CN110423725A - A kind of strain of human mesothelial cell vicious transformation and its application of chrysotile induction - Google Patents
A kind of strain of human mesothelial cell vicious transformation and its application of chrysotile induction Download PDFInfo
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- 210000005033 mesothelial cell Anatomy 0.000 title claims abstract description 58
- 229910052620 chrysotile Inorganic materials 0.000 title claims abstract description 31
- CWBIFDGMOSWLRQ-UHFFFAOYSA-N trimagnesium;hydroxy(trioxido)silane;hydrate Chemical compound O.[Mg+2].[Mg+2].[Mg+2].O[Si]([O-])([O-])[O-].O[Si]([O-])([O-])[O-] CWBIFDGMOSWLRQ-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 230000009466 transformation Effects 0.000 title claims abstract description 29
- 230000006698 induction Effects 0.000 title abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 77
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Abstract
The invention discloses the strain of human mesothelial cell vicious transformation and its application of a kind of induction of chrysotile, the human mesothelial cell vicious transformation strain classification namings are as follows: human mesothelial cell, strain number are as follows: AS-T, deposit number are as follows: CCTCC NO.C201923.The present inventor's pleural mesothelial cell vicious transformation strain is by human mesothelial cell (MeT-5A) after chrysotile multistage repeatedly contamination processing, vicious transformation occurs to obtain, has a good application prospect in many aspects such as the cell models studied as carcinogenic substance carcinogenic mechanism.
Description
Technical field
The present invention relates to biology and oncology technical field, more particularly to a kind of people's pleural mesothelium of chrysotile induction
Malignant transformation of cells strain and its application.
Background technique
Asbestos are the general names of the silicates minerals of natural threadiness, have high-tensile, high flexible, chemically-resistant
With thermal etching, electrical isolation and with spinnability.Asbestos mainly include crocidolite, chrysotile, amosite etc..IARC (international cancer
Research institution) all types of asbestos have been set to I class carcinogenic substance, i.e., all types of asbestos all can lead to the hair of malignant tumour
It is raw.Asbestos exposure results in the generation of 80% or more malignant mesothelioma.Malignant mesothelioma (malignant mesothelioma,
MM) incidence of occult, clinical manifestation lack specificity, and misdiagnosis rate is higher, and mostly middle and advanced stage, most of patient are making a definite diagnosis not when making a definite diagnosis
It will be dead in 1 year time.The developing Asian countries such as China, India, Thailand are the big countries of asbestos inlet and outlet, by inference
The trend that the disease incidence of the malignant mesothelioma of many decades rises year by year presentation from now on.Due to the pathogenesis to malignant mesothelioma
It is unclear that causing clinically to lack effective early diagnosis, treatment method.
Malignant transformation of cells model is the material of not only fabulous research carcinogenic substance carcinogenic mechanism, can be also used for pernicious
The basic research of rind gall Clinics and Practices.But since the genome comparison of human body cell is stable and the effective DNA repair mechanism of tool,
There is a stronger resistance to extrinsic damage, it is especially sensitive not as good as some zooblasts to carcinogenic damage, therefore it is relative to dynamic
More difficult conversion for object cell, therefore previously the general selection animal fibroblast cell of research active to cell carcinogenic carries out, such as
NIH/3T3 cell.We use people's normal rnesothelial cells, and gained cell model can directly reflect that chrysotile makees human body target cell
Actual conditions.Cell transformation is a kind of variation that hereditary change occurs for cell, is related to the change of DNA or gene, is one
A multifactor, multistage, multipath process.Thus, no matter use the processing normal human such as chemistry, physics, virus and oncogene
Cell is difficult to happen vicious transformation through 1 processing.
Therefore mesothelial cell's vicious transformation of chrysotile induction is extremely difficult, current rare report.(the Yang such as Yang H
H,Bocchetta M,Kroczynska B,et al.TNF-alpha inhibits asbestos-induced
cytotoxicity via a NF-kappaB-dependent pathway,a possible mechanism for
asbestos-induced oncogenesis.Proc Natl Acad Sci U S A.2006.103(27):10397-
10402.) with chrysotile induction of the vicious transformation of mesothelial cell, specific method is: mesothelial cell HM is total to macrophage
Culture, is added 5 μ g/cm after being pre-processed with the TNF-α of 10ng/ml2Chrysotile handle 48 hours, then continue with containing
The culture solution of TNF-α carries out culture 4 weeks, and as a result visible cell aggregation stove (foci) is formed.This method in the conversion process, uses
The technology co-cultured with macrophage, and joined the toxicity that TNF-α reduces chrysotile, this method is only with chrysotile independent
Processing does not use other methods or reagent；The foci that this method only has detected the culture cell in culture plate is formed, and without soft
Agar colony forms the not dependent growth that anchors of experimental verification transformed cells, and soft-fractrue rock mass experiment is verifying cell
The whether successful major criterion of vicious transformation, therefore this method not can prove that and have become function conversion.
Summary of the invention
The present invention mentions aiming at the problem that lacking mesothelial cell's malignant conversioning cell model of chrysotile induction in the prior art
The strain of human mesothelial cell vicious transformation and its application of a kind of chrysotile induction are supplied.
A kind of human mesothelial cell vicious transformation strain, classification naming are as follows: human mesothelial cell, strain number are as follows: AS-T, in
On January 23rd, 2019 is preserved in the China typical culture collection center (CCTCC) of the Wuhan University positioned at Wuhan, China, preservation
Number are as follows: CCTCC NO.C201923.The present inventor's pleural mesothelial cell AS-T is by human mesothelial cell (MeT-5A) through fire stons
The cotton multistage repeatedly occurs vicious transformation, has harvested one plant of cell, it is thin to be named as human mesothelial cell AS-T after contamination processing
Born of the same parents, the more original MeT-5A vitro growth rates of the cell are accelerated, and obtain layer-by-layer growth characteristic.
Invention further provides the progeny cells of the human mesothelial cell vicious transformation strain.
Invention further provides the human mesothelial cell vicious transformation strains to study as carcinogenic substance carcinogenic mechanism
Application in cell model.Wherein, the carcinogenic substance is chrysotile.
Invention further provides the human mesothelial cell vicious transformation strains to grind as malignant mesothelioma genesis mechanism
The application in cell model studied carefully.
Invention further provides the human mesothelial cell vicious transformation strains to extract malignant mesothelioma specific tumour
Application in marker.
Invention further provides the human mesothelial cell vicious transformation strains to screen or assess treatment malignant mesothelioma
Application in drug.
The present invention also provides the human mesothelial cell vicious transformation strains in exploitation malignant mesothelioma detection kit
In application.
It adopts international standards chrysotile dust, interruption contamination processing is carried out to normal person mesothelial cell, then removes fire stons
Cotton continues to cultivate, and is verified using not dependent experiment is anchor.As a result, after 28 contaminations and continuing to cultivate 15W, carefully
Born of the same parents obtain layer-by-layer growth, anchor the malignant characteristics such as not dependent.Detect the Apoptosis of chrysotile transformed cells, the cell cycle,
Mitochondrial membrane potential, activated oxidized water are averagely changed.The present invention by for the Mechanism Study of malignant mesothelioma caused by chrysotile,
The basic research offer experiment cell model of early diagnosis, treatment.
Detailed description of the invention
Fig. 1 is cytoactive detection result figure, wherein * indicates that p < 0.05, * * indicate that p < 0.01, * * * indicate p < 0.001
(similarly hereinafter).
Fig. 2 is cell layer-by-layer growth microscopic findings figure, wherein figure A is MeT-5A cell, and figure B is AS-T MeT-5A
Cell.
Fig. 3 is that cell anchors not dependent growth experiment testing result figure, wherein figure A is MeT-5A cell, and figure B is AS-
T MeT-5A cell, figure C are the statistical result of two kinds of cells.
Fig. 4 is that cellscan Electronic Speculum observes result figure, wherein figure A is MeT-5A cell, and figure B is that AS-T MeT-5A is thin
Born of the same parents.
Fig. 5 is flow cytomery changes of cell apoptosis result figure, wherein figure A is MeT-5A cell, and figure B is AS-T
MeT-5A cell, figure C are statistical result.
Fig. 6 is the flow cytomery cell cycle to change statistical results chart.
Fig. 7 is mitochondrial membrane potential flow cytometer detection result figure, wherein figure A is MeT-5A cell, and figure B is AS-T MeT-5A
Cell, figure C are statistical results chart.
Fig. 8 is the horizontal testing result figure of active oxygen ROS, wherein figure A is MeT-5A cell, and figure B is that AS-T MeT-5A is thin
Born of the same parents, figure C are statistical results chart.
Specific embodiment
Embodiment 1
Fiber preparation: chrysotile comes from Japanese mineral fibres association.5mg chrysotile asbestos fibre is weighed when use is suspended from 1ml
In PBS liquid, it is made into the mother liquor of 5mg/ml, ultrasonic treatment becomes uniform suspension in ice water.Ultrasound condition: 180W (watt),
10s is opened, and 5s is closed, totally 30 circulations.High pressure sterilization after ultrasound.
Cell culture: human mesothelial cell (MeT-5A) with contain 10% fetal calf serum M199 culture solution, 37 DEG C,
5%CO2It cultivates, passes on 1 time in the environment of saturated humidity within cell every 3-4 days, by 1: 3 inoculation.Cell is divided into conversion group and feminine gender
Control group, every group sets 3 holes and does parallel control.
Transformation experiment: the cell of logarithmic growth phase is digested through the pancreatin that mass fraction is 0.25%, is counted after piping and druming dispersion
Number, is diluted to 1~2 × 10 with full nutrient solution5The density of a cells/well is inoculated on 6 well culture plates, is put into 37 DEG C of incubators
In.After cell inoculation for 24 hours, old culture solution is discarded, conversion group, which is added, uses the diluted 5 μ g/cm of PBS2Chrysotile, control group are added same
Isometric PBS, every group sets three Duplicate Samples.After for 24 hours, culture medium and chrysotile asbestos fibre are absorbed, is changed after being rinsed 3 times with PBS often
Advise culture medium.With chrysotile be interrupted contamination processing human mesothelial cell for 24 hours/it is secondary, once a week, totally 28 times, no longer use thereafter
Chrysotile contamination continues to cultivate 15W (week) with the M199 containing 10% fetal calf serum.Micro- sem observation is used in experimentation and is remembered
Record cell growth and metamorphosis.
After the experimental method that multistage is repeatedly contaminated carries out vicious transformation to MeT-5A cell, one plant of cell has been harvested,
Classification naming are as follows: human mesothelial cell, strain number are as follows: AS-T was preserved in big positioned at Wuhan, China Wuhan on January 23rd, 2019
China typical culture collection center (CCTCC), deposit number are as follows: C201923, the more original MeT-5A cell growth of the cell
Speed accelerates (Fig. 1), and obtains layer-by-layer growth characteristic (Fig. 2).
Anchor not dependent growth experiment: cell takes the every hole of 6 orifice plates to be added after processing 72 times and persistently culture 2 months
It is complete that 0.35% agar-of the top layer 3ml containing 1000 cells to be measured is added in 0.7% bottom-layer agar of 3ml-full culture medium after to be solidified
Culture medium, every group of cell set 3 Duplicate Samples, cultivate 3 weeks, count the colony-forming efficiency of every group of cell, meters more than 50 cells
For 1 colony.The blank control group cell of synchronous culture equally carries out cultivating in soft agar according to the above method and observes colony shape
At situation.Cell shows layer-by-layer growth and anchors not dependent growth characteristics (Fig. 3) after multiple contamination processing, successfully obtains evil
Property transformed cells model.
And the variation occurred with scanning electron microscopic observation malignant conversioning cell AS-T cell interior, compared to MeT-5A cell,
Cell without chrysotile conversion, visible cell edge is observed under scanning electron microscope protrusion, and organelle is clear (Fig. 4 A)；Through temperature
The cell of asbestos conversion, it is seen that cell edges are smooth, and cell rounding, nucleus becomes larger, and organelle significantly reduces, it is seen that abnormal wire
Plastochondria and meat net (Fig. 4 B).
Comparative example 1
By 1 same procedure culture cell of embodiment, but transformation experiment process is different, is turned in the following manner respectively
Change experiment:
1, chrysotile discontinuity treatment 28W (week), without chrysotile exposure remove after continue culture (i.e. relative to reality
Cultural method in example 1 is applied, no longer carries out subsequent culture in 15 weeks here).
2, it while chrysotile discontinuity treatment, is added 800ng/ml carcinogenic promoting agent phorbol exters (TPA), after processing 28W (week), into
It goes or without continuing to cultivate 15W (week) after removing processed material.
3, while chrysotile discontinuity treatment, be added 0.5 μ g/ml TNF-α, processing 28W (week) after, progress or without
Continue to cultivate 15W (week) after removing processed material.
Equally using the growth of micro- sem observation cell and metamorphosis in experimentation, and carry out soft-fractrue rock mass reality
It tests, as a result the above each group all forms clone not on soft agar, i.e., does not obtain cell transformation.
Embodiment 2
Flow cytomery Apoptosis changes.
Cell is paved with through secondary culture to single layer, is collected in centrifuge tube after pancreatin digestion, and 1000g is centrifuged 5min, abandons training
Nutrient solution；PBS, which suspends, washs cell, 1000g, 5min centrifuge cell；Annexin is added in the combination buffer suspension cell of 500 μ l
15 μ l of V-FITC/PI, is placed at room temperature for 5min；Carry out flow cytometry analysis.The results show that compared with MeT-5A cell, through temperature
There is resistance to apoptosis characteristic in the AS-T MeT-5A cell of asbestos conversion, and Apoptosis substantially reduces (Fig. 5).
Embodiment 3
The variation of flow cytomery cell cycle.
Cell is paved with through secondary culture to single layer, is collected in centrifuge tube after pancreatin digestion, 1000g is centrifuged 5min；PBS is outstanding
Floating washing is washed cell 2 times, and 1000g, 5min are collected after centrifugation；Again with 0.3ml PBS again abundant suspension cell, cell is avoided to have knot
Group's phenomenon；The straight alcohol of pre-cooling, fixed at least 2h are added with final concentration 70-75% ratio；1000g is centrifuged 5min, discards ethyl alcohol；
1ml PBS, which suspends, cleans cell precipitation, places 1min, and 1000g is centrifuged 5min；It is outstanding with 500 μ l PI/Triton X-100 dye liquors
Cell precipitation is floated, is detected after 37 DEG C of placement 15min using flow cytometer.As a result as shown in fig. 6, MeT- compared to the control group
5A cell, chrysotile transformed cells G1 phase ratio shorten, and G2+M phase ratio increases, and illustrate that chrysotile transformed cells are arrested in the G1 phase
Ratio reduce, show chrysotile transformed cells proliferation accelerate.
Embodiment 4
Mitochondrial membrane potential (MMP) flow cytometer detection.
Cell is paved with through secondary culture to single layer, and pancreatin digests group of cells；Culture is added by every group of 1 × 105 cell
Liquid 1mL is washed 2 times, and supernatant is abandoned after centrifugation；0.5mL cell culture fluid is added to be resuspended.0.5mL JC-1 dyeing working fluid is added, overturns
It mixes for several times.37 DEG C of incubation 20min in cell incubator；Cell (1000rpm/min, 5min) is collected by centrifugation and abandons supernatant afterwards.With
1mL JC-1 dye solution washs 2 times；It is added after 0.5mL JC-1 staining buffer is resuspended with standardization program flow cytometer
Detection, mercury excitation wavelength 488nm count 104A cell.
As a result as shown in fig. 7, MeT-5A cell, AS-T MeT-5A mitochondrial membrane potential in anoxic slightly increase compared to the control group
Height is in significant difference.
Embodiment 5
The horizontal testing result of active oxygen ROS.
Cell is paved with through secondary culture to single layer, discards culture solution, and the diluted DCFH-DA 1mL of serum-free medium is added
(1: 1000 dilution)；20min is incubated in 37 DEG C of cell incubators.Cell is washed three times with serum-free cell culture medium；Pancreatin disappears
Change 2 groups of cells；Cell (1000rpm/min, 5min) is collected by centrifugation and abandons supernatant afterwards；Flow cytomery, 488nm excitation, meter
Number 104A cell.As a result as shown in figure 8, MeT-5A cell, AS-T MeT-5A cell ROS level reduce compared to the control group, it is in
Significant difference.
Claims (8)
1. a kind of human mesothelial cell vicious transformation strain, which is characterized in that it is named as human mesothelial cell, strain AS-T,
Deposit number is CCTCC NO.C201923.
2. the progeny cell of human mesothelial cell vicious transformation strain as described in claim 1.
3. human mesothelial cell vicious transformation strain as described in claim 1 is in the cell membrane studied as carcinogenic substance carcinogenic mechanism
Application in type.
4. application as claimed in claim 3, which is characterized in that the carcinogenic substance is chrysotile.
5. human mesothelial cell vicious transformation strain as described in claim 1 is as the thin of malignant mesothelioma genesis mechanism research
Application in born of the same parents' model.
6. malignant mesothelioma specific tumour marker is being extracted in human mesothelial cell vicious transformation strain as described in claim 1
In application.
7. human mesothelial cell vicious transformation strain as described in claim 1 is in screening or assessing treatment malignant mesothelioma drug
Application.
8. human mesothelial cell vicious transformation strain as described in claim 1 answering in exploitation malignant mesothelioma detection kit
With.
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