CN107541496A - A kind of human esophageal carcinoma cell line and its application - Google Patents
A kind of human esophageal carcinoma cell line and its application Download PDFInfo
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Abstract
The invention discloses a kind of human esophageal carcinoma cell line and its application.The human esophagus cancer cell is deposited in China typical culture collection center, and deposit number is CCTCC NO:C201661.The human esophageal carcinoma cell line is on the premise of Major Clinical biological property is retained, with tumor formation rate height, incubation period is short, the features such as homogeneity is good, enrich human esophagus cancer cell storehouse, to provide strong scientific research data based on the research that Chinese population genetic background is carried out, also new test material is provided for experiment in vitro in new drug preclinical study body.
Description
Technical field
The invention belongs to cell line field, more particularly to a kind of human esophageal carcinoma cell line and its application.
Background technology
The cancer of the esophagus refers to the malignant tumour in epithelium of esophagus source between being combined from hypopharynx to gastric and esophageal.Oesophagus
Cancer is one of most common malignant tumour of the mankind, but Incidence level height differs greatly, and has obvious region
Characteristic.The country occurred frequently of China's especially cancer of the esophagus, the hat in its morbidity and mortality Jun Ju worlds.
According to statistics, about more than 20 ten thousand people die from the cancer of the esophagus every year in the whole world, and wherein China dies from oesophagus every year
The people of cancer person about 150,000, accounts for global 3/4ths.95% above is squamous in China, the cancer of the esophagus
Cell cancer, minority is the gland cancer originating from oesophagus body of gland or ectopic gastric mucosa, accidental to have adenosquamous carcinoma or gland spine
Cancer.
The normal unobvious of cancer of the esophagus early symptom, but may have different degrees of discomfort when swallowing thick and stiff food
Feel, including swallow food choking feeling, sample, acupuncture sample or drawing friction sample pain are burnt after breastbone.In
Late period typical symptom is the food of progressive dyscatabrosis, before this difficult dry throat, is then semiliquid diet,
Last water and saliva can not be swallowed.Mucoid phlegm often is told, it is the saliva swallowed and the secretion of oesophagus.
Patient gradually becomes thin, is dehydrated, be powerless.Continue pectoralgia or backache and be expressed as late period symptom, cancer invaded to
Organized outside oesophagus.Therefore, middle and advanced stage has been belonged to when most patient is made a definite diagnosis, and due to the size of tumour, portion
Position etc. influences, it is impossible to is resistant to surgery excision, the treatment hand that chemicotherapy can only be used to be combined with operative treatment
Section.
It is the treatment means using surgical operation as standard in treatment very long a period of time of the cancer of the esophagus, but by
In postoperative higher recurrence rate, 90% Patients on Recurrence transfer there are about.Even patient by stages very early
(T1) patient for, still having nearly 50% was recurred in 5 years.Therefore, total pre- of primary esophageal carninomatosis people
Bad afterwards, there is an urgent need to seek significantly more efficient treatment method.
Lack at present and given birth to for what cancer of the esophagus genesis mechanism and anti esophageal cancer medicine were developed close to clinical tumor
The experiment material of thing characteristic.And the success rate that cell line is directly established with clinical tumor tissue is relatively low,
Therefore, by first establishing animal model, and then the people established by original cuiture with clinical tumor sample
Source tumour cell has the Clinical Biological closer to tumour, to the drug resistance and sensitiveness of medicine
There to be preferably predictability, and be to study oesophagus carcinogenesis molecular mechanism and the ideal of screening anti-tumor medicine
Experiment material.
The content of the invention
Two packing spaces of the technical problem to be solved in the present invention aiming at existing human esophageal carcinoma cell line
Degree is low, larger deficiency is differed with the biological character of the clinically cancer of the esophagus for property, there is provided a kind of new people
Esophageal carcinoma cell line and its application.
The human esophageal carcinoma cell line has tumor formation rate high on the premise of Major Clinical biological property is retained
The features such as, human esophagus cancer cell storehouse is enriched, the research to be carried out based on Chinese population genetic background is provided
Strong scientific research data, also provides new test material for experiment in vitro in new drug preclinical study body.
Technical scheme is used by the present invention solves above-mentioned technical problem：A kind of human esophageal carcinoma cell line,
It is deposited in China typical culture collection center, and deposit number is CCTCC NO:C201661.
The present invention also provides the daughter cell of above-mentioned human esophageal carcinoma cell line.
The present invention also provides the purposes of above-mentioned human esophageal carcinoma cell line, and it is used to prepare in the inhuman food in one's mouth
The reagent of the cancer of the esophagus is produced in newborn animal.
In the present invention, described inhuman mammal is the conventional inhuman mammal in this area,
Preferably immunodeficient mouse, described immunodeficient mouse are the conventional immunodeficient mouse in this area.
The immunodeficient mouse preferably transformed, such as the immunodeficient mouse of immunity function restructuring.More preferably wrap
Include nude mouse or SCID mouse.It is preferred that described esophageal carcinoma cell line is squamous cell carcinoma system.
The present invention also provides a kind of method for building up of above-mentioned human esophageal carcinoma cell line, and it comprises the following steps,
1) fresh clinical Operation on Esophageal Cancer Operated Specimens are obtained, are cut into 20~50mg fritter, subcutaneously
Percutaneous puncture-inoculation mammal；
2) percutaneous puncture-inoculation put to death tumor animal after 70~90 days, took out tumor tissues, and it is thin to carry out cancer
The original cuiture and Secondary Culture of born of the same parents.
Wherein, described mammal, the described cancer of the esophagus are all as described above.
In the present invention, described fresh clinical human esophagus cancer Operated Specimens preferably use mammalian cell
It is inoculated with again after nutrient solution or physiological saline rinsing.More preferably rinsed with fresh HBSS buffer solutions,
The HBSS buffer solutions contain 500U/mL benzyl penicillins, 500 μ g/mL streptomycin sulphates and 1.25
μ g/mL amphotericin Bs.
In the present invention, described primary culture method can be the original cuiture of conventional mammalian cell
Method.It is preferred that it comprises the following steps：Tumor tissues are resuspended in digestion, filter, and purifying, obtain scattered
Cell, scattered cell is resuspended in PC-1 nutrient solutions, is seeded in blake bottle, in 37 DEG C of incubators
5% (v/v) CO2Under the conditions of quiescent culture to cell cover with blake bottle bottom more than 80%.Original cuiture is every
Change liquid once within 3~4 days.The PC-1 nutrient solutions are the conventional PC-1 nutrient solutions in this area, preferably
Purchased from the PC-1 of Lonza companiesTMNutrient solution.
In the present invention, described Secondary Culture method can be the Secondary Culture side of conventional mammalian cell
Method.Preferably described Secondary Culture method comprises the following steps：Inhale the blake bottle abandoned and carry out original cuiture
In nutrient solution, into blake bottle add 0.05% (w/w) trypsin solution, after cell detachment,
Digestion is terminated, fresh PC-1 nutrient solutions are added into bottle., will after passage to the tenth generation
Nutrient solution is replaced by RPMI1640 nutrient solutions.The RPMI-1640 nutrient solutions include 10% (w/w)
Hyclone, 10 μ g/mL benzyl penicillins and 2 μM of hydrocortisones, the percentage are quality percentage
Than.
The present invention provides experimental method inside a kind of screening treatment cancer of the esophagus drug candidate, it is described inside
Experimental method comprises the following steps：Test compound is applied to animal model, the cancer of the esophagus is caused after
The test compound that symptom improves or cured is exactly to treat the candidate compound of the cancer of the esophagus, wherein described is dynamic
Thing model has the oesophagus tumor caused by human esophageal carcinoma cell line as described above.
It is preferred that experimental method comprises the following steps inside described：
(1) described esophageal carcinoma cell line or its daughter cell are prepared into cell suspension, are inoculated in the food in one's mouth
Under newborn Animal Skin, raised, obtain human esophagus cancer animal model；
(2) test compound is applied to animal model, causes cancer of the esophagus symptom to improve or control after
More test compound is exactly to treat the candidate compound of the cancer of the esophagus.
In the present invention, described animal model is the conventional animal model in this area, preferably nude mouse.
Described nude mouse is the conventional nude mouse in this area, preferably Nu/Nu nude mouses.Establish animal mould
The method of type is the conventional method in this area.It is preferred that animal mould can be established using pallium cell injection
Type.In step of applying, by test compound by tail vein injection, oral, intraperitoneal injection or in swollen
The modes such as knurl local application are applied to cancer of the esophagus tumor animal.It is preferred that methods described is also including the use of right
According to the step of.The control is the conventional control in this area, it is preferred that for without the molten of test compound
Agent is applied to cancer of the esophagus tumor animal.
The present invention also provides a kind of ill vitro test method for screening treatment cancer of the esophagus drug candidate, described body
Outer experimental method comprises the following steps：Test compound is directly applied to tumour cell with various concentrations,
According to the inhibition to tumor cell proliferation, anti-tumor capacity of the test compound to the cancer of the esophagus is judged.
Specifically, ill vitro test method of the present invention comprises the following steps：
(1) described esophageal cancer cell or its daughter cell are inoculated in 96 porocyte culture plates holes,
Culture 24 hours；
(2) cell is applied to after test compound is diluted, medicine is thin by determining after acting on 72 hours
The vigor of born of the same parents, by the Proliferation Ability ability of compound on intracellular, the half for calculating compound suppresses dense
Degree, so as to judge the anti-tumor capacity of test compound.
In the present invention, above-mentioned optimum condition can be combined on the basis of common sense in the field is met, and be produced
The preferred embodiments of the invention.
Raw material or reagent used in the present invention is commercially available in addition to special instruction.
It is as follows compared to prior art, beneficial effects of the present invention：The human esophagus cancer cell character of the present invention
It is stable, can stablize and repeatedly pass on, for cancer of the esophagus research provide newly closer to clinical tumor biological characteristics
The experiment material of property.With Tumor formation, oesophagus carcinoma animal model, obtained animal can be successfully prepared
Model can be used for basic research and drug screening.By compared with nude mouse interior generation parent's tumour,
Can be used to analyze the correlation of external, internal drug susceptibility and drug resistance, so can establish it is external,
Internal two associated anti esophageal cancer medicine Screening Platforms.It can also be used for studying the morbidity of Metastasis of Esophageal Carcinoma
The resistance mechanism of mechanism, the cancer of the esophagus, and then the feature biological marker of Metastasis of Esophageal Carcinoma and resistance can be found
Thing.It is the ideal cell line of human esophagus cancer basic research and preclinical phase application.
Biomaterial preservation information
The human esophageal carcinoma cell line of the present invention, Chinese Typical Representative culture is deposited on April 13rd, 2016
Collection (CCTCC), preservation address：Wuhan, China Wuhan University postcode 430072, preservation
Numbering is CCTCC NO：C201661, the entitled human esophagus cancer cell ESXC-006 of culture.
Brief description of the drawings
Fig. 1 is the result of the morphological observation (100 ×) of ESXC-006 cells.
Fig. 2A~Fig. 2 B are the result of the chromosome analysis of ESXC-006 cells.
Fig. 3 is ESXC-006 cell doubling time curves.
Fig. 4 is ESXC-006 cell growth curves.
Fig. 5 A~Fig. 5 F are that the suppression that the multiple cancer therapy drugs of testing in vitro are bred to ESXC-006 cells is made
Result.Wherein, A is oxaliplatin, and B is fluorouracil, and C is adriamycin, and D replaces for Yi Li
Health, E are Docetaxel, and F is cis-platinum.
Fig. 6 A~Fig. 6 I are short-movie section repetitive sequence (STR) analysis result of ESXC-006 cells.Its
Middle A~I be respectively STR bit point Amelogenin, THO1, TPOX, D13S317, vWA, D16S539,
D5S818, CSF1PO and D7S820.
Fig. 7 is the result detected the ESXC-006 cell cycles.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but is not therefore limited the present invention to
Among described scope of embodiments.The experimental method of unreceipted actual conditions in the following example, according to normal
Rule method and condition, or selected according to catalogue.
The preparation of the ESXC-006 cells of embodiment 1
From Provincial Hospital obtain fresh clinical Resection Esophagus Carcinoma sample (female, Chinese, Han nationality,
72 years old, cancer of the esophagus, pathological diagnosis result was：Esophagus and cardia Operated Specimens, esophageal squamous cell carcinoma I levels,
Invade and submucosa, oesophagus peripheral lymph node 0/4, orifice of the stomach：Gland cancer I-II levels, are confined to submucosa,
Orifice of the stomach peripheral lymph node 0/11, knuckle inferior gluteal lymph node 0/5), the sterile HBSS for immersing precooling immediately is buffered
(benzyl penicillin containing 500U/ml, 500 μ g/ml streptomycin sulphates and 1.25 μ g/ml anphotericins in liquid
B).In Biohazard Safety Equipment, with the fresh sterile HBSS wash buffers sample, it is cut into 20~
50mg fritter, it is (real purchased from Shanghai Si Laike that subcutaneous puncture is inoculated in immunodeficient animals SCID mouse
Test animal Co., Ltd).After animal inoculation pvaccination, health status is good, and behavior is normal.Inoculation 20 days
Afterwards, find subcutaneously there is tumor nodules section in inoculation position by touching, lesser tubercle is opened after being inoculated with 40 days
Growth of beginning is obvious, and when to after being inoculated with 60 days, gross tumor volume is more than 300mm3。
Subcutaneous puncture was inoculated with human esophagus cancer after 70~90 days, and lotus knurl immunodeficient SCID mice is excessive
Carbon dioxide anesthesia is put to death, and sterile dissection, takes out tumor tissues, carries out original cuiture.
Original cuiture：With HBSS buffer solutions (benzyl penicillin containing 500U/ml, 500 μ g/ml sulfate chains
Mycin and 1.25 μ g/ml amphotericin Bs) rinse tumor tissue 3 times, remove connective tissue and necrosis
Tissue；Tumor tissues are resuspended in digestion, filter, and purifying, obtain scattered cell, and scattered cell is resuspended
In PC-1TMNutrient solution (is purchased from Lonza companies), is seeded in blake bottle, in 37 DEG C of incubators 5% (v/v)
CO2Under the conditions of quiescent culture to cell cover with blake bottle bottom more than 80%；Original cuiture changes liquid in every 3~4 days
Once.
After the cell grown in by tissue block is covered with blake bottle bottom, passage is carried out.
Secondary Culture：The nutrient solution abandoned in the blake bottle for carrying out original cuiture is inhaled, is added into blake bottle
0.05% (w/w) trypsin solution, after cell detachment, digestion is terminated, adds fresh PC-1TM
Nutrient solution (is purchased from Lonza companies).After passage to the 10th generation, nutrient solution is replaced by
RPMI1640 nutrient solutions are [containing 10% (w/w) hyclone, 10 μ g/ml rh-insulins, 10 μ g/ml
Benzyl penicillin and 2uM hydrocortisones], cell growth is good, and form is more homogeneous.It is passaged to 50
It is more than generation.
In the present invention, the original cuiture and cultured cell line from tumor tissues are in Epithelial, carefully
Born of the same parents' form is more homogeneous, contactless suppression, and primary growth speed is more slow；With the increasing of passage number
Add, the speed of growth is progressively accelerated；The cell line is named as ESXC-006, April 13 in 2016
Day is deposited in China typical culture collection center (CCTCC), and deposit number is CCTCC NO:
C201661, the entitled human esophagus cancer cell ESXC-006 of culture.
The biological characteristics of the ESXC-006 cells of embodiment 2 and application
The present invention is first using the low albumen PC-1 without serumTMNutrient solution culture ESXC-006 cells,
After passage to the 10th generation, nutrient solution is replaced by the RPMI containing hyclone and insulin
1640 culture medium culture ESXC-006 cells, can long term growth and stable passage in vitro.Work as cell
It is more than generation to reach 20, cell quality is gradually stablized, and the biology, science of heredity and tissue for carrying out correlation come
Source is identified, until the 50th generation all had the stable character of identical.Through Germicidal efficacy with verifying, in vitro
The ESXC-006 cells of growth have typical Epithelial form, contact growth inhibition are lost, in pernicious
Growth.Genetics research confirms that the cell is heteroploid, and Numerical and structural chromsomal aberrations are serious, meet
The genetics characteristics of malignant tumour.The ESXC-006 cells can form tumour in nude mice, have
Oncogenicity.Passed in the ESXC-006 cells and the clinical cancer of the esophagus tumor specimen in its source, nude mice
Corresponding relation is formed for parent's tumour, can be to study external, internal and clinical anti-cancer drug susceptibility phase
Guan Xing, and generation, development, transfer and the biomarker of the cancer of the esophagus provide new test material.Tool
Body is as follows：
A. morphological observation
The blake bottle for cultivating ESXC-006 cells is placed under inverted microscope, clapped under bright-field
According to.As a result Fig. 1 is seen, the visible ESXC-006 cells of Fig. 1 lose contact inhibition, in malignancy,
The characteristics of with overlapping growth.Cell is in mainly polygonal, is differed in size, endochylema enriches, adherent growth
Part is in flat, based on irregular paving stone sample, the characteristics of meeting epithelioid cell.
B. the identification of chromosome
The ESXC-006 cells of culture are placed in 37 DEG C, 5% (v/v) CO2Under the conditions of cultivate 12 hours
Afterwards, colchicine is added, makes its final concentration of 0.4 μ g/ml, continues culture 10 in 37 DEG C of incubators
Hour.The cell of metaphase is gathered, is fixed with fixer, then drips cell suspension in precooling
Micro slide on, with Giemas dyeing liquors dye, the enumerating chromosomes number under microscope.As a result figure is seen
2.Fig. 2 is visible, and after ESXC-006 cell continuous passages, chromosome still keeps humanized's tumour cell to contaminate
The feature of colour solid, modal number (M) concentrate between 103~119, account for 45.05%, majority be present
Center and submetacentric chromosome (Fig. 2A, 1000 ×)；And the chromosome number 2n=40 of nude mouse,
And be top centromere (Fig. 2 B, 1000 ×), it can be distinguished accordingly with human chromosomal.It can be seen that should
ESXC-006 cells are heteroploid, and Numerical and structural chromsomal aberrations are serious, meet the something lost of malignant tumour
Pass and learn feature.
C. cyto-dynamics is tested
ESXC-006 cells are seeded in 96 orifice plates with the density in 2000/hole, 37 DEG C, 5% (v/v)
CO2Under the conditions of cultivate, cultivate respectively 12 hours, 24 hours, 36 hours, 48 hours, it is 72 small
When and 96 hours fixed cells, and carry out PI dyeing, measured with high intension cell screening instrument Acumen
Per hole cell number the specification of high intension cell screening instrument (assay method referring to).Wherein, the doubling time
Calculation formula be：Doubling time=Log2/ the slopes of curve.
As a result for shown in Fig. 3.Doubling time (X) and the curve of Log fluorescence signals (Y) in Fig. 3
Equation is：Y=0.0076X+5.1155；Wherein R2=0.9597.Tied by Fig. 3 cyto-dynamics research
Fruit shows that cyto-dynamics result of study is shown, the population doubling time of ESXC-006 cells is 39.6
Hour.
D. cell growth curve
By ESXC-006 cells with 1000,2000,3000,4000,5000,6000,7000,8000,
9000th, the density in 10000/hole is seeded in 96 orifice plates, in 37 DEG C of incubator 5% (v/v) CO2
Under the conditions of cultivated.Used respectively at 0 day, 1 day, 2 days, 3 days, 4 days, 5 days and 6 days
Cell viability of the mtt assay measure per hole.
As a result as shown in figure 4, when cell-seeding-density is up to more than 4000/hole, can be arrived at the 4th day
Up to plateau, cell-seeding-density 1000,2000,3000/hole enters exponential growth on the 5th day
Phase.
E. cell in vitro poison experiment
External test cancer of the esophagus clinic Common Chemotherapy medicine：Oxaliplatin (being purchased from Sigma, article No. O9512),
Fluorouracil (being purchased from Sigma, article No. 47576), adriamycin (being purchased from Sigma, article No. D1515),
Irinotecan (being purchased from Sigma, article No. I1406), Docetaxel (being purchased from Fluka, article No. 01885)
With the antiproliferative of cis-platinum (being purchased from Jiangsu Haosen Pharmaceutical Co., Ltd, Chinese medicines quasi-word H20040813)
Effect.Subject cell is seeded in 96 orifice plates with the density in 3000/hole, the medicine to various concentrations
After three days, lived with the cell under each drug concentration of CellTiter Glo kit measurements of Promega companies
Power, IC is calculated through XLFit softwares50(half-inhibition concentration).The step of determining cell viability is according to examination
The specification of agent box is carried out.Its result is as shown in Fig. 5 and table 1~2.As a result ESXC006 cells are illustrated
Sensitive to chemotherapeutics Docetaxel (Fig. 5 E), its IC50 is less than 0.032.Adriamycin (Fig. 5 C),
Irinotecan (Fig. 5 D) and cis-platinum (Fig. 5 F) are stronger to ESXC-006 cell growth inhibitions,
IC50 is in the range of 0.1-10 μM；And oxaliplatin (Fig. 5 A) and fluorouracil (Fig. 5 B) are right
The inhibitory action of ESXC-006 cells is then weaker, and its IC50 is all higher than 200.
The inhibitory action that the multiple cancer therapy drugs of the testing in vitro of table 1 are bred to ESXC-006 cells
Half-inhibition concentration (IC of the 2 various medicines of table to ESXC-00650)
| Medicine name | Oxaliplatin | Fluorouracil | Adriamycin | Irinotecan | Docetaxel | Cis-platinum |
| IC50 | >200 | >200 | 0.159 | 5.702 | <0.032 | 2.390 |
F. short-movie section repetitive sequence (STR) is identified
STR (short tandem repeat, STR) is also known as microsatellite DNA, refers to
On chromosome, by several base-pairs as core unit (2-6 base-pair), tandem sequence repeats formed one
Class DNA sequence dna (number of repetition is more than 10~60 times, and genetic fragment is below 400 base-pairs)；Each
Individual difference occurs in the number that core unit repeats, so as to form the different allele of fragment length.
Therefore, the number of repetition of one group of STR sequence is almost unique in Different Individual, is the base of individual
Because of identity characteristic, and the main method that cell biology is identified cell identity and source.
The ESXC-006 cells of fresh cultured are collected, with AxyPrep genomic DNA small volume of reagent boxes
(it is purchased from the AxyPrep of Axygen companiesTMMultisource Genomic DNA Miniprep Kit,
Article No. is AP-MN-MS-GDNA) extraction cell genomic dna, with the primer of 5 ' end fluorescence labelings
Enter performing PCR amplification, products therefrom is sequenced, analysis include Amelogenin, THO1, TPOX,
Each STR bit such as D13S317, vWA, D16S539, D5S818, CSF1PO and D7S820
Primer sequence (its nucleotide sequence such as sequence table SEQ ID of the sequence repeat number, wherein STR bit point of point
Shown in No.1~18) and copy number as shown in table 3~4 and Fig. 6.Above-mentioned sequence and ATCC, DSMZ
The database that storehouse is preserved Deng cell carries out inquiry contrast, does not return to identical genetic map, in U.S. typical case
In culture collection (ATCC) database, STR sequence retrievals are carried out, do not find identical STR
Testing result, it is possible thereby to prove its uniqueness, and do not occur during original cuiture and other
The cross pollution of cell.
The primer sequence in table 3STR sites
The primer sequence and copy number in table 4STR sites
G. the cell cycle is detected
ESXC-006 cells are seeded in 96 orifice plates with the density in 1000/hole, 37 DEG C of incubators 5%
(v/v)CO2Under the conditions of, after cultivating 24 hours, collect about 106Individual cell in 1.5mL centrifuge tubes,
Supernatant is abandoned in centrifugation.Cell precipitation is resuspended with 1mL-20 DEG C 75% (v/v) ethanol, and room temperature fixes 1
Hour.Supernatant is abandoned in centrifugation, after cell precipitation is washed with PBS, adds 500 μ L PI dyeing liquors.Mix,
Incubation at room temperature 30 minutes.Measured with high intension cell screening instrument Acumen per hole cell number and each thin
(the STb gene content of each cell and total PI fluorescence intensities of the cell are in just for the STb gene content of born of the same parents
Than)；And during according to the different cell cycles, the change of cell STb gene content, calculate each cell cycle
TCS.As a result as shown in fig. 7, each period profile ratio is as shown in table 5.Bred according to cell
Index (PI) formula PI=(G2/M+S) ÷ (G0/G1+S+G2/M) × 100%, calculates to obtain ESXC-006
Cell PI=55.58%.
Table 5ESXC-006 cell cycle distribution ratios
| Cycle | Ratio (%) |
| G0/G1 | 44.42 |
| S | 48.45 |
| G2/M | 7.13 |
It should be understood that after the above of the present invention has been read, those skilled in the art can be to this hair
Bright to make various changes or modifications, these equivalent form of values equally fall within the application appended claims and limited
Scope.
Claims (10)
1. a kind of human esophagus cancer cell, it is characterised in that it is deposited in China typical culture collection
The heart, deposit number are CCTCC NO:C201661.
2. the daughter cell of human esophagus cancer cell as claimed in claim 1.
3. the purposes of human esophagus cancer cell as claimed in claim 1 or 2, it is characterised in that be used for
Prepare the reagent that oesophagus tumor is produced in inhuman mammal.
4. the purposes of human esophagus cancer cell as claimed in claim 3, it is characterised in that the described food in one's mouth
Newborn animal is immunodeficient mouse.
5. the purposes of human esophagus cancer cell as claimed in claim 4, it is characterised in that described exempts from
Epidemic disease deficient mice is the immunodeficient mouse of immunity function restructuring.
6. the purposes of human esophagus cancer cell as claimed in claim 4, it is characterised in that described exempts from
Epidemic disease deficient mice includes nude mouse or SCID mouse.
7. a kind of method for building up of human esophagus cancer cell as claimed in claim 1 or 2, its feature exist
In, comprise the following steps,
1) fresh clinical Operation on Esophageal Cancer Operated Specimens are obtained, are cut into 20~50mg fritter, skin
Lower percutaneous puncture-inoculation mammal；
2) percutaneous puncture-inoculation put to death tumor animal after 70~90 days, took out tumor tissues, and it is thin to carry out cancer
The original cuiture and Secondary Culture of born of the same parents.
8. method as claimed in claim 7, before carrying out the subcutaneous puncture inoculation, described is fresh
Clinical human esophagus cancer Operated Specimens with HBSS buffer solutions rinse, described HBSS buffer solutions include 500
The amphotericin B of U/ml benzyl penicillin, 500 μ g/ml streptomycin sulphate and 1.25 μ g/ml；
Described primary culture method comprises the following steps：Tumor tissues are cut into fritter, insert culture
In bottle, cultivated under 37 DEG C of incubators；Next day, blake bottle is slowly overturn and kept flat, PC-1 is added into bottle
Nutrient solution, quiescent culture；When cell spread into account for culture bottle surface 70% it is full when carry out Secondary Culture and pure
Change；
The Secondary Culture comprises the following steps：Old nutrient solution is abandoned in suction, and fresh 0.05% is added into bottle
Trypsin solution, after cell detachment, fresh PC-1 nutrient solutions are added into bottle, when cell passes
After generation to the tenth generation, nutrient solution is replaced by RPMI1640 nutrient solutions, the RPMI-1640 cultures
Liquid includes 10% (w/w) hyclone, 10 μ g/mL benzyl penicillins and 2 μM of hydrocortisones, institute
It is mass percent to state percentage；The method of the purifying is differential velocity adherent.
9. experimental method inside one kind screening treatment cancer of the esophagus drug candidate, it is characterised in that it is wrapped
Include following steps：Test compound is applied to animal model, cause after cancer of the esophagus symptom improve or
The test compound of healing is exactly to treat cancer of the esophagus candidate compound, and described animal model has right will
Seek the oesophagus tumor caused by the human esophagus cancer cell described in 1 or 2.
10. a kind of ill vitro test method for screening treatment cancer of the esophagus drug candidate, it is characterised in that it is wrapped
Include following steps：Test compound is directly applied to various concentrations to the people described in claim 1 or 2
Esophageal cancer cell, according to the inhibition to tumor cell proliferation, judge test compound to the cancer of the esophagus
Anti-tumor capacity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019196606A1 (en) * | 2018-04-13 | 2019-10-17 | Primordial Biotech. Co. | Method for obtaining an animal model from conditionally reprogrammed cells and use of the animal model for screening anti-tumor drugs |
| CN111662874A (en) * | 2020-06-18 | 2020-09-15 | 上海市胸科医院 | Chinese esophageal squamous carcinoma cell line and application thereof |
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| CN112210538A (en) * | 2020-10-23 | 2021-01-12 | 中国医学科学院肿瘤医院 | Human esophageal squamous carcinoma cell line NCCE1, and establishment method and application thereof |
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