CN107828729A - A kind of human lung cancer A549 derived cell strains and its preparation method and application - Google Patents
A kind of human lung cancer A549 derived cell strains and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of human lung cancer A549 derived cell strains, the cell line is named as Non-small cell lung carcinoma cell line A549 SE06, and its deposit number is CGMCC No.14315.In addition, the invention also discloses the preparation method and its usage of the cell line.The change of morphology and biological phenotype that the present invention can be used for after analysis lung carcinoma cell mutation；Research influences molecular mechanism and the developed by molecule change of tumor cell proliferation invasive procedure；Contacting between research correlation molecule albumen and patients with clinical manifestations and its influence to patient's prognosis；It is included in research lung cancer tumor cell propagation molecular mechanism and specific protein molecule to the application in clinical prognosis influence；Application in lung cancer tumor animal model is prepared；Application in pulmonary cancer diagnosis reagent is prepared；And the application in lung cancer tumor drug target is developed.Experiments verify that the present inventor's lung cancer A549 derived cells strain A549 SE06 have a faster multiplication rate and stronger multiplication capacity than A549 cell line, the speed of growth is accelerated, and nude mice shortened into the knurl time.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of lung cancer cell line, more particularly to a kind of human lung cancer
A549 derived cell strains, the invention further relates to a kind of preparation method and purposes of human lung cancer A549 derived cell strains in addition.
Background technology
Lung cancer is as a kind of common malignant tumour, and in annual newborn cancer crowd, patients with lung cancer proportion is about
For 12.7%, men and women patient's proportion is 2.1:1, the age was at 60 years old or more when about 75% patient makes a definite diagnosis.Patients with lung cancer
Generally there is poorer prognosis, patient's five year survival rate is between 6-18%.Further to study the occurrence and development mechanism of lung cancer,
Applicant buys A549 cell lines in Chinese Academy of Sciences's cell bank, and animal experiment study is carried out on nude mice.The A549 cell lines by
D.J.Griad transplants culture by cancerous lung tissue, and patient is 58 years old Caucasian male, and the correlative study for being mainly used in lung cancer is worked as
In, medicine is such as studied to the lethal effect of lung carcinoma cell or for nude mice into knurl.It is stable to test initial tumor formation rate, through repeatedly passage
Afterwards, one-tenth knurl ability progressively weakens, until completely not into knurl, we be inoculated with May, 2,016 5 nude mice armpits subcutaneously carry out into
Knurl is tested, and is showed no into knurl in 2 months, and mouse is untreated, prepare April to find during processing mouse to have in 5 nude mices four into
Knurl, continuing observation and find that tumour growth is swift and violent, put to death mouse, tumour grinding is cultivated, when cell growth state is good, monoclonal
Tumor cell line is sorted, the cell after separation carries out check experiment with former A549 cells and done cellular identification, finds the cell
For A549 mutation stains, western bloting detections find mutantion line cellular portions protein abnormal expression, mutantion line cell
Zoopery is carried out with former A549 cells and finds that the growth of mutantion line cell tumour is swift and violent, and former A549 cells tumor formation rate is extremely low, growth
Heterogeneity.This A549 mutantion line can be used for substituting former A549 cells, for being studied before lung cancer clinical.
The content of the invention
One of the technical problem to be solved in the present invention is to provide a kind of human lung cancer A549 derived cell strains.
Present invention solves the technical problem that two be to provide a kind of preparation method of human lung cancer A549 derived cell strains.
Present invention solves the technical problem that three be to provide a kind of purposes of human lung cancer A549 derived cell strains.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
In one aspect of the invention, there is provided a kind of human lung cancer A549 derived cell strains, cell line name is people's non-small cell lung
Cancerous cell line A549-SE06, its deposit number are CGMCC No. 14315.Human lung cancer A549 derived cells strain is in 2017
On August is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 18（Deposit number is CGMCC No.
14315. preservation place：China, Beijing）.
The biological characteristics of the Non-small cell lung carcinoma cell line A549-SE06 is：Cell is in Epithelial, polygonal,
Adherent growth, cell is mellow and full full, and growth is rapid, and the cell percentage of the cell cycle each phase is respectively G0-G1Phase
35.45%, S phase 47.25%, G2- M the phases 17.30%, show tumor cell proliferation characteristic.
In the second aspect of the present invention, there is provided a kind of preparation method of human lung cancer A549 derived cell strains, including following step
Suddenly：Step 1, typeⅡ pneumocyte system are inoculated in nude mice by subcutaneous, into knurl after the several months；Step 2, institute is taken to disappear into tumor mass, shearing
After change, add in 1640 culture mediums and cultivate；Step 3, by repeatedly it is adherent purifying, select monoclonal after obtain single cell, i.e.,
For human lung cancer A549 derived cell strains.
As currently preferred technical scheme, the preparation methods of above-mentioned human lung cancer A549 derived cell strains specifically include as
Lower step：
（1）TypeⅡ pneumocyte system is inoculated in nude mice by subcutaneous, into knurl after the several months；
（2）Taking A549 nude mice by subcutaneous institute, after shearing digestion, centrifugation removes trypsase, is transferred in Tissue Culture Flask into tumor mass,
Add 1640 culture mediums；
（3）Culture a period of time, after there are a large amount of cell attachments, bottle inner tissue is removed, adds 1640 culture medium；
（4）When cell growth to 70-80% or so, with tryptic digestive juice vitellophag, the training without serum is used after centrifugation
Base is supported to be resuspended；Cell suspension is added in blake bottle, 15-20min is stood, is observed under inverted microscope, sees part cell patch
Wall, slightly shake when also not floating, cell suspension is imported in another blake bottle；
（5）Above steps may be repeated multiple times（4）In process, gradually fibroblast and other cellular compartments are opened, to last
Criticize the adherent obtained cell picking monoclonal of suspension, the human lung cancer A549 derived cell strains isolated and purified.
As currently preferred technical scheme, step（2）、（3）、（4）、（5）Condition of culture be： 37℃、5%CO2、
Cultivated under 95% damp condition.
In the third aspect of the present invention, there is provided the purposes of human lung cancer A549 derived cell strains, be included in study of lung cancerous swelling
Tumor cell proliferation molecular mechanism and specific protein molecule are on the application in clinical prognosis influence；Preparing lung cancer tumor animal model
In application；Application in pulmonary cancer diagnosis reagent is prepared；And the application in lung cancer tumor drug target is developed.The present invention
Change available for morphology and biological phenotype after analysis lung carcinoma cell mutation；Research influences tumor cell proliferation and attacked
Molecular mechanism and the developed by molecule change of journey；Contacting and its to patient between research correlation molecule albumen and patients with clinical manifestations
The influence of prognosis.
Morphological observation and Identification of Biological Characteristics are carried out to the A549 derived cells strain that this method is established, as a result shown
A549 derived cell strain cellular morphologies have a slight change, and the speed of growth is accelerated, and nude mice shortened into the knurl time.Experiments verify that this
Invention human lung cancer A549 derived cell strain A549-SE06 have faster multiplication rate and stronger propagation energy than A549 cell line
Power.
Brief description of the drawings
Fig. 1 is the observation figure of A549 cells under inverted microscope in the embodiment of the present invention 2（×100）；
Fig. 2 is the observation figure of A549-SE06 cells under inverted microscope in the embodiment of the present invention 2（×100）；
Fig. 3 is the growth curve figure of A549 cells and A549-SE06 cells in the embodiment of the present invention 2；
Fig. 4 is the cycle result figure of A549 cells in the embodiment of the present invention 2；
Fig. 5 is the cycle result figure of A549-SE06 cells in the embodiment of the present invention 2；
Fig. 6 is the animal of A549 cells and A549-SE06 cells in the embodiment of the present invention 2 into knurl result figure.
The Non-small cell lung carcinoma cell line that code name is A549-SE06 is deposited in the micro- life of China on the 18th in August in 2017
Thing culture presevation administration committee common micro-organisms center（Deposit number is CGMCC No. 14315；Preservation place：Chinese north
The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1, Institute of Microorganism, Academia Sinica）.
Embodiment
The following examples can make those skilled in the art that the present invention be more fully understood, but not limit in any way
The present invention.
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used, is normal unless otherwise specified in following embodiments
Rule biochemical reagents shop is commercially available.
Embodiment 1
The present invention establishes human lung cancer A549 derived cell strains using multiple purification process that is adherent, selecting monoclonal.Specific steps
It is as follows：
（1）TypeⅡ pneumocyte strain（Purchased from Chinese Academy of Sciences's Shanghai cell bank, frozen by this laboratory）, DMEM is used after recovery
Nutrient solution（Containing 10% hyclone, penicillin 100U/ml, the μ g/ml of streptomysin 100）In 37 DEG C, 5%CO2, under 95% damp condition
Culture；
（2）The digestion of the growth period A549 that takes the logarithm cells trypsinised liquid, is counted, adjustment density for every nude mice 5 ×
106/ 200 μ l, mix in physiological saline, are inoculated in nude mice by subcutaneous.Nude mice does not grow tumor mass in experiment setting time, continues
After raising the several months, nude mice is suddenly into knurl.After taking tumor mass to carry out shearing digestion, 1640 culture mediums are added（Containing 10% hyclone, green grass or young crops
Mycin 100U/ml, the μ g/ml of streptomysin 100）In 37 DEG C, 5%CO2, cultivate under 95% damp condition；
（3）Culture a period of time, it was observed that after there are a large amount of cell attachments, bottle inner tissue is removed, adds new 1640 culture medium；
（4）When cell growth is to 80% or so, with tryptic digestive juice vitellophag, the 1640 of not increase serum is used after centrifugation
Culture medium（Gibco, 61870036）It is resuspended.Cell suspension is added in blake bottle, 20min is stood, is seen under inverted microscope
Examine, see part cell attachment, slightly shake when also not floating, cell suspension is imported in another blake bottle.
（5）Above steps may be repeated multiple times（4）In process, gradually fibroblast and other cellular compartments are opened, to most
The adherent obtained cell picking monoclonal of later batch suspension, the human lung cancer A549 derived cell strains isolated and purified.
After cell growth is stable, part cell is frozen in liquid nitrogen, frozen stock solution presses FBS：1640 culture mediums：DMSO ratios
5:4:1 proportioning obtains, and freezes process and progressively cools, and takes 4 DEG C 30 minutes → 20 DEG C 1 hour → -80 DEG C overnight → liquid nitrogen
Order is carried out.The recovery of freeze-stored cell is carried out by following procedure：At least 10ml is added in 10cm Tissue Culture Dish to contain
The nutrient solution of 10% hyclone；The cryopreservation tube equipped with cell is taken out from liquid nitrogen, being immediately placed in 37 DEG C of water-baths and gently shaking makes
Freeze thing to thaw in 1 minute, wiped with cotton ball soaked in alcohol and taken after freezing pipe outer wall into super-clean bench；By the cell suspension after defrosting from
The heart, frozen stock solution is removed, is resuspended with 1640 culture medium.Cell suspension is moved into the culture dish for having added nutrient solution, slightly shaken
Mix, keep flat in CO2gas incubator, in 37 DEG C, 5% CO2, cultivate under 95% damp condition, about 24 hour cells are adherent
After change fresh medium 5-10ml and continue to cultivate.
The human lung cancer A549 derived cells strain that embodiment 2 is established to the method for the application present invention carries out morphological observation and biology
Learn CHARACTERISTICS IDENTIFICATION：
1. micro- sem observation cellular morphology
Take the logarithm growth period A549 cell and each one bottle of A549 derived cells, change to observe under inverted phase contrast microscope after liquid and live
Cellular morphology simultaneously takes pictures.A549 cells are as shown in figure 1, A549-SE06 derived cells are as shown in Figure 2.It is as shown in Fig. 2 of the invention
Non-small cell lung carcinoma cell line A549-SE06 cell is in Epithelial, and polygonal, adherent growth, cell is mellow and full full, growth
Rapidly.
2. CCK8 colorimetric method for determining cell proliferation rates
To take the logarithm respectively growth period A549 cell and A549 derived cells, cell dissociation, counting, adjustment cell density be 3 ×
104Individual/mL, it is inoculated in 96 well culture plates, 100 μ l/ holes.Put 37 DEG C, CO2Volume fraction is to be cultivated in 5% constant incubator.
Add 10 μ l CCK8 detection reagents into hole in 0h, 24h, 48h and 72h respectively, be incubated 3 hours in incubator, use all-wave
Long ELIASA determines absorbance（A450 and A620）, experiment is repeated 3 times, averages.Using the time as abscissa, absorbance
Value（A450-A620）Mapped for ordinate, obtain the cell growth curve of two kinds of cells, as a result show, after cultivating 24h, A549-
The strain of SE06 derived cells has faster multiplication rate than A549 cell line, and CCK8 growth curves are shown in Fig. 3.
3. the flow cytomery cell cycle
Take the logarithm growth period A549 cell and A549-SE06 cells paving 6cm culture dishes, use trypsin digestion and cell after 24h,
Cell is centrifuged to 1.5mlEP bottom of the tube.Add 70% ethanol of 1ml -20 DEG C of precoolings（PBS is configured）Fixed cell, will be fixed
Cell is positioned over -20 DEG C overnight, takes out within second day cell, and 1000rpm x 5min centrifugations remove ethanol and rinsed once with PBS,
Remove the ethanol of remaining.Remaining a little PBS, adds the PI solution that 300ul contains 10ug/ml RNase in pipe after cell is mixed
Cell is resuspended, flow cytometer detection is carried out after lucifuge 30min.Cell cycle, cell cycle distribution was shown in Table 1 as shown in Figure 4 and Figure 5：
As a result the S phase cell showed increaseds of A549-SE-06 cell lines are shown, are 47.25%, and A549 cells are 45.85%; A549-
The G0-G1 phase cells of SE-06 cells significantly reduce, and prompt A549-SE-06 cells to have stronger multiplication capacity.
A549 the and A549-SE-06 cell cycle distribution tables of table 1
4. nude mice by subcutaneous inoculation observation cell one-tenth knurl ability
Take the logarithm growth period A549 cell and A549-SE06 cells, count, adjustment density is every nude mice 5 × 106/ 200 μ l,
Mix in physiological saline, subcutaneous vaccination is in the left and right sides of same nude mice respectively.Compare two kinds of cell one-tenth knurl abilities, such as scheme
Shown in 6, A549-SE06 cells are more than A549 cells into knurl into knurl, as a result show that A549-SE06 cells of the present invention accelerate knurl
The speed of growth, nude mice into the knurl time shorten.
Claims (9)
1. a kind of human lung cancer A549 derived cell strains, it is characterised in that the cell line is named as Non-small cell lung carcinoma cell line
A549-SE06, its deposit number are CGMCC No. 14315.
2. human lung cancer A549 derived cell strains as claimed in claim 1, it is characterised in that the Non-small cell lung carcinoma cell line
A549-SE06 biological characteristics is：Cell is in Epithelial, and polygonal, adherent growth, cell is mellow and full full, and growth is rapid, should
The cell percentage of cell cycle each phase is respectively G0-G1Phase 35.45%, S phases 47.25%, G2- M the phases 17.30%, show to swell
Tumor cell proliferation characteristic.
A kind of 3. preparation method of human lung cancer A549 derived cell strains according to claim 1, it is characterised in that including with
Lower step：Step 1, typeⅡ pneumocyte system are inoculated in nude mice by subcutaneous, into knurl after the several months；Step 2, institute is taken to be cut into tumor mass
After cutting digestion, add in 1640 culture mediums and cultivate；Step 3, by repeatedly adherent purifying, select monoclonal after obtain it is single thin
Born of the same parents, as human lung cancer A549 derived cells strain.
4. according to the method for claim 3, it is characterised in that specifically comprise the following steps：
TypeⅡ pneumocyte system is inoculated in nude mice by subcutaneous, into knurl after the several months；
Taking A549 nude mice by subcutaneous institute, after shearing digestion, centrifugation removes trypsase, is transferred in Tissue Culture Flask, adds into tumor mass
Enter 1640 culture mediums；
Culture a period of time, after there are a large amount of cell attachments, bottle inner tissue is removed, adds 1640 culture medium；
When cell growth to 70-80% or so, with tryptic digestive juice vitellophag, the culture without serum is used after centrifugation
Base is resuspended；Cell suspension is added in blake bottle, 15-20min is stood, is observed under inverted microscope, see part cell attachment,
Slightly shake when also not floating, cell suspension is imported in another blake bottle；
Above steps may be repeated multiple times（4）In process, gradually fibroblast and other cellular compartments are opened, last batch of is hanged
The adherent obtained cell picking monoclonal of liquid, the human lung cancer A549 derived cell strains isolated and purified.
A kind of 5. preparation method of human lung cancer A549 derived cell strains according to claim 4, it is characterised in that step
（2）、（3）、（4）、（5）Condition of culture be： 37℃、5%CO2, cultivate under 95% damp condition.
6. a kind of human lung cancer A549 derived cells strain according to claim 1 is in research lung cancer tumor cell propagation molecule machine
System and specific protein molecule are on the application in clinical prognosis influence.
A kind of 7. human lung cancer A549 derived cells strain according to claim 1 answering in lung cancer tumor animal model is prepared
With.
A kind of 8. application of the human lung cancer A549 derived cells strain according to claim 1 in pulmonary cancer diagnosis reagent is prepared.
A kind of 9. human lung cancer A549 derived cells strain according to claim 1 answering in lung cancer tumor drug target is developed
With.
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Cited By (2)
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| CN111304085A (en) * | 2020-03-31 | 2020-06-19 | 中国科学院合肥物质科学研究院 | Biological sample culture device suitable for static magnetic field environment and application thereof |
| CN115197915A (en) * | 2022-08-01 | 2022-10-18 | 首都医科大学附属北京胸科医院 | Malignant phenotype large cell lung cancer cell strain and application thereof |
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| CN111304085A (en) * | 2020-03-31 | 2020-06-19 | 中国科学院合肥物质科学研究院 | Biological sample culture device suitable for static magnetic field environment and application thereof |
| CN115197915A (en) * | 2022-08-01 | 2022-10-18 | 首都医科大学附属北京胸科医院 | Malignant phenotype large cell lung cancer cell strain and application thereof |
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