CN107006429A - Stomach cancer Lung metastases mouse model and its method for building up - Google Patents
Stomach cancer Lung metastases mouse model and its method for building up Download PDFInfo
- Publication number
- CN107006429A CN107006429A CN201710155642.2A CN201710155642A CN107006429A CN 107006429 A CN107006429 A CN 107006429A CN 201710155642 A CN201710155642 A CN 201710155642A CN 107006429 A CN107006429 A CN 107006429A
- Authority
- CN
- China
- Prior art keywords
- stomach cancer
- lung
- mouse model
- metastases
- mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 55
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 51
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 51
- 206010027458 Metastases to lung Diseases 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000010172 mouse model Methods 0.000 title claims abstract description 19
- 206010027476 Metastases Diseases 0.000 claims abstract description 28
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 26
- 238000011579 SCID mouse model Methods 0.000 claims abstract description 16
- 230000001394 metastastic effect Effects 0.000 claims abstract description 16
- 206010061289 metastatic neoplasm Diseases 0.000 claims abstract description 16
- 230000003902 lesion Effects 0.000 claims abstract description 15
- 238000004043 dyeing Methods 0.000 claims abstract description 10
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 8
- 206010061598 Immunodeficiency Diseases 0.000 claims abstract description 7
- 208000029462 Immunodeficiency disease Diseases 0.000 claims abstract description 7
- 230000007813 immunodeficiency Effects 0.000 claims abstract description 7
- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 210000003462 vein Anatomy 0.000 claims abstract description 7
- 238000011156 evaluation Methods 0.000 claims abstract description 6
- 230000003833 cell viability Effects 0.000 claims abstract description 3
- 210000004072 lung Anatomy 0.000 claims description 36
- 238000011580 nude mouse model Methods 0.000 claims description 16
- 206010028980 Neoplasm Diseases 0.000 claims description 12
- 230000009401 metastasis Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 239000012188 paraffin wax Substances 0.000 claims description 5
- 238000009826 distribution Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims 3
- 241000699670 Mus sp. Species 0.000 claims 2
- 230000002950 deficient Effects 0.000 claims 2
- 238000012546 transfer Methods 0.000 abstract description 18
- 238000002474 experimental method Methods 0.000 abstract description 13
- 238000011160 research Methods 0.000 abstract description 6
- 238000010171 animal model Methods 0.000 abstract description 4
- 230000007812 deficiency Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 38
- 210000004027 cell Anatomy 0.000 description 33
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 22
- 201000011510 cancer Diseases 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 230000009545 invasion Effects 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 239000001993 wax Substances 0.000 description 5
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 208000010749 gastric carcinoma Diseases 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000002784 stomach Anatomy 0.000 description 4
- 201000000498 stomach carcinoma Diseases 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 201000002313 intestinal cancer Diseases 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 208000011581 secondary neoplasm Diseases 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010057315 Daydreaming Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 206010027457 Metastases to liver Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283965 Ochotona princeps Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 240000002825 Solanum vestissimum Species 0.000 description 1
- 235000018259 Solanum vestissimum Nutrition 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 208000018554 digestive system carcinoma Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000013210 hematogenous Diseases 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- -1 pH6.0) Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Classifications
-
- 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
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/13—Tumour cells, irrespective of tissue of origin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
-
- 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
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to stomach cancer Lung metastases mouse model and its method for building up, belong to technical field of experimental animals；The present invention cultivates BGC 823 and the stomach cancer cells of MKN 45 in vitro, and when cell viability is very vigorous, appropriate concentration is resuspended into sterile PBS；Be inoculated in by tail vein after being raised in the immunodeficiency type NUDE of immune deficiency and SCID mice body dyed by the statistics of surface Lung neoplasm, HE, IHC dyeing is identified and successfully obtains stomach cancer Lung metastases mouse model；Experiment condition and easy to operate required for the mouse model resume of the present invention, substantially, transfer efficiency is high for Lung metastases focus after experiment, and the mouse survival cycle formed after metastatic lesion is moderate；In the evaluation and statistically of transfer result, improvement is counted for the Positive Cell Counts of the IHC dyeing on microcosmic by Lung neoplasm macroscopically, it is as a result more objective accurate；The animal model of the metastases will may be widely used in scientific research and teaching.
Description
Technical field
The present invention relates to stomach cancer Lung metastases mouse model and its method for building up, belong to technical field of experimental animals.
Background technology
Stomach cancer（Gastric Cancer, GC）It is the fifth-largest common malignant tumour in the whole world（Lung cancer, intestinal cancer, breast cancer,
Liver cancer）One of.In World Developed Countries, stomach cancer is arranged in cancer neopathy number the 5th, positioned at lung cancer, breast cancer, intestinal cancer,
After liver cancer；And in developing country's row second, it is only second to lung cancer.Foreign literature is counted, and stomach cancer accounts for Digestive knurl 50%, state
Interior report accounts for 62%.According to statistics, there are 952000 new cases within 2012（Account for all cancer morbidities 7%）With 723000 deaths
（Account for all cancer mortalities 9%）.In China, the stomach cancer newly made a definite diagnosis every year has 300,000, dies from stomach cancer person up to 160,000, accounts for whole
The 23% of mortality of malignant tumors number, accounts for the 50% of digestive system carcinoma death toll.China's gastric cancer mortality 20 years（70 years 20th century
Generation to 1990s）Situation of change analysis result shows that mortality of gastric carcinoma general trend is that male is higher than women, and rural area is higher than
City；Compared with countries in the world, China male, women stomach cancer world death rate occupy the first.
Invasion and attack（Or invade profit）（invasion）With transfer（metastases）It is the main life of malignant tumour life threatening
Thing characteristic.The invasion and attack of so-called tumour are substantially that tumour cell destroys normal surrounding tissue structure by various modes, depart from former
Swell knurl, surrounding tissue and its process in gap are distributed in singularly, be the prerequisite steps that DISTANT METASTASES IN occurs for malignant tumour.But
The part benign tumour characteristic that also oriented surrounding tissue and its gap infiltrate.Occur invasion and attack or invasion and attack degree more for no
The malignant tumour of limitation, is generally effected a radical cure with treatment means such as operations.Transfer be malignant tumour be different from it is carcinoid most
One of key property.So-called transfer refers to that malignant cell departs from its original site.In vivo by various transporting pathway, arrive
Grown up to continuing to rise in value with the discontinuous tissue of original site, and form the secondary tumor of pathologic feature same with primary tumo(u)r
Overall process.Local challenge can suppress by the compressing to local normal structure organ, damage and influence the work(of normal structure organ
Energy.And the appearance shifted, then the crucial turnover of tumor development, DISTANT METASTASES IN are indicated once occurring, and often mean that tumour is entered
Enter late stage, be difficult to reach healing purpose only according to local treatment.Transfer it is main by way of including：1. hematogenous metastasis；2. drench
Ba Dao is shifted；3. Implantation matastasis.
The Blood route metastasis of stomach cancer is the process of a multi-step, be can be summarized as：Cancer cell departs from from primary tumor, invades cyclic system
System, passes through blood vessel, adheres to vascular wall, propagation generation secondary tumor.Lung metastases account for 20% ~ 40% in the Organ relative weight of stomach cancer,
It is only second to hepatic metastases.Tumour cell enters portal vein, reaches lung by liver, is forming transfer stove；Or retroperitoneal around stomach
Tumour cell in metastatic lymph node, above pass through ductus thoracicus, vein enter blood, form Lung metastases；Or tumour cell is through Lymphatic channel
Through diaphram, vertical diaphragm is up to, in hilus pulumonis or the formation lymphatic metastasis of vertical diaphragm, lung is further transferred to.Tumour cell can also be through
Diaphram goes directly pleura, and then involves lung through hilus pulumonis, vertical diaphragm, and such a transfer is less.
Zoopery studied as human diseases in an important step, can it is more objective simulation mankind's body in ring
Border, this is that experiment in vitro is completely irreplaceable.With the development of physics and chemistry, animal model is achieved very in recent years
Big progress, various human disease models are perfectly replicated, so as to provide broader platform for the disease research of the mankind.
It is used as a high aggressive and fatefulue malignant tumour, invasion and tumor suppressor gene, oncogene, the growth of stomach cancer
The mutation such as the factor and its acceptor is relevant.It is also few up to now effectively to control for metastases or the patients with terminal of recurrence
Treatment scheme.Therefore, the gastric carcinoma mouse metastasis model for setting up a kind of efficient stable is extremely crucial,
Traditional Metastasis of Gastric Cancer method, uses orthotopic transplantation more.Stomach cancer cell line well prepared in advance is needed, or finds stomach cancer trouble
The cancerous tissue that person's surgery excision gets off is expelled to the coat of the stomach of nude mice, the method for constructing human gastric cancer Orthotopic implantation in nude mice knurl model,
But the model has incubation period long, the problem of rate of transform is low.Also professional and technical personnel is directly embedded in stomach organization block
At the greater curvature of nude mice or small crook, although can stablize and set up Metastasis of Gastric Cancer model, but operation risk is larger, and it is postoperative right
The health of nude mice exerts a certain influence.Also there are some advanced laboratories, aid in microsurgical equipment so that the original of stomach cancer
The damage that displacement plants to nude mice is preferably minimized.But because experiment condition requires higher, this method also fails to be popularized.
Also the laboratory having is attempted by way of subcutaneous transplantation, it is desirable to by the transfer of muscle membranes, sets up simple
Metastasis of Gastric Cancer model.It is difficult to penetrate muscle membranes, and be transferred to flesh although the hypodermic tumour of transplanting can invade profit and arrive muscle layer
The situation on meat top layer also is difficult to accurate statistics.
The content of the invention：
It is an object of the invention to overcoming technological deficiency present in prior art there is provided a kind of stomach cancer Lung metastases mouse model and
Its method for building up.
Present invention firstly provides a kind of stomach cancer Lung metastases mouse model, the mouse is that immunodeficiency type NUDE or SCID are small
Mouse.
The present invention also provides a kind of method for building up of stomach cancer Lung metastases mouse model, and methods described concrete operations are as follows：
BGC-823 and MKN-45 stomach cancer cells are cultivated in vitro, when cell viability is very vigorous, are resuspended with sterile PBS
Into appropriate concentration；It is inoculated in after being raised in the immunodeficiency type NUDE of immune deficiency and SCID mice body and is obtained by tail vein
Stomach cancer Lung metastases mouse model.
BGC-823 the and MKN-45 stomach cancer cells in vitro culture is in containing 10% hyclone and Pen .- Strep
In RMPI-1640 culture mediums；.
It is described that 1x10 is resuspended into PBS7The concentration of individual/mL cells；
The stomach cancer cell tail vein inoculum concentration is 200 μ L, i.e. 200x104Individual cell/only.
Tail bone is inoculated with to NUDE and SCID mice breeding observing several weeks of BGC-823 and MKN-45 stomach cancer cells, Deng Dao little
When obvious adverse reaction occurs in mouse, mouse is died suddenly, and dissected；Now, it is found that lung tissue has significantly
Metastatic lesion；Lung tissue to mouse is cleaned, and is taken pictures, and is fixed with 10% formalin, makes paraffin section；Carry out HE
Dyed with IHC, and systematic assess is carried out to transfer efficiency.
The present invention also provides a kind of method of metastases efficiency evaluation, and resulting stomach cancer Lung metastases mouse is solved
Cut open, take out the lung for having had obvious metastatic lesion, paraffin section is made, using specific anti-human HLA（It is interior
Source property）Antibody（Anti-HLA class I ABC）IHC dyeing is done, the point of three relative distributions of histotomy is randomly selected, united
Count in every 100 cells, the number of cells accounting of positive reaction is metastases efficiency.
Compared with prior art, beneficial effects of the present invention are as follows：
Compared with prior art, the present invention is in experiment difficulty（Need not be performed an operation orthotopic transplantation）, experiment condition（Need not be outer
Section's microscopy apparatus auxiliary）, and experimental period（Stomach organization block need not be prepared in advance or waits patients with gastric cancer to carry out hand
Art）Several respects, are improved.And transfer efficiency is high, transfer method science is assessed, scientific research is highly suitable for and new drug is opened
Hair.
After the model of the present invention is successfully established, a variety of evaluation mechanism are employed, the result after Lung metastases is evaluated.
Dyed including two kinds of traditional approach, the i.e. statistics of surface Lung neoplasm and HE；Secondly, inspired by immunology, the cell in people source
Strain is transferred to after the lung of mouse, employs a species specific anti-human HLA（Endogenous）Antibody（Anti-
HLA class I ABC）IHC dyeing is done, under conditions of microcosmic, to evaluate the efficiency of Lung metastases.The present invention is from macroscopical lung knot
Microcosmic HE and IHC dyeing is saved, has all accomplished qualitative and quantitative.The mobility self-evaluation mechanism of system, it will thin to need to do stomach cancer
There is provided more convictive accurate proof for the experiment of born of the same parents' metastasis models.This is also the important step of the present invention, is compared
In the mode of traditional single calculating lung surface nodules, subjective factor will be smaller, counts more science, and it is more objective to evaluate
Accurately.
The present invention is by screening several conventional gastric carcinoma cell lines：BGC-823, MKN-45, AGS and SGC-7901, finally
Determine that BGC-823 and MKN-45 set up neoplasm lung metastasis mould with two kinds of conventional immunodeficiency type NUDE and SCID mice
Type.Experiment condition and operation required for Cheng Mo is relatively simple, and Lung metastases focus is obvious after experiment, and transfer efficiency is high, is formed
Mouse after metastatic lesion, time to live is moderate（1 to 1.5 month）, it is well suited for being used for scientific research and teaching.
The position that the present invention forms metastatic lesion is a larger histoorgan（Lung）, it is transferred to relative to some focuses
The model of lymph node or peritonaeum, metastatic lesion is easy to observe and easily win.In addition, also overcoming traditional gastric carcinoma mouse transfer mould
The number of drawbacks of type, the operating difficulty of experiment is reduced, after experiment（Post operation）The survival rate of mouse is significantly improved（Substantially can be with
Reach 100%）, test the equipment needed（Stereomicroscope is not needed）Simply, the cycle time that experiment needs（It need not shift to an earlier date
Prepare tissue block）, and later stage evaluation transfer efficiency index, all far superior to traditional methods.
The foundation of the present invention, can effectively screen medicine, strong data supporting is provided for preclinical research.In medicine
While thing is screened, it is possible to use the active drug screened reacts on the model, and in the different periods, taking-up, which has, to be turned
The lung of focus is moved, the mechanism in terms of molecular action is further inquired into.With molecular biology and the swift and violent hair of cell biology
Open up, especially genomics, protein science, the problems such as bioinformatics can be obtained on the basis of this model
Further explore or verify.This model can also be relied on to find the novel targets for the treatment of stomach cancer, so as to accelerate anti-gastric cancer tumour
The research and development of diversion medicaments.
Brief description of the drawings：
Fig. 1 is the performance figure directly perceived of mouse lung before and after NUDE Establishment of mouse model；Wherein, figure a is that NUDE mouse do not form transfer
The lung of focus, figure b and c is the lung of formation metastatic lesion after NUDE mouse injection BGC-823 and MKN-45 stomach cancer cell lines respectively.
Fig. 2 is the performance figure directly perceived of mouse lung before and after SCID mice model is set up；Wherein, figure a is that SCID mice is not formed
The lung of metastatic lesion, figure b and c is to form metastatic lesion after SCID mice injects BGC-823 and MKN-45 stomach cancer cell lines respectively
Lung.
Fig. 3 is that metastases focus does not occur and occurs the lung HE stain control figures of metastases focus for NUDE mouse.
Fig. 4 is that metastases focus does not occur and occurs the lung HE stain control figures of metastases focus for SCID mice.
Fig. 5 is NUDE and metastases focus does not occur and occurs the lung IHC dyeing of metastases focus for SCID mice
Compares figure.
Fig. 6 is NUDE and metastases focus does not occur and occurs lung's rate of transform system of metastases focus for SCID mice
Meter figure.
Embodiment
Preferably to make skilled in the art realises that technical scheme, below in conjunction with the accompanying drawings and specific embodiment
It is described in detail.
Embodiment 1：
S1. cell culture
BGC-823 and MKN-45 cells (American type culture collection) in vitro culture is in containing 10% tire ox
, it is necessary to often be preordained in the RMPI-1640 culture mediums (Life Technologies companies, the U.S.) of serum and Pen .- Strep
Phase observes cell growth state, to be passed in time when cell covers with blake bottle（It is general attached cell surface area account for 80% and more than
Shi Chuandai）.Next step experiment is carried out when cell culture and passage work well.
Cell is inoculated with
BGC-823 cells, 5min is centrifuged under 200g centrifugal force, with blood counting chamber, is calculated cell density, is then used PBS
1x10 is resuspended into7BGC-823, each of immunodeficiency type NUDE and SCID is inoculated into by tail vein by the concentration of individual/mL cells
In mouse（200 μ L, i.e. 200x104Individual/only）.Same operation, MKN-45 is also inoculated into NUDE and SCID mice respectively.Tool
Body cell is inoculated with details, is shown in Table 1.Immunodeficiency type NUDE and SCID mice are bought in Nanjing University's model animal research
Institute, raises in Jiangsu University SPF grades of isolation barrier.
The cell of table 1. is inoculated with situation
S3. die suddenly
The change of routine observation mouse, is decreased obviously when body weight occurs in mouse, absent-minded, the symptom such as body shakes when
Wait, vertebra dislocation is carried out to mouse and is died suddenly.Every mouse is from inoculating cell to sudden death elapsed cycles statistics such as upper table 1.
Dissection
The major organs of anatomic observation mouse, take out the lung for having had obvious metastatic lesion, as in culture dish, are rinsed with PBS
Several times, untill watery blood is rinsed well.
Take pictures
First complete lung tissue is taken pictures, front and back is respectively clapped once.By taking pictures, result can be clearly seen, BGC-
823 cells are transferred to the focus of lung（Lung neoplasm）Relatively relaxed with respect to the MKN-45 focuses for being transferred to lung, and BGC-823 transfer disease
Stove is small and uniform, influences little to the configuration of lung tissue；And MKN-45 metastatic lesions are larger, and mouse is had a strong impact on
The form of lung tissue.The lobe of the lung of normal mouse is without obvious metastatic lesion, and form is intact.Secondly, occurs the mouse of metastatic lesion
Lung tissue is relative to the mouse lung tissue without obvious metastatic lesion, and volume is bigger, refers to shown in Fig. 1 and Fig. 2.
It is fixed
Use 4% neutral formalin（10% formalin, plus excessive calcium carbonate, are made into saturated solution, take supernatant）Lung tissue is fixed
In the centrifuge tube being sized for（Liquid is about 20 times of tissue volume）, 1 to 2 h of middle interval change a fixer, put again
In slowly rocking 24h on shaking table, you can be placed in 4 DEG C of refrigerators preservations.
The making of paraffin section
After fixed 24h ~ 48h, you can take out the lobe of the lung tissue for having transfer stove, volume is of moderate size, and is placed in new centrifuge tube, plus steam
Distilled water puts slow shake 24h on shaking table in centrifuge tube（Distilled water twice is changed in centre, about every 8 h）.Then according to routine
Paraffin section step, is dehydrated, transparent, waxdip, is embedded, and section, mounted box is stand-by.
Comprise the following steps that：
Ethanol dehydration process：70% ethanol is stayed overnight（10h or so）, 85% ethanol 2h or so, 95% ethanol 1h or so, 95% ethanol 1h is left
The right side, 100% ethanol 1h, 100% ethanol 30 arrives 40min.
The transparent step of dimethylbenzene：About 40min transparent for the first time, with 100% dimethylbenzene；Second of time not necessarily, depending on
Depending on transparent effect, with 100% dimethylbenzene（General 20 arrive 30min）.
Waxdip step：3 wax cups are placed in 60 DEG C of insulating boxs, respectively numbering 1（52℃-54℃）、2（54℃-56℃）、
3（56℃-58℃）, wherein containing the wax stone of the corresponding fusing point of several piece respectively, allow it to be dissolved as transparency liquid shape in advance, it is transparent good to take
Tissue block is put into each hour in soft wax, and rapid take out is put into hard wax, same to place 1 hour, is divided into 3 waxdips, and wax stone melts
Point is from low to high
Embedding and section：FFPE and section are carried out according to operating instruction with the embedding machine and slicer of Lycra, 5 μ m-thicks are cut into
The slice, thin piece of degree, it is stand-by in slide box.
Haematoxylin eosin stains（HE）
Dry piece：90-95 DEG C of 0.5h, can preferably remove the bubble around tissue
Dewaxing：Dimethylbenzene 20min；Dimethylbenzene 20min；100% ethanol 30s；100% ethanol 30s；85% ethanol 10s；50% ethanol
10s；Distilled water 10s.
HE is dyed：Haematoxylin 10min, distilled water flushing to aubergine, hydrochloride alcohol（1:99）Break up 30s, circulating water punching
10min, 3 ~ 5min of eosin stain are washed, aubergine is washed to rapidly.
Dehydration：50% alcohol 30s, 85% alcohol 10s, 100% alcohol 10s, 100% alcohol 10s, dimethylbenzene 10s, dimethylbenzene
10s。
Mounting：Resinene mounting
It is observed that the lung tissue not shifted, space between cells is larger, the space between alveolar is high-visible.Generation Lung metastases
Lung tissue, grow intensive between cell and cell, without normal alveolar space, and nucleus is bigger than normal, with surrounding normal pneumonocyte
Size and the tightness degree of arrangement all have significant difference, refer to shown in Fig. 3 and Fig. 4.
Remarks：（Haematoxylin and Yihong come from：Wuhan bio tech ltd of Google）
S9. immunohistochemical staining（IHC）
Dry piece：90-95 DEG C of 0.5h, can preferably remove the bubble around tissue.
Dewaxing：Dimethylbenzene 20min, dimethylbenzene 20min, 100% ethanol 30s, 100% ethanol 30s, 85% ethanol 10s, 50% second
Alcohol 10s, distilled water 10s.
Citric acid is repaired：PH6.0 citric acid, boils 15min, pulls out power supply, opens lid, is put into ice bag, is cooled to
Room temperature（About 30min）.
Plus primary antibody：First rinsed one time with PBS, be then placed in 3min in PBS boxes, gauze dries moisture around, plus primary antibody
（Anti-HLA class Ⅰ ABC）, concentration 1:100, the μ L of volume 100,4 DEG C overnight（About 12 hours）.
Plus secondary antibody：First rinsed one time with PBS, be put into 3min in PBS boxes, gauze dries moisture around, plus the μ L of secondary antibody 50, room
Temperature is lower to be incubated 20min.
DAB is dyed：First rinsed one time with PBS, be put into 3min in PBS boxes, gauze dries moisture around, and DAB is added dropwise（A and B
According to 1:1 ratio, the μ L consumption proportions of every slice, thin piece about 50）, there is positive reaction and endures in dye liquor（About 20s）.
Core contaminates：Haematoxylin 2min, uses distilled water flushing.
Dehydration：50% alcohol 30s, 85% alcohol 10s, 100% alcohol 10s, 100% alcohol 10s, dimethylbenzene 10s, dimethylbenzene
10s。
Mounting：Resinene mounting
It is observed that occurring the section of Lung metastases, whole tissue positive reaction is fairly obvious, with significant people source characteristic
Cell line feature, refers to Fig. 5.
Remarks：The ABC of primary antibody Anti-HLA class I（Abcam companies）, sodium citrate pulvis（GT100202, is made into 2L
Sodium citrate repair liquid, pH6.0）, PBS pulvis (GT100102,2L standard dress), the general secondary antibody of pika
(GK500511A) GENE companies and DAB (GK34801) dye liquor, are all from.
Lung metastases rate is counted
The point of three relative distributions of histotomy is randomly selected, is counted in every 100 cells, the number of cells of positive reaction.
NUDE BGC-823 Lung metastases rate is 93.7% ± 8.7；NUDE MKN-45 Lung metastases rate is 94.3% ± 2.7；SCID BGC-823
Lung metastases rate is 93.7% ± 5.7；SCID MKN-45 Lung metastases rate is 94.3% ± 2 statistical result；Refer to Fig. 6.
Claims (10)
1. a kind of metastases mouse model, it is characterised in that the mouse model is stomach cancer Lung metastases mouse model.
2. metastases mouse model according to claim 1, it is characterised in that the mouse is immunodeficiency type NUDE
Or SCID mice.
3. metastases mouse model according to claim 1, it is characterised in that the mouse model is stomach cancer cell
BGC-823 inoculations NUDE mouse, stomach cancer cell BGC-823 inoculations SCID mice, stomach cancer cell MKN-45 inoculation NUDE mouse or
Stomach cancer cell MKN-45 is inoculated with SCID mice.
4. the method for building up of stomach cancer Lung metastases mouse model as claimed in claim 1, it is characterised in that methods described is specifically grasped
Make as follows：
Stomach cancer cell is cultivated in vitro, when cell viability is very vigorous, appropriate concentration is resuspended into sterile PBS；It is logical
Tail vein is crossed to be inoculated in immune-deficient mice body after raising；By the statistics of surface Lung neoplasm, HE dyeing, IHC dyeing mirror
It is fixed successfully to obtain stomach cancer Lung metastases mouse model.
5. method according to claim 4, it is characterised in that the stomach cancer cell is BGC-823 and MKN-45；It is described to exempt from
Epidemic disease deficient mice is NUDE and SCID mice.
6. method according to claim 4, it is characterised in that the stomach cancer cell in vitro culture is in containing 10% hyclone
And in the RMPI-1640 culture mediums of Pen .- Strep.
7. method according to claim 4, it is characterised in that described that 1x10 is resuspended into PBS7The concentration of individual/mL cells.
8. method according to claim 4, it is characterised in that the stomach cancer cell tail vein inoculum concentration is 200 μ L, i.e.,
200x104Individual cell/only.
9. a kind of metastases efficiency evaluation method, it is characterised in that using specific anti-human HLA（It is interior
Source property）Antibody（Anti-HLA class I ABC）IHC dyeing is done to be estimated.
10. appraisal procedure according to claim 9, it is characterised in that methods described concrete operations are：To claim 3
The obtained stomach cancer Lung metastases mouse of method dissected, take out the lung for having had obvious metastatic lesion, make paraffin section, adopt
With specific anti-human HLA（Endogenous）Antibody（Anti-HLA class I ABC）IHC dyeing is done, at random
The point of three relative distributions of histotomy is chosen, is counted in every 100 cells, the number of cells accounting of positive reaction is swollen
Tumor metastasis efficiency.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710155642.2A CN107006429A (en) | 2017-03-16 | 2017-03-16 | Stomach cancer Lung metastases mouse model and its method for building up |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710155642.2A CN107006429A (en) | 2017-03-16 | 2017-03-16 | Stomach cancer Lung metastases mouse model and its method for building up |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107006429A true CN107006429A (en) | 2017-08-04 |
Family
ID=59440714
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710155642.2A Pending CN107006429A (en) | 2017-03-16 | 2017-03-16 | Stomach cancer Lung metastases mouse model and its method for building up |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107006429A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621523A (en) * | 2020-06-09 | 2020-09-04 | 湖南昭泰生物医药有限公司 | ACE2 cell humanized mouse model and construction method and application thereof |
| CN111676243A (en) * | 2020-05-09 | 2020-09-18 | 厦门大学附属中山医院 | Preparation method of mouse model for specific expression of Cre enzyme in gastric mucosal epithelial cells |
| CN113875690A (en) * | 2021-09-30 | 2022-01-04 | 四川省肿瘤医院 | Lewis lung cancer mouse model based on tumor microenvironment and construction method thereof |
| CN110694050B (en) * | 2019-10-18 | 2022-07-12 | 江南大学附属医院（无锡市第四人民医院） | Application of PMCA4 protein in preparation of medicine for treating gastric cancer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101380478A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Installation method of fluorescent visual high-transfer human liver cancer nude mouse model |
| CN103320387A (en) * | 2013-07-16 | 2013-09-25 | 蚌埠医学院 | Method for establishing mice transplantable breast cancer experimental pulmonary metastasis model |
-
2017
- 2017-03-16 CN CN201710155642.2A patent/CN107006429A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101380478A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Installation method of fluorescent visual high-transfer human liver cancer nude mouse model |
| CN103320387A (en) * | 2013-07-16 | 2013-09-25 | 蚌埠医学院 | Method for establishing mice transplantable breast cancer experimental pulmonary metastasis model |
Non-Patent Citations (2)
| Title |
|---|
| 尚书恒: "非小细胞肺癌组织中人类白细胞抗原G的表达变化及意义", 《山东医药》 * |
| 曾知真等: "人胃癌细胞株在裸鼠体内不同种植部位的增殖、侵袭和转移", 《上海第二医科大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110694050B (en) * | 2019-10-18 | 2022-07-12 | 江南大学附属医院（无锡市第四人民医院） | Application of PMCA4 protein in preparation of medicine for treating gastric cancer |
| CN111676243A (en) * | 2020-05-09 | 2020-09-18 | 厦门大学附属中山医院 | Preparation method of mouse model for specific expression of Cre enzyme in gastric mucosal epithelial cells |
| CN111621523A (en) * | 2020-06-09 | 2020-09-04 | 湖南昭泰生物医药有限公司 | ACE2 cell humanized mouse model and construction method and application thereof |
| CN113875690A (en) * | 2021-09-30 | 2022-01-04 | 四川省肿瘤医院 | Lewis lung cancer mouse model based on tumor microenvironment and construction method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107006429A (en) | Stomach cancer Lung metastases mouse model and its method for building up | |
| CN106687152A (en) | Human liver scaffolds | |
| CN101185764A (en) | Orthotopic transplantation rat liver cancer model and preparing method and application thereof | |
| CN107137425A (en) | A kind of circulating tumor cell mouse model, its construction method and application | |
| Naito et al. | In vivo selection of human renal cell carcinoma cells with high metastatic potential in nude mice | |
| CN106367393B (en) | Prostate Carcinoma of Mice circulating tumor cell system and the separation of prostate cancer circulating tumor cell and cultural method | |
| CN104988122A (en) | Renal cancer sunitinib drug-resistant cell system and establishing method thereof | |
| CN102391992B (en) | Monoclonal antibody for resisting human NSCLC and application of monoclonal antibody | |
| CN101856496B (en) | Placenta stem-cell anti-tumor vaccine, preparation method and application thereof | |
| CN113667645B (en) | Canine breast cancer cell line, and establishment method and application thereof | |
| CN108685921A (en) | A kind of application of the quinoline of N isosteres iridin in medicines resistant to liver cancer | |
| CN107177551B (en) | Human intrahepatic bile duct cancer cell line with high tumor forming capability and application thereof | |
| CN101469320A (en) | Method for constructing oral squamous cell carcinoma animal model | |
| CN105062973B (en) | One plant carries HPV feminine gender penis squamous cell carcinomas system that TP53 is mutated and application thereof | |
| CN106399252A (en) | Oral squamous carcinoma cell strain as well as preparation method and applications thereof | |
| CN105969734A (en) | Esophageal cancer cell line and application thereof | |
| CN103013923A (en) | Method for creating animal model of human ovarian cancer | |
| CN101288602A (en) | Method for establishing radiation induced vitiligo mice model and application thereof | |
| CN110499290A (en) | One plant of people's Ewing sarcoma cell line | |
| CN110256364A (en) | A kind of quinazoline compounds and its preparation method and application | |
| CN103484425A (en) | Pseudosciaena crocea head kidney cell line and construction method thereof | |
| CN102154209B (en) | Human osteosarcoma cell strain and application thereof | |
| Sharma et al. | Etiologic relationship of skin tumors (skin leukosis) of chickens to Marek's disease | |
| CN102178568A (en) | Animal model constructing method for studying epithelial and stromal hyalinosis of hepatocellular carcinoma | |
| CN105669630A (en) | Extraction method of deoxydihydroxanthoangelol H chalcone suitable to serve as liver cancer targeted drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170804 |