CN109295156A - Chemicals induce the method for building up of the vitro detection model of human esophagus cancer - Google Patents
Chemicals induce the method for building up of the vitro detection model of human esophagus cancer Download PDFInfo
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Abstract
The invention discloses the method for building up that a kind of chemicals induce the vitro detection model of human esophagus cancer, comprising the following steps: provides the people's esophageal epithelial cell being seeded in in vitro culture container；The predetermined time is cultivated after applying the chemicals to be checked of multiple concentration within the scope of predetermined concentration respectively to people's esophageal epithelial cell, measures the curve that the cell viability of people's esophageal epithelial cell applies the variation of concentration with the chemicals to be checked；The concentration ranges of the corresponding chemicals to be checked of predetermined cell viability range in the curve are chosen as carcinogenic measurement concentration ranges, choose multiple concentration of the carcinogenic measurement concentration ranges as carcinogenic measurement concentration；And apply the chemicals to be checked of the carcinogenic measurement concentration respectively to people's esophageal epithelial cell, it carries out the chemicals to be checked and the carcinogenicity testing of people's esophageal epithelial cell is detected.
Description
Technical field
The present invention relates to field of biotechnology, and the vitro detection model of human esophagus cancer is induced more particularly to a kind of chemicals
Method for building up.
Background technique
Cancer occurs to be usually inherent cause and the interactive result of environmental factor, it is considered that 90% human cancer
It is related with environmental factor, wherein mainly chemicals factor, accounts for 90% or more.Currently, the long-term carcinogenic test of rodent
It is the chemicals carcinogenicity standard evaluation method uniquely received by regulatory agency of various countries.Although other test methods such as structure-activity relationship
Analysis, genetic toxicity test, cell malignant transformation test and crowd's epidemiology investigations of tumor etc. are used for chemical carcinogenesis
Evaluation, rodent carcinogenic test can be replaced but without a kind of ideal test macro.Chemicals are to induce people's food
The principal element of pipe cancer is based primarily upon zoopery to the research of human esophagus cancer currently based on chemicals.Based on zoopery
Time-consuming for carcinogenic test research, investment is big, is unfavorable for the high-throughput toxicity for detecting a large amount of chemicals.On the other hand, existing cause
Carcinous animal experiment, there is also many limitations, is mainly reflected in when being applied to the evaluation of chemicals Safety and toxicology: animal dye
It is horizontal that toxic dose is significantly larger than human body actual exposed；The carcinogenicity of smallest number experimental animal to a large amount of crowds have greatly not true
It is qualitative；Mixing exposure and the reciprocation etc. of compound can not be measured.
In recent decades, the substitution that is used with experimental animal, reduction, optimization " decades principle advocate with implement with
And the transformation of biomedical research mode, the external model research for substituting zoopery have become the important side of toxicology development
To based on human archeocyte, obtain academia, industry and regulatory agency by the conversion toxicologic study of core of toxicity access
Extensive concern.The external alternative of current toxicology test is answered in acute toxicity test such as Skin Irritation Test etc.
With, but meet difficulty in chronic toxicity and carcinogenic test or observation complexity effect terminal.Therefore, lack efficiently, accurately
Ill vitro test method carrys out clear chemicals and induces the relationship of human esophagus cancer.
Summary of the invention
Based on this, it is necessary to provide a kind of method for building up of the vitro detection model of chemicals induction human esophagus cancer, be used for
Efficiently, the relationship of accurate detection chemicals and induction human esophagus cancer.
A kind of method for building up of the vitro detection model of chemicals induction human esophagus cancer, comprising the following steps:
The people's esophageal epithelial cell being seeded in in vitro culture container is provided；
It is trained after applying the chemicals to be checked of multiple concentration within the scope of predetermined concentration respectively to people's esophageal epithelial cell
It supports the predetermined time, the cell viability that measurement is able to reflect people's esophageal epithelial cell applies concentration with the chemicals to be checked
The curve of variation；
The concentration ranges of the corresponding chemicals to be checked of predetermined cell viability range in the curve are chosen as cause
Cancer measures concentration ranges, chooses multiple concentration of the carcinogenic measurement concentration ranges as carcinogenic measurement concentration；And
The chemicals to be checked for applying the carcinogenic measurement concentration respectively to people's esophageal epithelial cell, described in progress
Chemicals to be checked detect the carcinogenicity testing of people's esophageal epithelial cell.
In one of the embodiments, inoculum density of the people's esophageal epithelial cell in the culture vessel be 8 ×
104/ ml~2 × 105/ml。
The minimum concentration of the predetermined concentration range is 0~100nmol/L in one of the embodiments, described predetermined
The maximum concentration of concentration range is 10mmol/L~100mmol/L.
The minimum cell viability within the scope of the predetermined cell viability is 90%~95% in one of the embodiments,.
Multiple concentration within the scope of the predetermined concentration are Geometric Sequence in one of the embodiments,.
The multiple carcinogenic measurement concentration is Geometric Sequence in one of the embodiments,.
The chemicals to be checked detect packet to the carcinogenicity testing of people's esophageal epithelial cell in one of the embodiments,
It includes ability of cell proliferation detection, cell migration and invasive ability detection and the chemicals to be checked induces animal tumor formation experiment inspection
One of survey is a variety of.
It in one of the embodiments, further include that the people is induced to chemicals to be checked described in carcinogenicity testing detection
Esophageal epithelial cell carries out gene expression analysis at the tumour cell of cancer, determines the gene and body metabolism access of differential expression
Relationship.
It in one of the embodiments, further include detection addition people's hepatomicrosome to the external of people's esophageal epithelial cell
The step of cell viability under culture influences.
The condition of culture of people's esophageal epithelial cell is 5% carbon dioxide, cultivation temperature in one of the embodiments,
It is 37 DEG C.
People's esophageal epithelial cell is inoculated in culture vessel and carries out in vitro culture by the present invention, by detecting people's oesophagus
It is described as may induce to choose predetermined cell viability range with the variation of chemical concentrations to be checked for the cell viability of epithelial cell
The carcinogenic measurement concentration ranges of people's esophageal epithelial cell generation canceration.It is described carcinogenic by applying to people's esophageal epithelial cell
The chemicals to be checked of multiple carcinogenic measurement concentration of measurement concentration ranges simultaneously carry out carcinogenicity testing detection, so that it is determined that described
The dosage of chemicals to be checked and the carcinogenic relationship for inducing people's esophageal epithelial cell.Due to the vitro detection letter of cell viability
It is single convenient, and the detection process of carcinogenicity testing is relatively cumbersome, is chosen and is caused according to the relationship of cell viability and chemical concentrations to be checked
Cancer measures concentration ranges, reduces the detection range of carcinogenic concentration, advantageously reduces experimental work amount, so that entire chemicals induce
The extracorporeal detection procedure of human esophagus cancer is more efficiently and accurately.
Compared with zoopery, the In vitro cell model the present invention is based on humanization is with people's esophageal epithelial cell for research
Object can more reflect human body actual conditions.The controllability of the experiment condition of In vitro cell model of the invention is stronger, can simulate
Influence of the chemicals to be checked of long-term low dose to people's esophageal epithelial cell can more reflect the actual environment for inducing human esophagus cancer.
The In vitro cell model has the detection means of intuitive and multiplicity, is suitable for research signal path and molecule in Carcinogenesis
In dynamic change.Meanwhile the In vitro cell model is suitble to large-scale culture, can satisfy the needs of high-throughput detection.Cause
This, vitro detection model of the invention can more accurately measure the relationship that chemicals induce human esophagus cancer.
Detailed description of the invention
Fig. 1 is the process for the method for building up that the chemicals of one embodiment of the invention induce the vitro detection model of human esophagus cancer
Schematic diagram；
Fig. 2A -2B is that the cell viability of reflection people's esophageal epithelial cell of one embodiment of the invention applies with chemicals to be checked
The curve of the variation of concentration, wherein Fig. 2A is the logarithm of chemical concentrations to be checked and the maximum inhibition of people's esophageal epithelial cell
Relationship, Fig. 2 B is cell viability and the control group people of experimental group people's esophageal epithelial cell under different chemical concentrations to be checked
The ratio of the cell viability of esophageal epithelial cell；
Fig. 3 is the chemicals to be checked of the reflection various concentration of one embodiment of the invention and the growth speed of people's esophageal epithelial cell
The curve of rate relationship；
Fig. 4 is the photo of the people's esophageal epithelial cell for the chemicals to be checked that one embodiment of the invention applies various concentration；
Fig. 5 be one embodiment of the invention people's esophageal epithelial cell cell specific gene mRNA expression rate with it is to be checked
Chemicals apply the curve of the relationship of concentration, wherein Fig. 5 A is Cyclin D1 gene, and Fig. 5 B is ki67 gene, and Fig. 5 C is PCNA
Gene；
Fig. 6 is that the cell of the people's esophageal epithelial cell for the chemicals to be checked that one embodiment of the invention applies various concentration moves
It moves and invasive ability photo；
Fig. 7 is the animal tumor formation experiment photo of one embodiment of the invention, wherein Fig. 7 A be animal tumor formation number and size with
Chemicals to be checked apply the photo of the relationship of concentration, and Fig. 7 B is the histopathology photo of the cell after tumor formation.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, by the following examples, it and combines attached
Figure, the method for building up for inducing the vitro detection model of human esophagus cancer to chemicals of the invention are further elaborated.It answers
Work as understanding, described herein specific examples are only used to explain the present invention, is not intended to limit the present invention.
Referring to Fig. 1, the embodiment of the present invention provides a kind of foundation of the vitro detection model of chemicals induction human esophagus cancer
Method, comprising the following steps:
S10 provides the people's esophageal epithelial cell being seeded in in vitro culture container；
S20 applies the chemicals to be checked of multiple concentration within the scope of predetermined concentration to people's esophageal epithelial cell respectively
After cultivate the predetermined time, measurement be able to reflect the cell viability of people's esophageal epithelial cell apply with the chemicals to be checked it is dense
The curve of the variation of degree；
S30, the concentration ranges for choosing the corresponding chemicals to be checked of predetermined cell viability range in the curve are made
For carcinogenic measurement concentration ranges, multiple concentration of the carcinogenic measurement concentration ranges are chosen as carcinogenic measurement concentration；And
S40 applies the chemicals to be checked of the carcinogenic measurement concentration respectively to people's esophageal epithelial cell, carries out
The chemicals to be checked detect the carcinogenicity testing of people's esophageal epithelial cell.
People's esophageal epithelial cell is inoculated in culture vessel and carries out in vitro culture by the embodiment of the present invention, by described in detection
With the variation of chemical concentrations to be checked, choosing predetermined cell viability range conduct may lure the cell viability of people's esophageal epithelial cell
Send out people's esophageal epithelial cell described and occur the carcinogenic measurement concentration ranges of canceration.By applying institute to people's esophageal epithelial cell
It states the chemicals to be checked of multiple carcinogenic measurement concentration of carcinogenic measurement concentration ranges and carries out carcinogenicity testing detection, thus really
The dosage of the fixed chemicals to be checked and the carcinogenic relationship for inducing people's esophageal epithelial cell.It is external due to cell viability
Detect simple and convenient, and the detection process of carcinogenicity testing is relatively cumbersome, according to the relationship of cell viability and chemical concentrations to be checked
Carcinogenic measurement concentration ranges are chosen, the detection range of carcinogenic concentration is reduced, advantageously reduce experimental work amount, so that entire chemistry
Object induces the extracorporeal detection procedure of human esophagus cancer more efficiently and accurately.
Compared with zoopery, the In vitro cell model the present invention is based on humanization is with people's esophageal epithelial cell for research
Object can more reflect human body actual conditions.The controllability of the experiment condition of In vitro cell model of the invention is stronger, can simulate
Influence of the chemicals to be checked of long-term low dose to people's esophageal epithelial cell can more reflect the actual environment for inducing human esophagus cancer.
The In vitro cell model has the detection means of intuitive and multiplicity, is suitable for research signal path and molecule in Carcinogenesis
In dynamic change.Meanwhile the In vitro cell model is suitble to large-scale culture, can satisfy the needs of high-throughput detection.Cause
This, vitro detection model of the invention can more accurately measure the relationship that chemicals induce human esophagus cancer.
Directly related cell of people's esophageal epithelial cell as the cancer of the esophagus, the chemicals as the embodiment of the present invention eat people
The research object of the In vitro cell model of the research of pipe cancer.
In one embodiment, people's esophageal epithelial cell can be selected from American type culture collection (ATCC
CRL-2692) the Het-1A cell line (people's esophageal epithelial cell) of preservation, state food security risk assessment central laboratory are protected
There is the Het-1A cell line.
First to the present embodiments relate to people's esophageal epithelial cell extracorporeal culturing method be introduced, people's oesophagus
Epithelial cell extracorporeal culturing method can be used for after step S10 people's esophageal epithelial cell is seeded in vitro culture container, apply to
Preculture before examining chemicals, step S20 apply the culture after chemicals to be checked and step S40 to people's esophageal epithelial cell
The cell culture that is related to of carcinogenicity testing detection process.
Preferably, the condition of culture of people's esophageal epithelial cell is 95% air, and 5% carbon dioxide, cultivation temperature is
37℃.The growth conditions of people's esophageal epithelial cell are more preferable under the condition of culture, can really grow closer to human body
Environment.Culture medium for cultivating people's esophageal epithelial cell can be bronchial epithelial cell basal medium
(Bronchial epithelial cell basal medium, BEGM).People's esophageal epithelial cell holds in the culture
It is adherent growth in device.
In the incubation of people's esophageal epithelial cell, it may include culture medium change liquid, cell passage, cell cryopreservation with
And cell recovery and etc..
People's esophageal epithelial cell can generate the harmful secondary metabolite of some pairs of cells during the cultivation process, described
Secondary metabolite has adverse effect on the growth rate of people's esophageal epithelial cell and metabolism, and the culture medium changes liquid
Step is used to exclude unfavorable metabolite, and the update of culture medium guarantees the good growth ring of people's esophageal epithelial cell
Border.Preferably, the time interval that the culture medium changes liquid can be 2.5 days to 3.5 days, can guarantee that people's epithelium of esophagus is thin
Born of the same parents have good continuous growth conditions.
In one embodiment, people's esophageal epithelial cell is preferably the area for growing to the inner surface of the culture vessel
70% or more when carry out cell passage.The cell passage can be to pass through people's esophageal epithelial cell of Primary Growth
Be added after the substance digestions such as pancreatin new culture medium continue on for cell culture formed passage cell, the passage cell with it is primary
People's esophageal epithelial cell have phase homokaryotype.
In one embodiment, the ultralow temperature that frozen stock solution is stored in -80 DEG C can be added in people from part esophageal epithelial cell
Environment, in case subsequent experimental.The frozen stock solution can be the mixed-culture medium of dimethyl sulfoxide and BEGM, preferably include quality point
Number is respectively 10% dimethyl sulfoxide, and (BEGM culture solution is raw for Lonza company for 10% fetal calf serum and 80%BEGM culture solution
Produce), be conducive to the preservation quality for improving people's esophageal epithelial cell, reduction freezes damage.
In step slo, preferably be seeded in after culture vessel first to cultivate by people's esophageal epithelial cell makes one to eat for a period of time
Pipe epithelial cell adherent growth, preferably 22 hours to 24 hours.People's esophageal epithelial cell in one of the embodiments,
Inoculum density in culture vessel can be 8 × 104/ ml~2 × 105/ ml, preferably 1 × 105/ml。
In step S20, the cell viability is hundred shared by living cells in the total cell in a certain moment culture vessel
Divide ratio, the growth conditions of the cell viability reflection cell, cell viability is higher, and cell growth state is better.In cell culture
In the process, some reasons, such as applying toxic chemicals, cell and itself generating harmful metabolite will lead to the dead of cell
It dies, cell activity is suppressed.In embodiments of the present invention, cause mainly due to the chemicals to be checked for applying various concentration
The cell viability of people's esophageal epithelial cell declines.Can by the cell viability of cytoactive inhibiting rate or experimental group with it is right
Reflect the cell viability according to the ratio of the cell viability of group.The chemistry to be checked of the experimental group addition various concentration
Object, the control group do not add the chemicals to be checked.The cytoactive inhibiting rate indicates the degree that cell viability inhibits, institute
State the calculation formula of cytoactive inhibiting rate are as follows:
In one embodiment, the range of the predetermined concentration of selection is unsuitable narrow, guarantees to obtain in wider concentration range
The cell viability of the people's esophageal epithelial cell arrived applies the curve of the variation of concentration with the chemicals to be checked.It is described predetermined
Concentration range can be determined according to the type of specific chemicals to be checked.The minimum concentration of the predetermined concentration range can be with
For 0~100nmol/L, preferably 0.The maximum concentration of the predetermined concentration range can be 10mmol/L~100mmol/L, excellent
It is selected as 10mmol/L~20mmol/L.Preferably, multiple concentration within the scope of the predetermined concentration are to change by Geometric Sequence
Concentration, it is ensured that the cell viability variation of people's esophageal epithelial cell is only related with concentration, reduces measurement accidental error.Deng
Ratio than ordered series of numbers is preferably 2 to 5.
In one embodiment, it is preferably to the predetermined time of people's esophageal epithelial cell culture after applying chemicals to be checked
20 hours to 26 hours, within the scope of the predetermined time, the compound to be checked fully penetrated can arrive people's epithelium of esophagus
The inside of cell, into the metabolism system of people's esophageal epithelial cell to induce canceration or lethal.
In step s 30, the predetermined cell viability ranges preferably from cell viability greater than 90%, and the carcinogenic concentration is surveyed
Determining section is preferably the concentration range that the cell viability is greater than the 90% corresponding chemicals to be checked.Cancer cell has unlimited
The property of division, quickly, after canceration occurs for cell, cancerous tumor cell can be proliferated proliferative capacity with cracking speed, due to
The number for the living cells that dead cell caused by cell metabolism increases rapidly relative to cell division is very little, so that the people of canceration
The cell viability of esophageal epithelial cell is close to 100%.When the cell viability is greater than 90%, people's esophageal epithelial cell base
Originally will not be lethal, but there is a possibility that carcinogenic.When cell viability is lower, the concentration of corresponding chemicals to be checked is more likely to
Keep people's esophageal epithelial cell lethal.It is furthermore preferred that the minimum cell viability within the scope of the predetermined cell viability is 90%
~95%, highest cell viability can be 100%.The measurement for first carrying out the cell viability, according to the survey of the cell viability
Determining result selects the concentration ranges of the corresponding chemicals to be checked of the predetermined cell viability range as the chemistry to be checked
The research range of the carcinogenic test can be reduced, advantageously reduce the work of complex experiment by the research section of object carcinogenicity testing
It measures, improves working efficiency, targetedly studied.
Multiple carcinogenic measurement concentration are selected to carry out further carcinogenic test in the carcinogenic concentration mensuration section.
In one embodiment, the multiple carcinogenic measurement concentration can be the concentration point changed by Geometric Sequence, guarantee institute
It states that carcinogenic test result is only related with concentration value, reduces the error of disturbing factor.The carcinogenic measurement concentration is preferably at least 3
It is a.The ratio of Geometric Sequence is preferably 10 to 20.
In step s 40, the chemicals to be checked may include cell to the carcinogenicity testing of people's esophageal epithelial cell
Proliferative capacity detection, cell migration and invasive ability detection and the chemicals to be checked induce one of animal tumor formation experiment
Or it is a variety of.
In one embodiment, the ability of cell proliferation detection may include: to apply respectively to people's esophageal epithelial cell
Add the different carcinogenic measurement concentration, and carries out cell growth rate detection, cell clonal formation experiment detection and cause
One of cancer correlation mRNA ability to express detection is a variety of.In identical detection time, the faster people of cell growth rate
The carcinogenic effect of the corresponding carcinogenic measurement concentration of esophageal epithelial cell is stronger；The more people's epithelium of esophagus of cell clone spot is thin
The carcinogenic effect of the corresponding carcinogenic measurement concentration of born of the same parents is stronger；The more people's esophageal epithelial cells of carcinogenic correlation mrna expression amount are corresponding
Carcinogenic measurement concentration carcinogenic effect it is stronger.
Specifically, the cell growth rate can be carried out the step of detection by the methods of decoration method, chemical reaction method
Detection.The decoration method may include: thin to the people's epithelium of esophagus for the chemicals to be checked for applying different carcinogenic measurement concentration
The step of born of the same parents apply cell dyeing liquid, make one the dead cell coloring of esophageal epithelial cell.The cell dyeing liquid can expect for platform
Blue dyeing liquor dyes people's esophageal epithelial cell, and dead cell is coloured, and living cells is not colored, so as to distinguish dead cell
With living cells, and then the cell growth curve of the number of living cells is obtained.People's epithelium of esophagus of different carcinogenic measurement concentration is thin
The growth rate of born of the same parents is different, and the growth rate carcinogenic effect for measuring concentration carcinogenic faster is stronger.
The decoration method can be adapted for the detection of the cell viability in the step S20.
The chemical reaction method may include: people's oesophagus to the chemicals to be checked for applying different carcinogenic measurement concentration
The step of epithelial cell applies specific chemical reagent, and specific chemical reagent and living cells is made to react.It is described specific
Chemical reagent can be CCK8 reagent, and the yellow first a ceremonial jade-ladle, used in libation product for reacting generation high water soluble of CCK8 reagent and living cells is raw
At first a ceremonial jade-ladle, used in libation product quantity it is directly proportional to the quantity of living cells.The chemical reaction method may further include detection reaction and produce
The step of absorbance of object.Absorbance is higher, and the number of living cells is more.The growth rate of the cell of canceration occurs than non-canceration
Cell growth rate faster, the chemicals to be checked of the carcinogenic measurement concentration of various concentration are obtained according to cell growth rate
Carcinogenic effect it is strong and weak.
Since the monoclonal that people's esophageal epithelial cell forms class bacterium colony needs intensive dividing cell, cancer cell is improper
Cell, the ability with infinite multiplication, is capable of the carry out cell division of rapid, high volume, and in the same time, cancer cell generates Dan Ke
Grand density is bigger.The colony formation may include: by the institute for the chemicals to be checked for applying different carcinogenic measurement concentration
It states people's esophageal epithelial cell to cultivate in the culture dish containing BEGM culture medium, counts the list in the culture dish of contemporaneity
Colony counts.When contemporaneity, the carcinogenic effect of the corresponding carcinogenic measurement concentration of the more culture dishes of monoclonal number is stronger.
After canceration occurs for people's esophageal epithelial cell, the ability to express of the mRNA of specific gene relevant to cell carcinogenesis
It can be remarkably reinforced.The carcinogenic correlation mRNA ability to express detection may include: that difference is carcinogenic to measure the to be checked of concentration to applying
The step of expression quantity of the mRNA of the specific gene of people's esophageal epithelial cell of chemicals detects.The specific gene
MRNA the more people's esophageal epithelial cells of expression quantity it is corresponding it is carcinogenic measurement concentration carcinogenic effect it is stronger.In an embodiment
In, the specific gene includes one of Cyclin D1, ki57 and PCNA or a variety of.
In one embodiment, the cell migration and invasive ability detection can by the cell Transwell detection method into
Row detection.Specifically, the detection of the cell Transwell may comprise steps of: the cell Transwell is put into culture plate,
It is used as upper chamber above the cell Transwell, lower room is used as below the cell Transwell, the cell Transwell upper layer is under
The culture solution of layer is separated by by polycarbonate membrane；By the institute of the chemicals processing to be checked of the carcinogenic measurement concentration of various concentration
The cell suspension inoculation of people's esophageal epithelial cell is stated to the upper chamber of the cell Transwell.Polycarbonate membrane has permeability,
People's esophageal epithelial cell can be attached to the lower surface of the polycarbonate membrane across polycarbonate membrane.It is attached by cell count
The lower surface of the polycarbonate membrane people's esophageal epithelial cell number, the more polycarbonate membranes of cell number are corresponding
Carcinogenic measurement concentration cell migration and invasive ability it is stronger, carciongenic potency is also stronger.Specifically, in the cell suspension
The concentration of people's esophageal epithelial cell can be 300/ μ L to 400/ μ L.The method for cell count can by Yihong or
Crystal violet carries out cell dyeing.
In one embodiment, the experimental animal that the chemicals to be checked induce animal tumor formation experiment can be nude mice.It is described
Animal tumor formation experiment may include: to implement described in the chemicals to be checked processing of the different carcinogenic measurement concentration of subcutaneous injection to nude mice
People's esophageal epithelial cell suspension induces the nude mice tumor formation.Record the chemistry to be checked of the carcinogenic measurement concentration of various concentration
People's esophageal epithelial cell suspension of object processing induces the parameters such as tumor formation time and the tumor formation rate of nude mice tumor formation.Further, also
The step of may include the tumour progress pathology detection to nude mice, judges the described to be checked of the carcinogenic measurement concentration of various concentration
Carcinogenic effect of the chemicals to people's esophageal epithelial cell.
It in one of the embodiments, further include that the chemicals to be checked are induced with people's esophageal epithelial cell into cancer
Tumour cell carries out the step of gene expression analysis.Gene expression analysis is used to determine that the gene of differential expression and body metabolism to be logical
The relationship on road, to predict that the chemicals to be checked induce the toxicity access of the cancer of the esophagus, for described in the chemicals induction to be checked
People's esophageal epithelial cell provides theories integration at the research of the disease treatment at cancer mechanism and human esophagus cancer of cancer.
It in one embodiment, further include detection addition people's hepatomicrosome under the in vitro culture of people's esophageal epithelial cell
Metabolic process in cell viability influence the step of, this is preferably a step carries out between the step S10 and S20.People liver
Microsome be with the immediate In vitro metabolism platform of human body, by detect whether addition people's liver particle to people's esophageal epithelial cell
The influence of the cell viability of in vitro culture, characterize the embodiment of the present invention In vitro cell model and human body true environment close to journey
Degree.
Embodiment
(1) cell culture of people's esophageal epithelial cell
People normal esophageal cell strain Het-1A (ATCC) is incubated at Bronchial epithelial cell basal
Medium (BEGM) culture medium.Culture vessel: 25cm2Culture bottle, 0.1% gelatin use after being coated with.Condition of culture: 95% is empty
Gas, 5% carbon dioxide, 37C.Every 3 days replacement culture solutions during cell culture, when bottle 80% area to be grown to confluent cultures into
Row passage.When cell passes on, after PBS washes cell twice, digested with pancreatin to cell rounding, BEGM culture medium is received after terminating reaction
Collect cell, supernatant is abandoned in 500g centrifugation after five minutes, and new culture solution is added with 1:3 and carries out secondary culture.When cell cryopreservation, choosing life
It after long status is good, eugonic cell washes cell twice with PBS, is digested with pancreatin to cell rounding, with containing 10% tire ox blood
Clear normal incubation medium terminates reaction, and cell 500g centrifugation abandons supernatant after five minutes, frozen stock solution is added.Frozen stock solution ingredient: 10%
DMSO, 10% fetal calf serum, 80%BEGM culture solution.Cell is sub-packed in cryopreservation tube, be put in program temperature reduction box be stored in-
In 80C ultra low temperature freezer, it is transferred in liquid nitrogen after 24 hours.When recovery cell, the cell frozen is taken out in liquid nitrogen, is set rapidly
37C water-bath, constantly shaking cryopreservation tube makes cell fast melt 70%, then cell is transferred in culture dish, normal incubation medium is added
Overnight incubation is carried out, liquid is changed after cell is adherent and continues to cultivate, passage when cell grows to 90% density.
(2) cell viability of preparation people's esophageal epithelial cell applies the curve of the variation of concentration with chemicals to be checked
The people normal esophageal epithelial cell strain Het-1A of above-mentioned culture is taken, with 105/ ml concentration is inoculated in 25cm2Culture bottle.
After 24 hour cell adherent growths, the chemicals Methyl jasmonate (NMBzA) to be checked of various concentration is added, NMBzA's
Concentration is respectively 0,0.5mM, 1.0mM, 2.0mM, 4.0mM, 8.0mM, 12.0mM (mM mmol/L).Culture 24 hours, measurement
Half lethal concentration LC50 of the NMBzA to Het-1A cell.Compare simultaneously and carries out metabolism work in 1%, 5% people's hepatomicrosome of addition
To the influence of LC50 under the conditions of change.
Please refer to Fig. 2A and Fig. 2 B, it can be seen that whether add people's hepatomicrosome to the cell viability of people's esophageal epithelial cell
Substantially without influence, illustrate that the In vitro cell model of people's esophageal epithelial cell and human body true environment are close.LC50=5.635mM.
Fig. 2A is reflected in the cytoactive inhibiting rate of people's esophageal epithelial cell under the conditions of the NMBzA of addition various concentration, it can be seen that
NMBzA concentration is higher, and cytoactive inhibiting rate is higher, illustrates the higher cell viability to people's esophageal epithelial cell of NMBzA concentration
Extent of the destruction it is higher.The cell of the NMBzA and the people's esophageal epithelial cell for not adding NMBzA of Fig. 2 B reflection addition various concentration
The ratio of vigor, it can be seen that NMBzA concentration is higher, and cell viability ratio is smaller, illustrates that NMBzA concentration is higher on people's oesophagus
The extent of the destruction of the cell viability of chrotoplast is higher.Do not cause cell toxicant substantially in Fig. 2 B discovery NMBzA < 200uM concentration range
Property effect, cell viability ratio is 100% to 95%, final to determine the model foundation examination that three concentration are carried out with 20 times of spacing
It tests, carcinogenic measurement concentration is selected as 500nM, and 10 μM, 200 μM.
(3) ability of cell proliferation detects
Various concentration Methyl jasmonate is added in the BEGM culture medium of people's esophageal epithelial cell to be contaminated for a long time
Culture, Methyl jasmonate concentration is respectively 500nM, 100M, 200MM；Solvent 0.1%DMSO generation is added in negative control cell
For Methyl jasmonate.The culture solution of replacement in every 3 days, passage is primary weekly, freezes the people of one group of contamination culture every two weeks
Esophageal epithelial cell cell.Timing observation cell growth state under the microscope, detects weekly cellular morphology, survival rate, growth
Rate etc..Carry out cell growth curve drafting, colony formation and the detection of carcinogenic correlation mRNA ability to express.
Cell growth rate detection: digesting the good contamination people esophageal epithelial cell pancreatin of cultivation conditions and count,
Cell is uniformly inoculated in 96 holes version, 96 orifice plates four according to the standard containing 4000 cells in every 200 μ L cell suspension of hole
The micropore on side not inoculating cell, is closed with culture solution.96 orifice plates are placed in 37 DEG C, 5%CO in incubator2Culture, 24 hours
After 10 μ L CCK-8 reagents are added, be incubated for 2 hours under cell culture condition, in 450nm ultraviolet waves strong point measure absorbance value,
And continuous 7 days were added CCK-8 reagent in the same time, absorbance value at same time measurement 450nm, and by first day extinction
Angle value is standardized as 100%, by several later days according to the absorbance value of different Cells On days progress Percentage Criterion
Change, and draws growth curve.Referring to Fig. 3, the application various concentration chemicals people oesophagus to be checked of Fig. 3 reflection different time measurement
The number of viable cells and inoculation viable count purpose ratio of epithelial cell.It can be seen that 10 μM of Methyl jasmonate processing people's foods
After pipe epithelial cell, people's esophageal epithelial cell obtains stronger growth ability, illustrates that 10 μM of Methyl jasmonates eat people
The carcinogenic effect of pipe epithelial cell is better than 500nM and 200 μM of Methyl jasmonate.
Cell clonal formation experiment: cultivation conditions good contamination people esophageal epithelial cell pancreatin is digested, is counted, is blown
Beating keeps cell fully dispersed at unicellular and according to the standard containing 500 cells in every hole 2mL cell suspension that cell is uniform
6 orifice plates are inoculated in, 37 DEG C, 5%CO in incubator are placed in2Culture, every 3 days one subcultures of replacement will be trained after culture 10 days
Feeding base discards, and forms dyeing liquor by 1:1 volume mixture with 0.5% crystal violet and 6% glutaraldehyde, and 2mL dyeing liquor is added in each hole,
6 orifice plates, are immersed in water clean dyeing liquor later, take pictures after drying by dyeing 45 minutes.Referring to Fig. 4, it can be seen that 10 μ
After M Methyl jasmonate handles people's esophageal epithelial cell, the number for clone's spot that people's esophageal epithelial cell is formed is more, says
Bright 10 μM of Methyl jasmonates are better than 500nM and 200 μM of methylbenzyl nitrous to the carcinogenic effect of people's esophageal epithelial cell
Amine.
Carcinogenic correlation mRNA ability to express detection: using the carcinogenic related mrna expression amount of Immunohistochemical Method detection.It will culture
Contamination people esophageal epithelial cell in good condition is digested with pancreatin, is counted, and piping and druming keeps cell fully dispersed at unicellular.And according to
Cell is uniformly inoculated in 6 orifice plates by the standard containing 500 cells in every hole 2mL cell suspension, is arranged 3 groups, is placed in culture
37 DEG C, 5%CO in case2Culture, is separately added into the labeling of monoclonal antibody of Cyclin D1, ki57 and PCNA in 3 groups of 6 orifice plates, so
High power lens observation is carried out afterwards, and the cell number of recording mark calculates the expression rate of Cyclin D1, ki57 and PCNA.Please refer to figure
5A-5C, it can be seen that after 10 μM of Methyl jasmonate processing people's esophageal epithelial cells, people's esophageal epithelial cell DE
The expression rate of Cyclin D1, ki57 and PCNA are higher, illustrate 10 μM of Methyl jasmonates to the carcinogenic of people's esophageal epithelial cell
Effect is better than 500nM and 200 μM of Methyl jasmonate.
(4) cell migration and invasive ability detection
500uL culture medium is added in 24 orifice plates, and the cell Transwell is put into, is incubated for 30 in 37 DEG C of incubators
Minute, the good contamination people esophageal epithelial cell pancreatin of cultivation conditions is digested and is counted, and with serum free medium according to
In the cell Standard entertion Transwell of 70000 cells of every 200uL cell suspension, the upper and lower of the cell Transwell
It is separated by with polycarbonate membrane, 24 orifice plates is placed in 37 DEG C, 5%CO in incubator2Eosin stains people oesophagus is used in culture after 18 hours
Epithelial cell 10 minutes, and use ddH2O rinsing, wipes polycarbonate membrane upper cell with cotton swab, takes pictures after drying.Please refer to figure
6, it can be seen that after 10 μM of Methyl jasmonate processing people's esophageal epithelial cells, the migration and invasion of people's esophageal epithelial cell
Ability is stronger, illustrates that 10 μM of Methyl jasmonates are better than 500nM and 200 μM of first to the carcinogenic strong fruit of people's esophageal epithelial cell
Base benzyl nitrosamine.
(5) tumor formation in nude mice
Using the male nude mouse (8 week old tie up experimental animal technology company of tonneau China purchased from Beijing) of BALB/c strain, animal
It adapts to environment and carries out tumor cell transplantation experiment after a week.Will contamination people's esophageal epithelial cell PBS adjust concentration to 1.0 ×
107Contamination 200 μ l of people's esophageal epithelial cell suspension is subcutaneously injected in nude mice oxter in/ml, and inoculating cell sum is 2 × 106It is a.With
Subcutaneous tumors tubercle diameter is more than that 0.5cm is that tumour forms standard, records tumor formation time and tumor formation rate.Nude mice is put to death after 3 weeks, is collected
Nude mice by subcutaneous tumour carries out histopathological examination.
Please refer to Fig. 7 A, it can be seen that after 10 μM of Methyl jasmonate processing people's esophageal epithelial cells, on people's oesophagus
The number that chrotoplast induces nude mice tumor formation is more, illustrates 10 μM of Methyl jasmonates to the carcinogenic effect of people's esophageal epithelial cell
It is better than 500nM and 200 μM of Methyl jasmonate.Fig. 7 B is please referred to, Study On Cytopathology is carried out, finds the cell of tumor formation
Characteristic still persistently exists after removing Methyl jasmonate, illustrates that irreversible vicious transformation has occurred for the tumor cells,
As dermoid cancer cell.
(6) metabolic pathway is analyzed
After clear Methyl jasmonate induces concentration and the temporal characteristics of the canceration of people's esophageal epithelial cell, collect different
The cell model sample at concentration and time point (each 3) carries out transcription group analysis, (is purchased from Affymetrix transcript profile chip
Affymetrix company of the U.S.) difference expression gene and protein phosphorylation horizontal analysis are detected, pass through different in cell model contaminate
The gene expression profile at time point (for 24 hours, 10 weeks, 30 weeks), which changes analysis, has the identical gene for changing rule, determines differential expression
The relationship of the specific metabolic pathway of gene and human body induces people's esophageal epithelial cell cancer for further research Methyl jasmonate
The toxicity access of change is provided fundamental basis.Table 1 is please referred to, the metabolism of the people's esophageal epithelial cell for canceration as the result is shown of contaminating for 24 hours
It is related with following 10 metabolic pathways.The bonding of the contamination metabolism of people's esophageal epithelial cell of canceration and cell as the result is shown in 10 weeks
It is related to connect associated metabolic access.Please refer to table 2,30 weeks contamination as the result is shown the metabolism of people's esophageal epithelial cell of canceration with
Lower 10 metabolic pathways are related.
The metabolism and the correlation of metabolic pathway of the people's esophageal epithelial cell of the contamination of table 1 for 24 hours
| Metabolic pathway | Correlation |
| The interaction of cell factor-cytokine receptor | 20.0% |
| NF-kappaB Cell signal propagation pathways | 12.5% |
| Hematopoietic cell lineage | 10.0% |
| Rheumatoid arthritis | 10.0% |
| Amcbiasis | 10.0% |
| Legionaires' disease | 7.5% |
| NOD sample recipient cell signal transduction pathway | 7.5% |
| ARVC disease | 7.5% |
| Pertussis | 7.5% |
| Hypertrophic cardiomyopathy | 7.5% |
The metabolism and the correlation of metabolic pathway for people's esophageal epithelial cell that table 2 is contaminated 30 weeks
| Metabolic pathway | Correlation |
| Glycosyl sphingolipid biosynthesis | 9.7% |
| Folic acid biological synthesis | 6.5% |
| It is thio to thank | 6.5% |
| Cytoskeleton regulation | 19.4% |
| Shigella | 9.7% |
| Cholinergic synapse | 12.9% |
| Adipocyte Factor signal transduction pathway | 9.7% |
| Galactose metabolism | 6.5% |
| The bacterium of epithelial cell invades | 9.7% |
| PPAR signal path | 9.7% |
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. the method for building up that a kind of chemicals induce the vitro detection model of human esophagus cancer, comprising the following steps:
The people's esophageal epithelial cell being seeded in in vitro culture container is provided；
It is cultivated in advance after applying the chemicals to be checked of multiple concentration within the scope of predetermined concentration respectively to people's esophageal epithelial cell
It fixes time, the cell viability that measurement is able to reflect people's esophageal epithelial cell applies the variation of concentration with the chemicals to be checked
Curve；
The concentration ranges of the corresponding chemicals to be checked of predetermined cell viability range in the curve are chosen as carcinogenic survey
Determine concentration ranges, chooses multiple concentration of the carcinogenic measurement concentration ranges as carcinogenic measurement concentration；And
The chemicals to be checked for applying the carcinogenic measurement concentration respectively to people's esophageal epithelial cell, carry out described to be checked
Chemicals detect the carcinogenicity testing of people's esophageal epithelial cell.
2. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In inoculum density of the people's esophageal epithelial cell in the culture vessel is 8 × 104/ ml~2 × 105/ml。
3. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In the minimum concentration of the predetermined concentration range is 0~100nmol/L, and the maximum concentration of the predetermined concentration range is
10mmol/L~100mmol/L.
4. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In the minimum cell viability within the scope of the predetermined cell viability is 90%~95%.
5. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In multiple concentration within the scope of the predetermined concentration are Geometric Sequence.
6. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In the multiple carcinogenic measurement concentration is Geometric Sequence.
7. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In the chemicals to be checked include ability of cell proliferation detection, cell to the carcinogenicity testing detection of people's esophageal epithelial cell
Migration and invasive ability detection and the chemicals to be checked induce one of animal tumor formation experiment detection or a variety of.
8. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In further including that induce people's esophageal epithelial cell to chemicals to be checked described in carcinogenicity testing detection thin at the tumour of cancer
Born of the same parents carry out gene expression analysis, determine the gene of differential expression and the relationship of body metabolism access.
9. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In, further include detection addition people's hepatomicrosome under the in vitro culture of people's esophageal epithelial cell cell viability influence step
Suddenly.
10. chemicals according to claim 1 induce the method for building up of the vitro detection model of human esophagus cancer, feature exists
In the condition of culture of people's esophageal epithelial cell is 5% carbon dioxide, and cultivation temperature is 37 DEG C.
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