CN109006694A - A kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model - Google Patents
A kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model Download PDFInfo
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- CN109006694A CN109006694A CN201810926880.3A CN201810926880A CN109006694A CN 109006694 A CN109006694 A CN 109006694A CN 201810926880 A CN201810926880 A CN 201810926880A CN 109006694 A CN109006694 A CN 109006694A
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- 201000007270 liver cancer Diseases 0.000 title claims abstract description 51
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 51
- 210000003711 chorioallantoic membrane Anatomy 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 28
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 34
- 210000004027 cell Anatomy 0.000 claims abstract description 26
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 17
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 17
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 17
- 239000006285 cell suspension Substances 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 101710172711 Structural protein Proteins 0.000 claims description 9
- 102000016611 Proteoglycans Human genes 0.000 claims description 7
- 108010067787 Proteoglycans Proteins 0.000 claims description 7
- 102000003886 Glycoproteins Human genes 0.000 claims description 2
- 108090000288 Glycoproteins Proteins 0.000 claims description 2
- 240000002853 Nelumbo nucifera Species 0.000 abstract description 6
- 235000006508 Nelumbo nucifera Nutrition 0.000 abstract description 6
- 235000006510 Nelumbo pentapetala Nutrition 0.000 abstract description 6
- 241000287828 Gallus gallus Species 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 4
- 210000004204 blood vessel Anatomy 0.000 abstract description 3
- 238000009825 accumulation Methods 0.000 abstract description 2
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000000386 microscopy Methods 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 31
- 235000013601 eggs Nutrition 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 230000005740 tumor formation Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 238000010171 animal model Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005748 tumor development 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
- A01K67/0271—Chimeric vertebrates, e.g. comprising 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
- 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
- A01K2227/30—Bird
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to life sciences, the method for building up of its specifically a kind of liver cancer chick chorioallantoic membrane M-CDX model, method for building up includes being incubated for embryo egg 2-4 days in the incubator that temperature is 35 DEG C -40 DEG C, humidity is 60-80%, cuts off 1cmx1cm window on embryo egg top；By (0.5-8) × 106The liver cancer cells of quantity mix extracellular matrix, and cell suspension is made, and are added drop-wise in the silica gel ring being put in chorioallantoic membrane in advance, 35 DEG C -40 DEG C, humidity be incubated for 2-4 days under the conditions of being 50%-65%, and microscopy is taken pictures；The present invention is that transplanting object constructs liver cancer chick chorioallantoic membrane M-CDX model with liver cancer cells using chicken embryo as model；The carrier of M-CDX lotus knurl is that one layer of chorioallantoic membrane with abundant blood vessel compared with conventional model has experimental period short, and feeding cost is low, and the unique advantage for facilitating operation etc.；Operation instrument, experiment equipment needed for this preparation method are easy to use, and cheap, common lab can be routinely equipped with, and producing process is easy to operate, are not necessarily to long-time experience accumulation.
Description
Technical field
The present invention relates to life science, the foundation of specifically a kind of liver cancer chick chorioallantoic membrane M-CDX model
Method.
Background technique
Duplication of the animal model for tumour as human tumor is indispensable means in tumor research, as modern age cures
Development, the especially needs of oncology and immunological investigation are learned, the appearance of immunodeficient animals solves the immune of transplantation tumor
Exclusive problem, human tumor are transplanted in immunodeficient animals, its biological characteristics is able to maintain, for studying the machine of human tumor
System, drug screening provide very big help.
CN102821600A provides cancerous disease state non-human animal model and its utilizes method, it is characterised in that: can reproduce
Hierarchical organization, cancer progression process or its biological characteristics of human cancer tissue.CN102821600A is in order to solve above-mentioned class
NOG is established cancer cell line first and migrated in NOG mouse by topic, its tissue construction of morphological observation.As a result, it was confirmed that this is inhuman
Animal model possesses the morbid state similar with human cancer disease (hierarchical organization, cancer progression process or its biology of cancer cell
Characteristic).
CN104771240A provides the tumor model for antineoplaston option screening, is used to prepare the tumor model
Method and using the tumor model screening antineoplaston scheme method.Wherein, tumor model is from the first biology
The tumor tissues formed in vivo, the tumor model are that the tumor tissues by being formed in first organism are being set forth in
It is proliferated and obtains in two organisms, wherein first organism is different from second organism.Utilize the tumor model
The therapy that can be used for the first organism can effectively be screened.
In order to understand malignant tumour in the biological behaviour feature of body, building tumour in vivo model simulation tumour is generally required
True living environment.Chick chorioallantoic membrane (chorioallantoic membrance, CAM) is not arranged in the early metaphase of development
Denounce tissue from heterogenous animal, cell transplantation, therefore can make to transplant the acceptor of human malignancies.CAM model is extensive
Research applied to new vessels generation, microcirculation, oncobiology etc..
Membrane-based cell-derived xenograft(M-CDX) cell origin based on film allosome move
It plants, that is, the liver cancer cell lines of heterologous species are transplanted to the model constructed in chick chorioallantoic membrane.This model can be used for blood vessel
The newborn interaction with tumor development, and the research of screening effective antitumor compound.
Traditional CDX model, lotus knurl carrier are usually nude mice, and transplanted tumor in nude mice can save original histology construction or each
Kind of function, and by the tissue culture culture transferring of human cancer tissue when nude mice, can reappear disappeared in tissue cultures it is original
Cancer structure.The property that these features help to analyze human tumor provides to push the work of various aspects for experimental oncology
New effective tool.But since the raising of nude mice and breeding requirement condition are stringenter, price is more expensive, it is related naked
There are certain limitations for mouse neoplasm transplantation.
Summary of the invention
In view of this, the present invention provides a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model, method for building up
After liver cancer cells are planted on chick chorioallantoic membrane, it was incubated under the proper conditions 3 days, and used sediments microscope inspection.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model, comprising:
Step 1: embryo egg being incubated for 2-4 days in the incubator that temperature is 35 DEG C -40 DEG C, humidity is 50%-70%, on embryo egg top
Cut off 1cmx1cm window；
Step 2: by (0.5-8) × 106The liver cancer cells of quantity mix extracellular matrix, and cell suspension is made, and are added drop-wise in advance
Be put in the silica gel ring in chorioallantoic membrane, 35 DEG C -40 DEG C, humidity be 50%-65% under the conditions of be incubated for 2-4 days after, obtain liver cancer
Chick chorioallantoic membrane M-CDX model.
Preferably, in step 1, it is 37 DEG C in temperature by embryo egg, is incubated for 3 days in the incubator that humidity is 60%.
Preferably, in step 2, using 5x106The liver cancer cells of quantity.
Preferably, in step 2, it is incubated for 3 days under the conditions of 37 DEG C of temperature, humidity 60%.
Preferably, in step 2, the ratio of the extracellular matrix and cell suspension is 1:(1-3).
Preferably, in step 2, the extracellular matrix is one of structural proteins, proteoglycans, adhesion glycoprotein
Or several mixing.
Preferably, in step 2, the extracellular matrix is structural proteins, proteoglycans with (3-5): the mixing of 1 ratio
Object.
Preferably, in step 2, the extracellular matrix is structural proteins, proteoglycans with the mixture of 4:1 ratio.
Preferably, the liver cancer chick chorioallantoic membrane M-CDX model of foundation is screened applied to medicines resistant to liver cancer.
It compared with prior art, is transplanting with liver cancer cells the beneficial effects of the present invention are: the present invention is using chicken embryo as model
Object constructs liver cancer chick chorioallantoic membrane M-CDX model；
The carrier of M-CDX lotus knurl is one layer of chorioallantoic membrane with abundant blood vessel, compared with conventional model, with experimental period
Short, feeding cost is low, and facilitate operation etc. unique advantage；
Operation instrument, experiment equipment needed for this preparation method are easy to use, and cheap, common lab can routinely match
It is standby, and producing process is easy to operate, it is not necessarily to long-time experience accumulation, and be substantially not subject to the limitation in terms of animal ethics.
Detailed description of the invention
Fig. 1 is liver cancer cells M-CDX lotus knurl produced by the present invention structural form figure under the microscope.
Specific embodiment
The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot
It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, described according to the literature in the art
Technology or conditions or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to
Cross the conventional products of commercially available acquisition.
Embryo egg 90 of the health of same period are selected, numbers respectively, is divided into tri- groups of A, B, C, every group 30.
Embodiment 1
A kind of liver cancer chick chorioallantoic membrane M-CDX model is had the chicken embryo structure of liver cancer cells by transplanting in chick chorioallantoic membrane
At method for building up are as follows:
(1) it is 37 DEG C in temperature by the embryo egg of 30 embryo eggs of A group, is incubated for 3 days in the incubator that humidity is 60%, embryo egg is complete
Without incompleteness, cracker is gently inserted into embryo egg top, struts bivalve at leisure, be careful not to destroy the integrality of outer embryo egg, used
Scissors is along shell edge to the window of each 1cm of shell top direction clip surplus；
(2) by 4 × 106In liver cancer cells incorporation extracellular matrix in range, at 5 DEG C with centrifuge, 1500r/ is kept
Min, centrifugation 5min prepare cell suspension, and extracellular matrix selects structural proteins, proteoglycans with the mixture of 4:1 ratio；
(3) cell suspension is added drop-wise in the silica gel ring being put in chorioallantoic membrane in advance, 37 DEG C of merging, the culture of 60% humidity
After being incubated for 3 days in case, a kind of liver cancer chick chorioallantoic membrane M-CDX model is obtained.
30 embryo eggs are divided into 10 group models, 3 embryo eggs of every group model；It is thin that the liver cancer transplanted in each embryo egg is observed in timing
Born of the same parents determine model foundation success when occurring tumor formation quantity on the chick chorioallantoic membrane in model greater than 1, occur in A group at
The model of tumor is 6 groups, tumor formation rate 60%.
M-CDX lotus knurl made from embodiment 1 is observed under the microscope, is recorded, digit microscope is taken pictures, and M-CDX is obtained
The structural form figure of lotus knurl, as shown in Figure 1, the morphologic localization and structure change to each layer tissue cell of outer embrane are observed,
Liver cancer cells and chorioallantoic membrane comparison are obvious；Visible each layer tissue eucaryotic cell structure of film is complete under microscope, organizes fine structure
Clearly, feature is obvious, can more accurately show the fine structure of each layer tissue cell and its distribution situation at each position of outer embrane.
Embodiment 2
A kind of liver cancer chick chorioallantoic membrane M-CDX model is had the chicken embryo structure of liver cancer cells by transplanting in chick chorioallantoic membrane
At method for building up are as follows:
(1) be 36 DEG C in temperature by 30 embryo eggs of B group, be incubated for 4 days in the incubator that humidity is 65%, embryo egg completely without incompleteness,
Cracker is gently inserted into embryo egg top, struts bivalve at leisure, with scissors along shell edge to each 1cm of shell top direction clip surplus
Window；
(2) by 4 × 106Taken out with slave liver cancer mouse in range in fresh liver cancer cells incorporation extracellular matrix, with from
Scheming keeps 2000r/min, centrifugation 3min to prepare cell suspension, extracellular matrix selects structural proteins at 6 DEG C；
(3) cell suspension is added drop-wise in the silica gel ring being put in chorioallantoic membrane in advance, 38 DEG C of merging, the culture of 55% humidity
After being incubated for 3 days in case, a kind of liver cancer chick chorioallantoic membrane M-CDX model is obtained.
30 embryo eggs are divided into 10 group models, 3 embryo eggs of every group model；It is thin that the liver cancer transplanted in each embryo egg is observed in timing
Born of the same parents determine model foundation success when occurring tumor formation quantity on the chick chorioallantoic membrane in model greater than 1, occur in B group at
The model of tumor is 6 groups, tumor formation rate 60%.
Embodiment 3
Comparative example, liver cancer cells derive from the mouse of liver cancer mid-term.
A kind of liver cancer chick chorioallantoic membrane M-CDX model is had the liver cancer of liver cancer mouse by transplanting in chick chorioallantoic membrane
The chicken embryo of cell is constituted, method for building up are as follows:
(1) be 36 DEG C in temperature by 30 embryo eggs of C group, be incubated for 4 days in the incubator that humidity is 65%, embryo egg completely without incompleteness,
Cracker is gently inserted into embryo egg top, struts bivalve at leisure, with scissors along shell edge to each 1cm of shell top direction clip surplus
Window；
(2) by 4 × 106Taken out with slave liver cancer mouse in range in fresh liver cancer cells incorporation extracellular matrix, with from
Scheming keeps 2000r/min, centrifugation 3min to prepare cell suspension, extracellular matrix selects structural proteins at 6 DEG C；
(3) cell suspension is added drop-wise in the silica gel ring being put in chorioallantoic membrane in advance, 38 DEG C of merging, the culture of 55% humidity
It is incubated for 4 days in case, obtains a kind of liver cancer chick chorioallantoic membrane M-CDX model.
30 embryo eggs are divided into 10 group models, 3 embryo eggs of every group model；It is thin that the liver cancer transplanted in each embryo egg is observed in timing
Born of the same parents determine model foundation success when occurring tumor formation quantity on the chick chorioallantoic membrane in model greater than 1, occur in C group at
The model of tumor is 7 groups, tumor formation rate 70%.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (8)
1. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model, it is characterised in that: include:
Step 1: embryo egg being incubated for 2-4 days in the incubator that temperature is 35 DEG C -40 DEG C, humidity is 50%-70%, on embryo egg top
Cut off 1cm × 1cm window；
Step 2: by (0.5-8) × 106The processed liver cancer cells of quantity mix extracellular matrix, and cell suspension is made, and are added dropwise
To being put in the silica gel ring in chorioallantoic membrane in advance, 35 DEG C -40 DEG C, after humidity is incubated for 2-4 days under the conditions of being 50%-65%, liver
Cancer chick chorioallantoic membrane M-CDX model.
2. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 1, feature exist
In: in step 1, by embryo egg temperature be 37 DEG C, humidity be 60% incubator in be incubated for 3 days.
3. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 1, feature exist
In: in step 2, the liver cancer cells derive from American Type Culture collection warehousing (ATCC), HepG2.
4. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 4, feature exist
In: in step 2, it is incubated for 3 days under the conditions of 37 DEG C of temperature, humidity 60%.
5. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 1, feature exist
In: in step 2, the ratio of the extracellular matrix and cell suspension is 1:(1-3).
6. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 1, feature exist
In: in step 2, the extracellular matrix is the mixing of one or more of structural proteins, proteoglycans, adhesion glycoprotein.
7. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 6, feature exist
In: in step 2, the extracellular matrix is structural proteins, proteoglycans with (3-5): the mixture of 1 ratio.
8. a kind of method for building up of liver cancer chick chorioallantoic membrane M-CDX model according to claim 7, feature exist
In: in step 2, the extracellular matrix is structural proteins, proteoglycans with the mixture of 4:1 ratio.
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Cited By (1)
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| CN109929797A (en) * | 2019-02-22 | 2019-06-25 | 南昌乐悠生物科技有限公司 | Chicken embryo Transplanted tumor model method for building up and its application in drug susceptibility detection |
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