CN108990900A - A kind of lung cancer chick chorioallantoic membrane M-CDX model and preparation method - Google Patents
A kind of lung cancer chick chorioallantoic membrane M-CDX model and preparation method Download PDFInfo
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- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 56
- 210000003711 chorioallantoic membrane Anatomy 0.000 title claims abstract description 49
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 39
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 34
- 210000004027 cell Anatomy 0.000 claims abstract description 27
- 201000005296 lung carcinoma Diseases 0.000 claims abstract description 17
- 239000006285 cell suspension Substances 0.000 claims abstract description 12
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 8
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 8
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 8
- 241000287828 Gallus gallus Species 0.000 claims abstract description 7
- 241000894007 species Species 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
- 238000010348 incorporation Methods 0.000 claims description 5
- 108090000288 Glycoproteins Proteins 0.000 claims description 4
- 102000003886 Glycoproteins Human genes 0.000 claims description 4
- 102000016611 Proteoglycans Human genes 0.000 claims description 4
- 108010067787 Proteoglycans Proteins 0.000 claims description 4
- 101710172711 Structural protein Proteins 0.000 claims description 3
- 239000003814 drug Substances 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
- 210000004204 blood vessel Anatomy 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000000386 microscopy Methods 0.000 abstract 1
- 235000013601 eggs Nutrition 0.000 description 22
- 206010028980 Neoplasm Diseases 0.000 description 20
- 238000000034 method Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 230000005740 tumor formation Effects 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000001154 Dermoid Cyst Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 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
- 230000036952 cancer formation Effects 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 231100000504 carcinogenesis Toxicity 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
- 238000010276 construction Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000001727 in vivo Methods 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
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 108010082117 matrigel Proteins 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
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0603—Embryonic cells ; Embryoid bodies
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2227/00—Animals characterised by species
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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Abstract
The present invention relates to life sciences, its specifically a kind of lung cancer chick chorioallantoic membrane M-CDX model and preparation method, the lung cancer chick chorioallantoic membrane M-CDX model is built-up in chick chorioallantoic membrane for the lung carcinoma cell of heterologous species to be transplanted to, preparation method 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 lung carcinoma cell of quantity mixes extracellular matrix, and cell suspension is made, and is 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 3 days under the conditions of being 50%-65%, and microscopy is taken pictures；The present invention is that transplanting object constructs lung cancer chick chorioallantoic membrane M-CDX model with lung carcinoma cell using chicken embryo as model；The carrier of M-CDX lotus knurl is one layer of chorioallantoic membrane with abundant blood vessel, and compared with conventional model, short with experimental period, feeding cost is low, facilitates the unique advantages such as operation；Operation instrument, experiment equipment needed for this preparation method are easy to use, cheap.
Description
Technical field
The present invention relates to life science, specifically a kind of lung cancer chick chorioallantoic membrane M-CDX model and preparation
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.
CN103239479A provides a kind of animal model of people's primary cutaneous dermoid cancer tumor stem cell
Method for building up is isolated this method comprises: single cell suspension is made in the clinical case patients with squamous cell carcinoma of collection
CD31-CD24-CD45-CD61-CD133+ tumor stem cell group, then with 1 × 106People's primary fibroblast, 1 × 106People is primary
Endothelial cell is outstanding to be mixed in matrigel, then is injected in the microenvironment through humanized nude mice by subcutaneous tumorigenesis position.
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 × enograft(M-CDX) cell origin based on film allosome move
It plants, that is, the lung cancer cell line of heterologous species is 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 lung cancer chick chorioallantoic membrane M-CDX model and preparation method, this model are
The lung carcinoma cell of heterologous species is transplanted to built-up in chick chorioallantoic membrane, preparation method includes by lung carcinoma cell kind
After being implanted 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 lung cancer chick chorioallantoic membrane M-CDX model has the lung cancer of heterologous species including transplanting in chick chorioallantoic membrane
The chicken embryo of cell.
Preferably, the heterologous species are behaved.
Preferably, the lung cancer chick chorioallantoic membrane M-CDX model is screened applied to anti-lung-cancer medicament.
A kind of preparation method of lung 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 60%-80%, on embryo egg top
Cut off 1cm × 1cm window；
Step 2: by (0.5-8) × 106The lung carcinoma cell incorporation extracellular matrix of quantity handled well, is made cell suspension, drips
It is added in the silica gel ring being put in chorioallantoic membrane in advance, is incubated for 3 under the conditions of 35 DEG C -40 DEG C of temperature, humidity are 50%-65%
It, obtains lung 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 70%.
Preferably, in step 2, using 4 × 106The lung carcinoma cell 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:(1-5).
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 the fibronectin in adhesion glycoprotein.
It compared with prior art, is transplanting with lung carcinoma cell the beneficial effects of the present invention are: the present invention is using chicken embryo as model
Object constructs lung 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 facilitates the unique advantages such as operation；
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, is not necessarily to long-time experience accumulation.
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 60 of the health of same period are selected, numbers respectively, is divided into tri- groups of A, B, C, every group 20.
Embodiment 1
A kind of lung cancer chick chorioallantoic membrane M-CDX model is had the lung carcinoma cell of patients with lung cancer by transplanting in chick chorioallantoic membrane
Chicken embryo constitute, method for building up are as follows:
(1) it is 37 DEG C in temperature by 20 embryo eggs of A group, is incubated for 3 days in the incubator that humidity is 60%, is gently inserted with cracker
Enter embryo egg top, strut bivalve at leisure, be careful not to destroy the integrality of outer embryo egg, with scissors along shell edge to shell top direction
The window of each 1cm of clip surplus；
(2) by 4 × 106In lung carcinoma cell incorporation structural proteins in range, with centrifuge at 4 DEG C, 1500r/min is kept,
Centrifugation 5min prepares cell suspension；
(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, lung cancer chick chorioallantoic membrane M-CDX model is obtained.
20 embryo eggs are divided into 10 group models, 2 embryo eggs of every group model；It is thin that the lung 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 5 groups, tumor formation rate 50%.
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,
Lung carcinoma cell 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 lung cancer chick chorioallantoic membrane M-CDX model is had the lung carcinoma cell of patients with lung cancer by transplanting in chick chorioallantoic membrane
Chicken embryo constitute, method for building up are as follows:
(1) be 36 DEG C in temperature by 20 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 5 × 106In lung carcinoma cell incorporation proteoglycans in range, with centrifuge at 6 DEG C, 2000r/min is kept,
Centrifugation 6min prepares cell suspension；
(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, lung cancer chick chorioallantoic membrane M-CDX model is obtained.
20 embryo eggs are divided into 10 group models, 2 embryo eggs of every group model；It is thin that the lung 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, lung carcinoma cell derive from the mouse of lung cancer mid-term.
A kind of lung cancer chick chorioallantoic membrane M-CDX model is had the lung cancer of lung cancer in mice 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 20 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 6 × 106In the mixed liquor of the 1:1 of lung carcinoma cell incorporation proteoglycans, adhesion glycoprotein in range, with centrifugation
Machine keeps 2000r/min, centrifugation 6min to prepare cell suspension 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, lung cancer chick chorioallantoic membrane M-CDX model is obtained.
20 embryo eggs are divided into 10 group models, 2 embryo eggs of every group model；It is thin that the lung 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 (7)
1. a kind of lung cancer chick chorioallantoic membrane M-CDX model, it is characterised in that: have including transplanting in chick chorioallantoic membrane different
The chicken embryo of the lung carcinoma cell of kind species.
2. a kind of lung cancer chick chorioallantoic membrane M-CDX model according to claim 1, it is characterised in that: the xenogenesis
Species are behaved.
3. a kind of lung cancer chick chorioallantoic membrane M-CDX model according to claim 1, it is characterised in that: the lung cancer
Chick chorioallantoic membrane M-CDX model is screened applied to anti-lung-cancer medicament.
4. a kind of preparation method of lung cancer chick chorioallantoic membrane M-CDX model according to claim 1 to 4, special
Sign is: including:
Step 1: embryo egg being incubated for 2-4 days in the incubator that temperature is 35 DEG C -40 DEG C, humidity is 60%-80%, on embryo egg top
Cut off 1cmx1cm window；
Step 2: by (0.5-8) × 106The lung carcinoma cell incorporation extracellular matrix of quantity handled well, is made cell suspension, is 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 3 days under the conditions of being 50%-65%, obtain
Lung cancer chick chorioallantoic membrane M-CDX model.
5. a kind of preparation method of lung cancer chick chorioallantoic membrane M-CDX model according to claim 4, feature exist
In: in step 1, by embryo egg temperature be 37 DEG C, humidity be 70% incubator in be incubated for 3 days；After transplanting, 37 DEG C of temperature,
It is incubated for 3 days under the conditions of humidity 60%.
6. a kind of preparation method of lung cancer chick chorioallantoic membrane M-CDX model according to claim 4, feature exist
In: in step 2, the ratio of the extracellular matrix and cell suspension is 1:(1-5).
7. a kind of preparation method of lung cancer chick chorioallantoic membrane M-CDX model according to claim 4, feature exist
In: in step 2, the extracellular matrix is the mixing of one or more of structural proteins, proteoglycans, adhesion glycoprotein.
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