung cancer malignant pleural effusion of embodiment 1 source
1.Experimental animal
The female SCID combined immunodeficiency mouse of 4-5 week old is ordered from supplier, is raised in SPF rank Animal Houses, it is real
Animal at least adaptability is raised three days before testing beginning.
2.Experimentation
The collection of 2.1 lung cancer clinical malignant pleural effusion samples
Each 200ml of non-small cell lung cancer malignant pleural effusion sample of fresh collection is collected in the test tube containing anti-coagulants,
Transport on ice, laboratory was transported in 24 hours.15 malignant pleural effusions are collected altogether.
The enrichment of tumour cell in 2.2 malignant pleural effusions
The PBS (HyClone companies, SH30256.01) accumulated with monoploid in Biohazard Safety Equipment dilutes pernicious chest
Chamber hydrops sample, 500g is centrifuged 10 minutes, removes supernatant, and PBS is resuspended, and repeated centrifugation is cleaned 2 times.
After PBS is resuspended, 70um strainer filterings, 500g is used to be centrifuged 10 minutes.Supernatant is removed, erythrocyte cracked liquid, cracking is added
3-5 minutes, plus the 2-3 times of PBS neutralization of volume, 500g centrifugations 10 minutes.Supernatant is removed, adds appropriate PBS resuspended.On cell suspension
Into tumor cell enrichment instrument ClearCell FX systems (Clearbridge BioMedics companies), tumour cell is carried out
Enrichment.
The cell of enrichment is taken into 30000 cells carries out paster, and H＆E dyeing, CK immunofluorescence dyeings determine what is be enriched with
Cell is tumour cell (result is shown in accompanying drawing 1), and is confirmed through professional pathologists.
2.3 prepare aseptic inoculation liquid
Mix the HBSS buffer solutions of 45 parts by volume, the Matrigel of the hyclone of 5 parts by volume and 50 parts by volume (SIGMA,
E1270-10ml) glue, and add ROCK protein inhibitors Thiazovivin (Selleck, S1459) to final concentration of 10uM.Will
The above-mentioned inoculation liquid sterilizing for preparing.
The foundation of 2.4 patients with lung cancer malignant pleural effusions source Replanting model mice
The tumour cell of enrichment is divided into two parts, portion is not added with aseptic inoculation liquid, portion is suspended in the aseptic of above-mentioned preparation
In inoculation liquid, subcutaneous transplantation to severe combined immunodeficiency right side of mice back is distinguished with syringe.After cell transplantation, see daily
Examine mouse health status, periodic detection tumour growth situation measures weekly the length and width of tumour with electronic vernier caliper, swells
Knurl volume computing formula：(* long is wide for TV=2)/2.Referring to accompanying drawing 2, (figure is that the growth of CTC15012 is bent to tumour growth situation
Line).
The pathological analysis of 2.5 tumor tissues
Routine pathology is carried out to the tumor tissues for being formed with hematoxylin eosin stain and detects (result is shown in accompanying drawing 3).
3.Experimental result
It is modeled by 15 malignant pleural effusions collected, is not added with aseptic inoculation liquid group, only into knurl 2, one is
ALK fusion gene lung cancer model, another example is the exon insertion mutation lung cancer models of EGFR 20, and tumor formation rate is 13.3%.And
Plus aseptic inoculation liquid group, 6 allograft mouse models are finally built up, wherein comprising two ALK fusion genes (wherein one life
Entitled CTC15012), an exon L858R of EGFR 21 is mutated (being named as CTC15008), and an extra of EGFR 20 shows
Sub- T790M mutation (being named as CTC15015), an exon insertion mutation of EGFR 20 and an EGFR wild type, into knurl
Rate reaches 40%.
The effect experiment that embodiment 2 is carried out on non-small cell lung cancer MODEL C TC15012
It is consistent with clinical response in order to verify the lung cancer catastrophic model that the present invention is set up, on the animal model set up
Carry out the corresponding effect experiment consistent with the medicine that clinical patient is taken.
The ALK fusion gene MODEL C TC15012 tumor mass in female NU/NU nude mouses subcutaneous vaccination people source.Non-small cell lung
Cancer MODEL C TC15012 is derived from 55 years old female patients, and the pathological diagnosis of the patient is broncho-pulmonary gland cancer.By tumour in
It is cut into HBSS and shreds into fritter and be inoculated into the subcutaneous of nude mice, treats that tumour is long to 500-700mm3When, tumour is cultivated in HBSS
The fritter of 10-15mg is cut into liquid is used for 20 experiment nude mice by subcutaneous inoculations.Specifically inoculation method is：Iodophor is to mouse back two
Side skin degerming, with No. 20 trochars by 2 × 2 × 2mm of total amount3Tumour be organized in soon back both sides piercing, syringe needle traveling in
Between skin and muscle, most tissue feeding forelimb shoulder back is subcutaneous at last, and compressing wound is to without bleeding.Routinely raised under SPF grades of environment
Support, treat that gross tumor volume is long to averagely about 100-300mm3When, 12 preferable tumor-bearing mices of tumour growth homogeneity are selected, according to
Tumor size and nude mice body weight are grouped administration at random, are grouped as follows：
First group：N=6, control group, gastric infusion, once a day；
Second group：N=6, gram Zhuo replace Buddhist nun, 25mg/kg, gastric infusion, once a day.
Every group of mouse is weighed be administered and observe mouse health status daily, and 2 mouse tumor volumes are investigated weekly.
Result as shown in figure 4, ALK fusion gene MODEL C TC15012 to ALK inhibitor gram Zhuo for Buddhist nun (Selleck,
S1068 it is) sensitive.
The effect experiment that embodiment 3 is carried out on non-small cell lung cancer MODEL C TC15008
The exon L858R catastrophic model CTC15008 tumours of EGFR 21 in female NU/NU nude mouses subcutaneous vaccination people source
Block.Non-small cell lung cancer MODEL C TC15008 is derived from 71 years old female patients, and the pathological diagnosis of the patient is broncho-pulmonary
Gland cancer.Tumour is cut into HBSS and is shredded into fritter and is inoculated into the subcutaneous of nude mice, treat that tumour is long to 500-700mm3When, will be swollen
The fritter that knurl is cut into 10-15mg in HBSS nutrient solutions is used for 20 experiment nude mice by subcutaneous inoculations.Specifically inoculation method is：Iodophor
To mouse back both sides skin degerming, with No. 20 trochars by 2 × 2 × 2mm of total amount3Tumour be organized in soon back both sides thorn
Enter, between skin and muscle, most tissue feeding forelimb shoulder back is subcutaneous at last, and compressing wound is to without bleeding for syringe needle traveling.SPF grades
Conventinal breeding under environment, treats that gross tumor volume is long to averagely about 100-300mm3When, select 12 tumour growth homogeneity preferable
Tumor-bearing mice, administration is grouped according to tumor size and nude mice body weight at random, is grouped as follows：
First group：N=6, control group, gastric infusion, once a day；
Second group：N=6, Gefitinib, 75mg/kg, gastric infusion, once a day.
Every group of mouse is weighed be administered and observe mouse health status daily, and 2 mouse tumor volumes are investigated weekly.
Result is as shown in figure 5, the exon L858R catastrophic models CTC15008 of EGFR 21 are lucky to EGFR inhibitor non-replaces
Buddhist nun (Selleck, S1025) is sensitive.
The effect experiment that embodiment 4 is carried out on non-small cell lung cancer MODEL C TC15015
The exon T790M catastrophic model CTC15015 tumours of EGFR 20 in female NU/NU nude mouses subcutaneous vaccination people source
Block.Non-small cell lung cancer MODEL C TC15015 is derived from 51 years old male patient, and the pathological diagnosis of the patient is broncho-pulmonary
Gland cancer, clinic is to Erlotinib resistance.Tumour is cut into HBSS and is shredded into fritter and is inoculated into the subcutaneous of nude mice, treat that tumour is long
To 500-700mm3When, the fritter that tumour is cut into 10-15mg in HBSS nutrient solutions is used for 30 experiment nude mice by subcutaneous inoculations.
Specifically inoculation method is：Iodophor to mouse back both sides skin degerming, with No. 20 trochars by 2 × 2 × 2mm of total amount3Tumour
The piercing of back both sides is organized in soon, and between skin and muscle, most tissue feeding forelimb shoulder back is subcutaneous at last, compressing for syringe needle traveling
Wound is to without bleeding.Conventinal breeding under SPF grades of environment, treats that gross tumor volume is long to averagely about 100-300mm3When, select 18 and swell
Knurl grows the preferable tumor-bearing mice of homogeneity, and administration is grouped at random according to tumor size and nude mice body weight, is grouped as follows：
First group：N=6, control group, gastric infusion, once a day；
Second group：N=6, Erlotinib, 50mg/kg, gastric infusion, once a day.
3rd group：N=6, AZD9291,5mg/kg, gastric infusion, once a day.
Every group of mouse is weighed be administered and observe mouse health status daily, and 2 mouse tumor volumes are investigated weekly.
Result is as shown in fig. 6, the exon T790M catastrophic models CTC15015 of EGFR 20 are replaced to EGFR inhibitor angstrom sieve
Buddhist nun (Selleck, S1023) is insensitive.Result is as shown in fig. 7, exon T790M catastrophic models CTC15015 pairs of EGFR 20
EGFR inhibitor AZD9291 (Selleck, S7297) is sensitive.
Claims (10)
1. a kind of aseptic inoculation liquid composition, comprising following components：Hank's balanced salt solutions/phosphate buffer PBS, tire ox
Serum, basement membrane matrix glue, and ROCK protein inhibitors.
2. aseptic inoculation liquid composition as claimed in claim 1, it is characterised in that wherein described basement membrane matrix glue is selected from
Matrigel glue or Cultrex BME glue；The ROCK protein inhibitors are selected from Thiazovivin, Y-27632, Pinacidil
(Pinacidil), Fasudil (Fasudil), Ripasudil, RKI-1447, GSK429286A, or their any combination.
3. aseptic inoculation liquid composition as claimed in claim 1, it is characterised in that the Hank's balanced salt solutions are
35-55 parts by volume, hyclone is 3-10 parts by volume, and basement membrane matrix glue is 40-60 parts by volume, and ROCK protein inhibitors
Final concentration of 5-20uM in the inoculation liquid composition.
4. a kind of method for building malignant pleural effusion source xenograft tumor animal model, comprises the following steps：
The clinical malignant pleural effusion sample of (a) collection；
B () is enriched with the tumour cell in the malignant pleural effusion sample；
C () is thin using the tumour being enriched with aseptic inoculation liquid composition resuspending step (b) as described in claim any one of 1-3
Born of the same parents；And
D () will be suspended in tumor cell inoculation in aseptic inoculation liquid composition to experimental animal as described in step (c), raise dynamic
Thing to tumour is formed, so as to obtain the xenograft tumor animal model in malignant pleural effusion source.
5. method as claimed in claim 4, it is characterised in that used in the step (b)FX systems are carried out carefully
Born of the same parents are enriched with.
6. method as claimed in claim 4, it is characterised in that the experimental animal is that severe combined immunodeficiency (SCID) is small
Mouse.
7. method as claimed in claim 4, it is characterised in that the malignant pleural effusion sample comes from lung cancer, breast cancer, pouring
Bar knurl or oophoroma.
8. application of the animal model according to any one of claim methods described structure in clinical or scientific research.
9. application as claimed in claim 8, it is characterised in that the application is selected from drug screening research, pharmacodynamic study, medicine
Pharmacological research, patient's response Journal of Sex Research, gene specific Journal of Sex Research, biomarker research, and/or biological mechanism research.
10. application as claimed in claim 8, it is characterised in that the animal model is applied to selected from following genotype：
EGFR20 exons T790M mutation, the exon L858R of EGFR 21 mutation, and/or ALK fusion gene.
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| CN108835051A (en) * | 2018-06-01 | 2018-11-20 | 郑州大学第附属医院 | Utilize immune deficiency nude mice model constructed by lymphoma cell strain |
| CN108866000A (en) * | 2018-07-16 | 2018-11-23 | 上海市肺科医院 | A kind of hEGF's tyrosine kinase inhibitor acquired resistance lung cancer cell line and its method for building up and application |
| CN112608999A (en) * | 2020-12-24 | 2021-04-06 | 深圳市罗湖区人民医院 | Method and reagent combination for predicting sensitivity to recombinant human endostatin |
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| CN108866000A (en) * | 2018-07-16 | 2018-11-23 | 上海市肺科医院 | A kind of hEGF's tyrosine kinase inhibitor acquired resistance lung cancer cell line and its method for building up and application |
| CN108866000B (en) * | 2018-07-16 | 2021-09-28 | 上海市肺科医院 | Human epidermal growth factor tyrosine kinase inhibitor acquired drug-resistant lung cancer cell line and establishment method and application thereof |
| CN112608999A (en) * | 2020-12-24 | 2021-04-06 | 深圳市罗湖区人民医院 | Method and reagent combination for predicting sensitivity to recombinant human endostatin |
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