CN103215224A - Laryngocarcinoma spontaneous cervical lymph nody metastasis animal model - Google Patents
Laryngocarcinoma spontaneous cervical lymph nody metastasis animal model Download PDFInfo
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Abstract
The invention relates to a laryngocarcinoma spontaneous cervical lymph nody metastasis animal model, a method for establishing the animal model, and a method for obtaining high-metastasis laryngocarcinoma cells.
Description
Technical field
The present invention relates to the animal model for cancer field; Particularly, the present invention relates to the spontaneous cervical lymph node of laryngocarcinoma and shift animal model.
Background technology
Laryngocarcinoma is one of incidence common cancer, the most common with squamous cell carcinoma, account for more than 95%, sickness rate is on the rise in recent years, local invasion and attack take place in laryngocarcinoma easily and cervical lymph node shifts, the same with other malignant tumor patients, the patients with laryngeal carcinoma majority is died from tumor recurrence and transfer, but laryngocarcinoma invasion and attack, metastasis it be not immediately clear, the present invention is by setting up the spontaneous cervical lymph node metastasis model of laryngocarcinoma, filter out high aggressive laryngeal cancer cell, for the invasion and attack of research laryngocarcinoma, metastasis lay the foundation.
Summary of the invention
The present invention relates to the spontaneous cervical lymph node of a kind of laryngocarcinoma and shift animal model, the establishment method of this animal model and the method that obtains high transitivity laryngeal cancer cell.
In one embodiment, the present invention relates to a kind of laryngeal cancer cell and tie up to the purposes for preparing in the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma.
In one embodiment, the present invention relates to a kind of laryngeal cancer cell and tie up to the purposes for preparing in the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, wherein said Human Laryngeal Cancer Cell is a larynx squamous cell carcinoma system.
In one embodiment, the present invention relates to a kind of laryngeal cancer cell and tie up to the purposes for preparing in the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, wherein said Human Laryngeal Cancer Cell is that people's larynx squamous cell carcinoma is HEP-2.
In one embodiment, the present invention relates to a kind of laryngeal cancer cell and tie up to the purposes for preparing in the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, wherein said Human Laryngeal Cancer Cell is that people's larynx squamous cell carcinoma is HEP-2, and wherein said non-human animal is a mouse.
In one embodiment, the present invention relates to a kind of laryngeal cancer cell and tie up to the purposes for preparing in the test kit that produces high transitivity laryngeal cancer cell, wherein said high transitivity laryngeal cancer cell has higher transitivity than described Human Laryngeal Cancer Cell, also comprises substratum, narcotic etc. in the described test kit.
In one embodiment, the present invention relates to a kind of method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal non-human animal tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, get non-human animal's poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion.
In one embodiment, the present invention relates to a kind of method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal non-human animal tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, get non-human animal's poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion;
Wherein said Human Laryngeal Cancer Cell is a larynx squamous cell carcinoma system.
In one embodiment, the present invention relates to a kind of method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal non-human animal tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, get non-human animal's poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion;
Wherein said larynx squamous cell carcinoma is is that people's larynx squamous cell carcinoma is HEP-2.
In one embodiment, the present invention relates to a kind of method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under the nude mouse lingual margin mucous membrane;
(b) treat that the nude mouse tongue becomes knurl, becomes thin when being at death's door, put to death nude mouse, get nude mouse poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion.
In one embodiment, the present invention relates to a kind of method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under the nude mouse lingual margin mucous membrane;
(b) treat that the nude mouse tongue becomes knurl, becomes thin when being at death's door, put to death nude mouse, get nude mouse poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion;
Wherein said Human Laryngeal Cancer Cell is that people's larynx squamous cell carcinoma is HEP-2.
In one embodiment, the present invention relates to a kind of method for preparing high transitivity laryngeal cancer cell, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal's tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, aseptic non-human animal's poke lymphoglandula of getting, separation is also cultivated the laryngeal cancer cell of transferring to cervical lymph node;
(c) the described laryngeal cancer cell repeated inoculation of transferring to cervical lymph node is arrived non-human animal's tongue, described in step (b), separate, cultivate the laryngeal cancer cell of transferring to cervical lymph node;
(d) randomly repeat (c) step one or many;
(e) filter out high transitivity laryngeal cancer cell, the checking laryngeal cancer cell that obtains comes from parental cell line;
Wherein said high transitivity laryngeal cancer cell has higher transitivity than Human Laryngeal Cancer Cell described in (a).
In one embodiment, the present invention relates to a kind of method for preparing high transitivity laryngeal cancer cell, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal's tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, aseptic non-human animal's poke lymphoglandula of getting, separation is also cultivated the laryngeal cancer cell of transferring to cervical lymph node;
(c) the described laryngeal cancer cell repeated inoculation of transferring to cervical lymph node is arrived non-human animal's tongue, described in step (b), separate, cultivate the laryngeal cancer cell of transferring to cervical lymph node;
(d) randomly repeat (c) step one or many;
(e) filter out high transitivity laryngeal cancer cell, detect with PCR and verify that the laryngeal cancer cell that obtains comes from parental cell line;
Wherein said high transitivity laryngeal cancer cell has higher transitivity than Human Laryngeal Cancer Cell described in (a).
In one embodiment, the present invention relates to a kind of method for preparing high transitivity laryngeal cancer cell, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal's tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, aseptic non-human animal's poke lymphoglandula of getting, separation is also cultivated the laryngeal cancer cell of transferring to cervical lymph node;
(c) the described laryngeal cancer cell repeated inoculation of transferring to cervical lymph node is arrived non-human animal's tongue, described in step (b), separate, cultivate the laryngeal cancer cell of transferring to cervical lymph node;
(d) randomly repeat (c) step one or many;
(e) filter out high transitivity laryngeal cancer cell, detect the human specific Alu sequence by PCT and verify that the laryngeal cancer cell that obtains comes from parental cell line;
Wherein said high transitivity laryngeal cancer cell has higher transitivity than Human Laryngeal Cancer Cell described in (a).
In one embodiment, the present invention relates to a kind of method for preparing high transitivity laryngeal cancer cell, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under the nude mouse lingual margin mucous membrane;
(b) treat that the nude mouse tongue becomes knurl, becomes thin when being at death's door, put to death nude mouse, the aseptic nude mouse poke lymphoglandula of getting, separation is also cultivated the laryngeal cancer cell of transferring to cervical lymph node;
(c) the described laryngeal cancer cell repeated inoculation of transferring to cervical lymph node is arrived the nude mouse tongue, described in step (b), separate, cultivate the laryngeal cancer cell of transferring to cervical lymph node;
(d) randomly repeat (c) step one or many;
(e) filter out high transitivity laryngeal cancer cell, detect the human specific Alu sequence by PCT and verify that the laryngeal cancer cell that obtains comes from parental cell line;
Wherein said high transitivity laryngeal cancer cell has higher transitivity than Human Laryngeal Cancer Cell described in (a).
Unless this paper has definition in addition, the relevant Science and Technology term that uses of the present invention has the implication of those of ordinary skills' common sense.And unless context has other regulation, the term of singulative should comprise plural number, and the term of plural form should comprise odd number.Usually, the name and the technology of relevant with tissue culture, molecular biology and immunology use with cell as herein described, be this area as everyone knows and generally use those.Except as otherwise noted, following term should be understood to have following implication:
Term " non-human animal " be meant remove the homo sapiens (
Homo sapiens) species other any model animals in addition, for example mouse, rat, cavy, dog, gorilla, chimpanzee etc.
Term " high transitivity laryngeal cancer cell " is meant that the ratio that obtains by the present invention is inoculated in that used Human Laryngeal Cancer Cell has higher metastatic laryngeal cancer cell under non-human animal's lingual margin mucous membrane.
Description of drawings
Fig. 1 is that the nude mouse tongue becomes knurl and poke lymphoglandula synoptic diagram.
Fig. 2 illustrates nude mouse tongue and cervical lymph node pathological section result.
Fig. 3 illustrates the high transitivity laryngeal cancer cell that nude mouse laryngocarcinoma lymphatic metastasis model obtains.
Embodiment
Further describe the present invention in following examples, described embodiment shows and implements concise and to the point method of the present invention, might modification but do not limit its institute.
The abbreviation implication is as follows: " min " expression minute, and " U " representation unit, " μ g " represent microgram, and " g " represent to restrain, and " μ l " represents microlitre, and " ml " represents milliliter, " bp " represents base pair.
Embodiment 1
The spontaneous cervical lymph node of squamous carcinoma of larynx shifts the foundation of animal model
The used laboratory animal of present embodiment is as follows: the Bal/Bc nude mouse, about 4 ages in week, 12-14g, male and female are not limit (Military Medical Science Institute of PLA animal provides), its experiment and raising are all at no specificity pathogenic bacteria (specific pathogen free, SPF), carry out in the ultra-clean laminar-flow rack under the constant temperature (20-26 ℃), constant humidity (45%-50%) condition, aqua sterilisa and sterilization feed are freely taken in for animal.
The used larynx squamous cell carcinoma of present embodiment is as follows: people's larynx squamous cell carcinoma HEP-2 clone is provided by Beijing consonance medical university preclinical medicine cell centre.The HEP-2 cell routine is incubated in the DMEM nutrient solution that contains 10% foetal calf serum, places 37 ℃, 5%CO
2Cultivate in the constant incubator, stablize and go down to posterity 3-4 after generation, the cell in vegetative period of taking the logarithm, with 0.25% tryptic digestion collecting cell, it is 1.5 * 10 that stroke-physiological saline solution is adjusted cell density
8/ ml is standby, and trypan blue dyeing determines that viable count is more than 95%.
Press 35mg/kg body weight abdominal injection 1.25% vetanarcol anesthesia nude mouse, successfully after the anesthesia, use the 50ul microsyringe, inject the 10ul cell suspension down, inoculate 20 at every turn in every nude mouse lingual margin mucous membrane.Selecting two nude mouses at random is left intact in contrast.Close observation nude mouse growth conditions.Treat that the nude mouse tongue becomes knurl, become thin, when being at death's door, put to death nude mouse, get the cervical lymph node of swollen thing of tongue and enlargement, 4% Paraformaldehyde 96 is fixed, paraffin embedding, serial section, conventional H E dyeing, light microscopic is observed the nude mouse tongue down and is become knurl and cervical lymph node metastases situation.
Tongue becomes the visual inspection result of knurl and poke lymphoglandula as follows: nude mouse lingual margin mucous membrane is down behind the injection tumour cell after about 4 weeks, the nude mouse tongue obviously becomes knurl, the feed difficulty, health is become thin, is at death's door, put to death nude mouse, aseptic condition is dissected neck, visible one to several pieces enlarged lymph nodes down.
Tongue becomes the pathological examination of knurl and poke lymphoglandula as follows: the lymphoglandula and the normal tongue of control group nude mouse of swollen thing of experimental group nude mouse tongue and enlargement are all collected with normal lymphoglandula, carry out conventional H E dyeing, ordinary optical microscope is observed tongue down and is become knurl and enlargement of lymph node tumor metastasis situation.The result shows that experimental group nude mouse lingual margin mucous membrane forms noumenal tumour down, and spherical in shape, laryngeal cancer cell fills the air arrangement, and volume is bigger, and form is various, and nuclear fission is unusual.The dyed back of enlarged lymph node mirror is observed down, visible tumour cell diffuse infiltrating, and normal lymphoglandula internal structure is destroyed (seeing Fig. 1).
Embodiment 2
Transfer to the cultivation of the laryngeal cancer cell of lymphoglandula--the acquisition of high transitivity larynx squamous cell carcinoma
As described in example 1 above people's larynx squamous cell carcinoma HEP-2 clone is injected under every nude mouse lingual margin mucous membrane, treat that nude mouse becomes thin when being at death's door, put to death nude mouse, the aseptic cervical lymph node of getting in the Bechtop shreds lymphoglandula on 150 eye mesh screens, the PBS collecting cell, centrifugal, contain 10% foetal calf serum DMEM(penicillin 100U/ml, Streptomycin sulphate 100ug/ml) the nutrient solution re-suspended cell, routine is incubated at 37 ℃, 5% CO
2In the incubator.With the laryngeal cancer cell culture repeated inoculation of transferring to lymphoglandula of gained, obtain easily to transfer to the laryngeal cancer cell of lymphoglandula with quadrat method.Inoculate into the knurl situation for twice and the nodus lymphoideus transferring rate ratio is seen Fig. 2 and table 1.
Table 1
.。
Embodiment 3
PCT amplification people Alu sequence--the checking of high transitivity larynx squamous cell carcinoma
Separate, cultivate the larynx squamous cell carcinoma of transferring to the nude mouse cervical lymph node, microscopically is observed and is found, compares with parent HEP-2 cell, and the tumour cell volume that is obtained slightly increases, and form slightly becomes the circle (see figure 3).
Application cell genome DNA extracting reagent kit (centrifugal column type, it root) extracts DNA in the last tumour cell that obtains, pcr amplification goes out 221bp human specific Alu sequence (see figure 3), the Alu sequence primer is: 5 '-CACCTGTAATCCCAGCAGTTT-3 ', 5 '-CGCGATCTCGGCTCACTGCA-3 ', cycling condition is 95 ℃ of sex change 5min, 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations, 72 ℃ are extended 5min.From pcr amplification product, draw 5 μ L through 1.5% agarose gel electrophoresis 20-30 minute, on the ultraviolet imagery instrument, observe.The result proves that the HEP-2 cell transfer in tumour cell behaviour source arrives the nude mouse cervical lymph node and separated, cultivation acquisition.
Claims (9)
1. a laryngeal cancer cell ties up to the purposes for preparing in the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma.
2. according to the purposes of claim 1, wherein said Human Laryngeal Cancer Cell is a larynx squamous cell carcinoma system.
3. according to the purposes of claim 2, wherein said larynx squamous cell carcinoma is is that people's larynx squamous cell carcinoma is HEP-2.
4. according to the purposes of claim 1, wherein said non-human animal is a mouse.
5. a laryngeal cancer cell ties up to the purposes for preparing in the test kit that produces high transitivity laryngeal cancer cell, and wherein said high transitivity laryngeal cancer cell has higher transitivity than described Human Laryngeal Cancer Cell.
6. method for preparing the spontaneous nodus lymphoideus transferring rate non-human animal model of laryngocarcinoma, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal's tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, get non-human animal's poke lymphoglandula;
(c) the applied pathology method identifies whether swollen thing of tongue and poke lymphoglandula are tumor cell invasion.
7. method for preparing high transitivity laryngeal cancer cell, it comprises the steps:
(a) Human Laryngeal Cancer Cell is inoculated under non-human animal's lingual margin mucous membrane;
(b) treat that non-human animal's tongue becomes knurl, becomes thin when being at death's door, put to death the non-human animal, aseptic non-human animal's poke lymphoglandula of getting, separation is also cultivated the laryngeal cancer cell of transferring to cervical lymph node;
(c) the described laryngeal cancer cell repeated inoculation of transferring to cervical lymph node is arrived non-human animal's tongue, described in step (b), separate, cultivate the laryngeal cancer cell of transferring to cervical lymph node;
(d) randomly repeat (c) step one or many;
(e) filter out high transitivity laryngeal cancer cell, the checking laryngeal cancer cell that obtains comes from parental cell line;
Wherein said high transitivity laryngeal cancer cell has higher transitivity than Human Laryngeal Cancer Cell described in (a).
8. according to the method for claim 7, the method for the checking laryngeal cancer cell that obtains is that PCR detects in the wherein said step (e).
9. method according to Claim 8, it is that PCT detects the human specific Alu sequence that wherein said PCR detects.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657129A (en) * | 2022-04-11 | 2022-06-24 | 中山大学孙逸仙纪念医院 | Bile duct cancer lymph node metastasis animal model and construction method thereof |
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| CN1850252A (en) * | 2006-02-28 | 2006-10-25 | 陈万军 | Method for treating throat cancer |
| CN101366950A (en) * | 2008-09-18 | 2009-02-18 | 武汉大学 | Uses of gamma-ray combined phTERT-HRP/IAA in medicament for treating cancer of larynx |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657129A (en) * | 2022-04-11 | 2022-06-24 | 中山大学孙逸仙纪念医院 | Bile duct cancer lymph node metastasis animal model and construction method thereof |
| CN114657129B (en) * | 2022-04-11 | 2023-12-01 | 中山大学孙逸仙纪念医院 | Bile duct cancer lymph node metastasis animal model and construction method thereof |
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Application publication date: 20130724 |